JP2020529605A - Filtration and chromatography pods and how to use them - Google Patents

Abstract

Methods for capturing and purifying recombinant proteins from liquid cell cultures, including dual-use filtration and chromatography pods, filtration and chromatography pods, and the use of these pods are provided herein.

Description

Cross-reference to related applications This application claims priority to US Patent Provisional Application No. 62 / 539,943 filed August 1, 2017, the entire contents of which are incorporated herein by reference.

Technical Field This disclosure relates to the field of biotechnology, and more specifically to the capture and purification of recombinant proteins.

Background Current techniques and tools for producing recombinant proteins are often labor-intensive, time-consuming, costly, and error-prone. Protein recovery can be compromised, especially for each process that requires user intervention.

Summary This disclosure is based, at least in part, on the generation of new filtration and chromatography pods for capturing and purifying recombinant proteins from liquid cell cultures. As detailed below and illustrated in the Examples, the filtration and chromatography pods of the present invention provide, for example, improved speed, ease of use, improved protein stability, and multiple culture vessels and other consumables. It offers several benefits over traditional methods of purifying recombinant proteins, such as eliminating the need for use, which allows for rapid capture and purification of the recombinant protein of interest. Other advantages of the invention claimed in this application are also described herein.

Accordingly, provided herein are pods for filtration and chromatography, including containers formed by walls and having top and bottom edges. The wall forms a seal with the bottom edge. And one or more of the walls, top, and bottom includes at least one area covered with a semi-porous or porous material that allows liquid to reversibly flow from outside the pod into the inside of the pod. .. In some embodiments of any of the pods described herein, the lower end comprises at least one area covered with a semi-porous or porous material.

In some aspects of any of the pods described herein, the semi-porous or porous material does not allow (a) cells to flow into the pod from outside the pod, and (b) resin. Is characterized by one or both of holding or substantially holding the inside of the pod. In some embodiments, the semi-porous or porous material does not allow cells to flow into the pod from outside the pod. In some embodiments, the semi-porous or porous material retains or substantially retains the resin inside the pod. In some aspects of any of the pods described herein, the semi-porous or porous material does not allow cells to flow from outside the pod into the pod, and retains the resin inside the pod. Or characterized by substantially retaining. In some embodiments of any of the pods described herein, the cells are mammalian cells. In some embodiments of any of the pods described herein, the cells are insect cells or yeast cells. In some embodiments of any of the pods described herein, the cells are bacterial cells (eg, E. coli cells). In any of the pods, methods, kits, or some aspect of any other aspect described herein, the cell is not a bacterial cell. In some embodiments, the semi-porous or porous material retains or substantially retains the resin inside the pod structure.

In some embodiments of any of the pods described herein, the material is a semipermeable or porous membrane. In some aspects of any of the pods described herein, the material is a semipermeable or porous mesh. In some embodiments, the mesh is coated with a hydrophilic or hydrophobic material. In some embodiments of any of the pods described herein, the mesh is made of nylon or polyester. In some aspects of any of the pods described herein, the material has a pore size of about 50 nm to about 100 μm. In some aspects of any of the pods described herein, the material has a pore size of about 50 nm to about 50 μm. In some aspects of any of the pods described herein, the material has a pore size of about 100 nm to about 50 μm. In some aspects of any of the pods described herein, the material has a pore size of about 10 μm to about 50 μm. In some aspects of any of the pods described herein, the material has a pore size of about 20 μm to about 50 μm. In some aspects of any of the pods described herein, the material has a pore size of about 100 nm to about 30 μm. In some aspects of any of the pods described herein, the material has a pore size of about 500 nm to about 20 μm. In some aspects of any of the pods described herein, the material has a pore size of about 1 μm to about 10 μm. In some aspects of any of the pods described herein, the material has a pore size of about 2 μm to about 5 μm.

In some embodiments of any of the pods described herein, the container has an internal volume of about 0.1 mL to about 500 mL. In some embodiments of any of the pods described herein, the container has an internal volume of about 0.1 mL to about 250 mL. In some aspects of any of the pods described herein, the container has an internal volume of about 0.1 mL to about 100 mL. In some embodiments of any of the pods described herein, the lower end comprises at least one area covered by the material. In some aspects of any of the pods described herein, the wall comprises at least one area covered by the material. In some aspects of any of the pods described herein, the bottom edge and the wall each include at least one area covered by the material. In some embodiments of any of the pods described herein, the upper end comprises at least one area covered by the material. In some aspects of any of the pods described herein, the top and bottom edges each include at least one area covered by the material. In some embodiments of any of the pods described herein, each of the at least one area has a surface area of about 1 mm ² to about 25 cm ² . In some embodiments of any of the pods described herein, each of the at least one area has a surface area of about 1 mm ² to about 20 cm ² . In some embodiments of any of the pods described herein, each of the at least one area has a surface area of about 1 mm ² to about 10 cm ² . In some embodiments of any of the pods described herein, each of the at least one area has a surface area of about 1 mm ² to about 5 cm ² . In some embodiments of any of the pods described herein, each of the at least one area has a surface area of approximately 1 mm ² and 2.5 cm ² . In some embodiments of any of the pods described herein, each of the at least one area has a surface area of about 1 mm ² and about 1 cm ² .

In some aspects of any of the pods described herein, the bottom edge is flat. In some embodiments, the lower end is hemispherical. In some embodiments of any of the pods described herein, the lower end has a conical shape. In some aspects of any of the pods described herein, the wall has a cylindrical shape. In some aspects of any of the pods described herein, the wall has a conical shape. In some aspects of any of the pods described herein, the walls are spherical. In some aspects of any of the pods described herein, the walls are hemispherical. In some aspects of any of the pods described herein, the wall has an hourglass shape.

In some aspects of any of the pods described herein, the pod further comprises a chromatographic resin placed inside the container. In some aspects of any of the pods described herein, the chromatographic resin is a commercially available chromatographic resin. In some aspects of any of the pods described herein, the chromatography resin is an affinity chromatography resin. In some embodiments of any of the pods described herein, the affinity chromatography resin is a protein A resin, a protein G resin, a protein L resin, a nickel resin, or a magnetic bead resin. In some aspects of any of the pods described herein, the chromatographic resin has a volume of about 0.1 mL to about 500 mL. In some aspects of any of the pods described herein, the chromatographic resin has a volume of about 0.1 mL to about 250 mL. In some aspects of any of the pods described herein, the chromatographic resin has a volume of about 0.1 mL to about 100 mL. In some aspects of any of the pods described herein, the chromatographic resin has a volume of about 0.1 mL to about 50 mL. In some aspects of any of the pods described herein, the chromatographic resin has a sterility assurance level of about 1 x 10 ^-6 to about 1 x 10 ^-10 .

Kits containing any of the pods described herein are also provided herein. Some aspects of these kits may further include instructions for using any of the pods described herein. Some aspects of any of the kits provided herein may further include means for stably positioning the pod inside the culture vessel. In some embodiments of any of the kits described herein, the culture vessel is a shaking flask, shaking tube, bioreactor, or multi-well plate. In some aspects of any of the kits described herein, the bioreactor is a perfusion bioreactor. In some embodiments of any of the kits described herein, the bioreactor is a feed batch bioreactor.

With any of the pods described herein; (b) each of the one or more areas on the wall to prevent fluid from leaking from inside the pod to outside the pod through the wall. Kits containing, with, one or more panels that can be securely secured on top, are also provided herein.

With any of the pods described herein; (b) at least one of the walls to prevent fluid from leaking from the inside of the pod to the outside of the pod through the at least one area of the wall. A kit including an outer sleeve, which can be securely secured on the pod to cover the area, is also provided herein. In some embodiments, the sleeve is an empty chromatographic column.

One or both of (a) pods of any of the pods described herein; (b) elution outlets that can be stably anchored on the bottom edge, and buffer reservoirs that can be stably anchored on the top edge. Kits containing, and are also provided herein. In some aspects of any of the kits described herein, the kit comprises a pod and an elution outlet. In some embodiments of any of the kits described herein, the kit comprises a pod and a buffer reservoir. In some embodiments of any of the kits described herein, the kit comprises a pod, an elution outlet, and a buffer reservoir. In some aspects of any of the kits provided herein, the elution outlet is a funnel. In some aspects of any of the kits provided herein, the elution outlet comprises a flat surface with a port. In some aspects of any of the kits provided herein, the elution outlet is configured to be able to regulate the flow of fluid through the elution outlet. In some aspects of any of the kits provided herein, the elution outlet is not configured to regulate the flow of fluid through the elution outlet. In some aspects of any of the kits provided herein, the elution outlet can be screwed or clicked onto the pod. In some aspects of any of the kits provided herein, the elution outlet uses one or more (eg, two or three) O-rings (eg, fixed to a pod or elution outlet). It may be sealed on the pod.

(A) With any of the pods described herein; (b) the movable outer sleeve rotates to prevent fluid from leaking from inside the pod to outside the pod through at least one area of the wall. A kit is also provided herein that includes a movable outer sleeve, which is then connected to the wall so that at least one area of the wall can be covered. In some aspects of any of the kits provided herein, the kit further comprises means for stably positioning the pod inside the culture vessel.

A kit comprising (a) any of the pods described herein; (b) means for stably positioning the long axis of the pod in a horizontal position within the culture vessel is provided herein. To do. Non-limiting examples of means for stably positioning the pod inside the culture vessel are shown in FIGS. 11-19, 36, and 37.

An inlet system having (a) any of the pods described herein; (b) an upper end, an elongated cylindrical body containing a fluid conduit, and a lower end, with the upper end of the inlet system providing the fluid inlet. Provided and configured to be stably attached to the top of the pod; and when stably connected to the pod, the flow of fluid through the fluid inlet and fluid conduit and out of the bottom edge of the inlet system into the pod. An inlet system in which the elongated cylindrical body of the inlet system is configured to fit inside the wall of the pod; (c) an outlet system with a fluid outlet, the lower end of the pod. The kit also includes an outlet system, which is configured to be stably mounted on the pod and allows the flow of fluid out of the pod through the fluid outlet when stably connected to the bottom edge of the pod. Provided in the statement. In some aspects of these kits, the top of the inlet system and the top of the pod include threading that allows the top of the pod to be stably attached to the top of the inlet system. In some aspects of these kits, threading at the top of the inlet system, or threading at the top of the pod, comprises an O-ring. In some aspects of these kits, the pods contain a chromatographic resin (eg, one of the exemplary types of chromatographic resins described herein); and the inlet system and pods are an inlet system and a pod. The lower end of the inlet system is configured to be close to the chromatographic resin when mounted stably. In some aspects of these kits, the inner surface of the pod and the lower end of the inlet system are configured to allow stable attachment of the pod to the lower end of the inlet system. In some aspects of these kits, the inner surface of the pod and the lower edge of the inlet system include threading that allows stable attachment of the pod to the lower edge of the inlet system. In some aspects of these kits, threading on the inner surface of the pod, or threading on the lower edge of the inlet system, comprises an O-ring. In some aspects of these kits, the outlet system and the bottom edge of the pod include threading that allows the outlet system to be stably attached to the bottom edge of the pod. In some aspects of these kits, threading in the outlet system, or threading at the bottom edge of the pod, comprises an O-ring. In some aspects of these kits, the fluid inlet can be attached to the tubing. In some aspects of these kits, the fluid outlet can be attached to the tubing. Some aspects of these kits further include (d) means for stably positioning the long axis of the pod in a horizontal position within the culture vessel (eg, any of the culture vessels described herein). .. Some aspects of these kits further include (d) means for stably positioning the major axis of the pod in a vertical position within the culture vessel (eg, any of the culture vessels described herein). ..

A method of capturing recombinant protein produced by liquid cell culture, comprising contacting any of the pods described herein with liquid cell culture for a period of time, wherein the contacting step is described above. The method is also described herein under conditions that result in contact of at least a portion of at least one area with liquid cell culture medium, and that contact results in capture of recombinant protein by a chromatographic resin. Provide to the book. In some embodiments of any of the methods described herein, the liquid cell culture medium is placed in a culture vessel.

A method of purifying a recombinant protein produced by liquid cell culture, wherein (a) any of the pods described herein is in contact with the liquid cell culture for a period of time, at least one of the above. The steps are performed under conditions that result in contact of at least a portion of one area with liquid cell culture, and that contact results in the capture of recombinant protein by a chromatographic resin; and (b) said period. Also provided herein is a method comprising eluting the recombinant protein from the chromatographic resin and thereby purifying the recombinant protein.

In some embodiments of any of the methods provided herein, the method comprises (b) followed by (c) performing one or more additional unit operations on the recombinant protein. Including further. In some embodiments of any of the methods provided herein, the method further comprises (c) followed by the step of formulating the recombinant protein into a recombinant protein formulation. In some aspects of any of the methods provided herein, the method further comprises washing the chromatographic resin between steps (a) and step (b). In some embodiments of any of the methods described herein, the contact step is performed under sterile conditions.

In some embodiments of any of the methods described herein, the period is from about 5 minutes to about 2 weeks. In some embodiments of any of the methods described herein, the period is from about 5 minutes to about 168 hours. In some embodiments of any of the methods described herein, the period is from about 5 minutes to about 96 hours. In some embodiments of any of the methods described herein, the period is from about 5 minutes to about 48 hours. In some embodiments of any of the methods described herein, the period is from about 5 minutes to about 24 hours. In some embodiments of any of the methods described herein, the period is from about 5 minutes to about 12 hours. In some embodiments of any of the methods described herein, the period occurs when the liquid cell culture is in an exponential growth phase. In some embodiments of any of the methods described herein, the period occurs when the liquid cell culture is in a steady growth phase. In some embodiments, the contact stage occurs when the liquid cell culture is in the dying phase.

In some embodiments of any of the methods described herein, at least 30% of the total surface area of the at least one area is in contact with the liquid cell culture medium over the period in step (a). In some embodiments of any of the methods described herein, at least 50% of the total surface area of the at least one area is in contact with the liquid cell culture medium over the period in step (a). In some embodiments of any of the methods described herein, at least 75% of the total surface area of the at least one area is in contact with the liquid cell culture medium over the period in step (a). In some embodiments of any of the methods described herein, at least 90% of the total surface area of the at least one area is in contact with the liquid cell culture medium over the period in step (a).

In some embodiments of any of the methods described herein, the liquid cell culture medium is placed in a culture vessel. In some embodiments of any of the methods described herein, the culture vessel is a plate (eg, a multi-well plate), a shaking tube, a shaking flask, or a bioreactor. In some embodiments of any of the methods described herein, the bioreactor is a perfusion bioreactor. In some embodiments of any of the methods described herein, the bioreactor is a fed batch bioreactor. In some embodiments of any of the methods described herein, the liquid cell culture medium comprises bacterial cells, yeast cells, insect cells, plant cells, or mammalian cells. In some embodiments of any of the methods described herein, mammalian cells are human cells. In some embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell or NS0 cell.

In some embodiments of any of the methods described herein, the one or more additional unit operations include virus inactivation, virus filtration, polishing, size exclusion chromatography, cation exchange chromatography, anions. It is selected from the group consisting of exchange chromatography, hydrophobic interaction chromatography, mixed mode chromatography, and ultrafiltration / diafiltration.

In some embodiments of any of the methods described herein, the recombinant protein is a secretory protein. In some embodiments of any of the methods described herein, the recombinant protein is a tag (eg, fluorescent tag, radioactive isotope, polyhistidine tag (HIS tag), c-myc tag, FLAG tag, maltose binding. Protein (MBP) tag, albumin binding protein (ABP) tag, bacteriophage T7 epitope (T7-tag), biotin carboxyl carrier protein (BCCP) tag, blue tongue virus tag (B-tag), carmodulin-binding peptide tag, cellulose binding Domain tag, chitin-binding domain tag, choline-binding domain tag, galactose-binding protein tag, Glu-Glu epitope tag, human influenza hemagglutinin tag, Halo Tag®, histidine affinity tag (HAT), polyarginine tag (Asp) -Tag), Polyphenylalanine tag (Phe-tag), S-TAG, Streptavidin-binding peptide, Streptavidin tag, Strep-tag, Enzymes (eg, alkali phosphatase, chloramphenicol acetyltransferase (CAT), glutathione S-transferase) ), Or any protein with or without the Fc tag). In some embodiments of any of the methods described herein, the recombinant protein is an antibody, antigen-binding antibody fragment, enzyme, or fusion protein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are exemplary only and have no limiting intent. All publications, patent applications, patents, sequences, database entries, and other references referred to herein are incorporated by reference in their entirety. In the event of any inconsistency, this specification, including the definitions, will prevail.

Other features and advantages of the present invention will become apparent from the following detailed description and drawings, as well as the claims.

FIGS. 1A-1E show different exemplary pods and kits provided herein. It is an exemplary embodiment of the pods provided herein that the vertical axis of the pod is approximately horizontal when the pod is placed in the bioreactor. FIG. 5 shows an exemplary pod in FIG. 2 after being removed from the bioreactor and rotated so that its vertical axis is approximately vertical and its lower end connected to an elution outlet. Optionally, the top edge of the pod may be connected to a buffer reservoir (not shown). FIG. 6 is an exemplary illustration of the pods described herein, arranged within a container (eg, a bioreactor) by rods traversing through holes in the side walls of the container and the pods. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) comparing the conventional batch capture method (Trad.) And the capture purification method (Cap. & Pure) of the present invention under reducing and non-reducing conditions. This is a typical image of analysis. E = 1 μg elution sample; FT = flow-through sample; W = wash sample; M = Biorad Precision Plus ladder marker; "-" = blank. It is a typical diagram comparing the experimental timelines of the AKTA purification method, the conventional batch capture method, and the capture purification method of the present invention starting from the 4th day after transfection. It is a representative graph showing high performance liquid chromatography (HPLC) traces under four different conditions outlined in Table 2. It is a representative graph showing the HPLC traces under four different conditions outlined in Table 3. It is a representative graph showing the HPLC traces under two different conditions outlined in Table 4. FIG. 6 is a two-dimensional drawing showing different exemplary means for stably positioning the pod inside the culture vessel. The symmetrical bifurcated claw can hold any of the exemplary pods described herein in a culture vessel. FIG. 6 is a two-dimensional drawing showing different exemplary means for stably positioning the pod inside the culture vessel. The symmetrical bifurcated claw can hold any of the exemplary pods described herein in a culture vessel. FIG. 6 is a two-dimensional drawing showing different exemplary means for stably positioning the pod inside the culture vessel. The symmetrical bifurcated claw can hold any of the exemplary pods described herein in a culture vessel. FIG. 6 is a two-dimensional drawing showing different exemplary means for stably positioning the pod inside the culture vessel. The symmetrical bifurcated claw can hold any of the exemplary pods described herein in a culture vessel. It is a two-dimensional drawing showing a part of an exemplary means for stably positioning a pod inside a culture vessel; the center of the part can hold any of the exemplary pods described herein in the culture vessel. It can be attached to a symmetrical bifurcated claw (as shown in Figures 16-19). It is a two-dimensional drawing showing a part of an exemplary means for stably positioning a pod inside a culture vessel; the center of the part can hold any of the exemplary pods described herein in the culture vessel. It can be attached to a symmetrical bifurcated claw (as shown in Figures 16-19). FIG. 6 is a two-dimensional drawing showing different exemplary means for stably positioning the pod inside the culture vessel. The symmetrical bifurcated claw can hold any of the exemplary pods described herein in a culture vessel. FIG. 6 is a two-dimensional drawing showing different exemplary means for stably positioning the pod inside the culture vessel. The symmetrical bifurcated claw can hold any of the exemplary pods described herein in a culture vessel. FIG. 6 is a two-dimensional drawing showing different exemplary means for stably positioning the pod inside the culture vessel. The symmetrical bifurcated claw can hold any of the exemplary pods described herein in a culture vessel. FIG. 6 is a two-dimensional drawing showing different exemplary means for stably positioning the pod inside the culture vessel. The symmetrical bifurcated claw can hold any of the exemplary pods described herein in a culture vessel. 2 μg protein samples injected onto an analytical TOSOH Supergel TSK mAb HTP column were overlayed with elution profiles by co-purification using a device similar to that shown in Figures 27 and 28, or by conventional batch capture and elution It is also a figure shown together. In each case, after removing the device or the free-floating resin from the clarified supernatant, the clarified supernatant containing the antibody was exposed to the protein A resin, and then washed and eluted from the protein A resin. The elution profile of a 2 μg sample injected onto an analytical TOSOH Supergel TSK mAb HTP column using the same clarified supernatant as in Figure 20, but with the AKTA system and the proA resin contained within the packed column. It is a figure shown. It is a figure which showed the exemplary pod provided in this specification. It is a figure which showed the exemplary pod provided in this specification. It is a figure which showed the exemplary kit provided in this specification. It is a figure which showed the exemplary kit provided in this specification. It is a figure which showed the exemplary kit provided in this specification. It is a figure which showed the exemplary kit provided in this specification. It is a figure which showed the exemplary kit provided in this specification. It is a figure which showed the exemplary pod provided in this specification. It is a figure which showed the exemplary pod provided in this specification. It is a figure which showed the exemplary pod provided in this specification. It is the figure which showed the elution profile by the device containing the protein A resin shown in FIG. 27 and 28; the protein A resin was exposed to the unfiltered supernatant, and was eluted using the AKTA system. It is the figure which showed the elution profile by the device containing the protein A resin shown in FIG. 27 and 28; the protein A resin was exposed to the unfiltered supernatant, and was eluted using the AKTA system. It is a figure which showed the exemplary adapter provided in this specification. FIG. 5 shows an exemplary adapter provided herein, attached to an exemplary pod described herein. It is a figure which showed the exemplary means for stably positioning a pod inside a culture vessel. It is a figure which showed the exemplary means for stably positioning a pod inside a culture vessel.

Detailed Description A filtration and chromatography pod is provided herein that includes a container formed by a wall and having an upper end and a lower end; the wall forms a seal with the lower end; and of the walls, upper end, and lower end. One or more include at least one area covered with a semi-porous or porous material that allows liquid to reversibly flow from the outside of the pod into the inside of the pod. Kits containing any of these pods, methods using these pods to capture recombinant proteins produced in liquid cell cultures, and these pods to purify recombinant proteins produced by liquid cell cultures. The method used is also provided herein.

The non-limiting aspects of these pods, kits, and methods are described below. And these may be used in any combination without limitation. Additional aspects of these pods, kits, and methods are known in the art.

The pods, kits, and methods provided herein use recombinant proteins from a protein expression system (eg, proteins produced by recombinant cells, such as stable cells expressing the recombinant protein). It makes it possible to bring about unexpected reductions in the time required for capture or purification. For example, recombinant protein is about 5 minutes to about 20 hours, about 5 minutes to about 18 hours, about 5 minutes to about 16 hours, about 5 minutes to about 14 hours, about 5 minutes to about 12 hours, about 5 minutes. ~ About 10 hours, about 5 minutes ~ about 8 hours, about 5 minutes ~ about 6 hours, about 5 minutes ~ about 4 hours, about 5 minutes ~ about 3 hours, about 5 minutes ~ about 2 hours, about 5 minutes ~ about 1 hour, about 5 minutes to about 30 minutes, about 5 minutes to about 20 minutes, about 5 minutes to about 10 minutes, about 10 minutes to about 20 hours, about 10 minutes to about 18 hours, about 10 minutes to about 16 hours , About 10 minutes to about 14 hours, about 10 minutes to about 12 hours, about 10 minutes to about 10 hours, about 10 minutes to about 8 hours, about 10 minutes to about 6 hours, about 10 minutes to about 4 hours, about 10 minutes to about 3 hours, about 10 minutes to about 2 hours, about 10 minutes to about 1 hour, about 10 minutes to about 30 minutes, about 10 minutes to about 20 minutes, about 20 minutes to about 20 hours, about 20 minutes ~ About 18 hours, about 20 minutes ~ about 16 hours, about 20 minutes ~ about 14 hours, about 20 minutes ~ about 12 hours, about 20 minutes ~ about 10 hours, about 20 minutes ~ about 8 hours, about 20 minutes ~ about 6 hours, about 20 minutes to about 4 hours, about 20 minutes to about 3 hours, about 20 minutes to about 2 hours, about 20 minutes to about 1 hour, about 20 minutes to about 30 minutes, about 30 minutes to about 20 hours , About 30 minutes to about 18 hours, about 30 minutes to about 16 hours, about 30 minutes to about 14 hours, about 30 minutes to about 12 hours, about 30 minutes to about 10 hours, about 30 minutes to about 8 hours, about 30 minutes to about 6 hours, about 30 minutes to about 4 hours, about 30 minutes to about 3 hours, about 30 minutes to about 2 hours, about 30 minutes to about 1 hour, about 1 hour to about 20 hours, about 1 hour ~ About 18 hours, about 1 hour ~ about 16 hours, about 1 hour ~ about 14 hours, about 1 hour ~ about 12 hours, about 1 hour ~ about 10 hours, about 1 hour ~ about 8 hours, about 1 hour ~ about 6 hours, about 1 hour to about 4 hours, about 1 hour to about 3 hours, about 1 hour to about 2 hours, about 2 hours to about 20 hours, about 2 hours to about 18 hours, about 2 hours to about 16 hours , About 2 hours to about 14 hours, about 2 hours to about 12 hours, about 2 hours to about 10 hours, about 2 hours to about 8 hours, about 2 hours to about 6 hours, about 2 hours to about 4 hours, about 2 hours to about 3 hours, about 3 hours to about 20 hours, about 3 hours to about 18 hours, about 3 hours to about 16 hours, about 3 hours to about 14 hours, about 3 hours to about 12 hours, about 3 hours ~ About 10 hours, about 3 hours ~ about 8 hours, about 3 hours ~ about 6 hours, about 3 hours ~ about 4 hours, about 4 hours ~ about 20 hours, about 4 hours ~ about 18 hours, about 4 hours ~ about 16 hours, about 4 hours to about 14 hours, about 4 hours to about 12 hours, about 4 hours ~ About 10 hours, about 4 hours ~ about 8 hours, about 4 hours ~ about 6 hours, about 6 hours ~ about 20 hours, about 6 hours ~ about 18 hours, about 6 hours ~ about 16 hours, about 6 hours ~ about 14 hours, about 6 hours to about 12 hours, about 6 hours to about 10 hours, about 6 hours to about 8 hours, about 8 hours to about 20 hours, about 8 hours to about 18 hours, about 8 hours to about 16 hours , About 8 hours to about 14 hours, about 8 hours to about 12 hours, about 8 hours to about 10 hours, about 10 hours to about 20 hours, about 10 hours to about 18 hours, about 10 hours to about 16 hours, about 10 hours to about 14 hours, about 10 hours to about 12 hours, about 12 hours to about 20 hours, about 12 hours to about 18 hours, about 12 hours to about 16 hours, about 12 hours to about 14 hours, about 14 hours ~ About 20 hours, about 14 hours ~ about 18 hours, about 14 hours ~ about 16 hours, about 16 hours ~ about 20 hours, about 16 hours ~ about 18 hours, or about 18 hours ~ about 20 hours capture or purification May be done.

A filtration and chromatography pod is provided herein that includes a container formed by a pod wall and having an upper end and a lower end; the wall forms a seal with the lower end; and one of the wall, upper end, and lower end. Alternatively, the plurality includes at least one area covered with a semi-porous or porous material that allows liquid to reversibly flow into the pod from outside the pod. The semi-porous or porous material may be attached to the walls, top and / or bottom of the pod by heat sealing. Other means for attaching the semi-porous or porous material to the walls, top and / or bottom of the pod are known in the art. Non-limiting examples of the pods and kits provided herein are shown in FIGS. 1A-1E, 2, 3, 3, 4, 22-31, and 35. In some embodiments, the pod design, eg, as shown in FIG. 1A, is one or more panels adapted to cover part or all of the side surface of the pod formed of a semi-porous or porous material. May be optionally included. The installation of such a covering may facilitate subsequent replication steps.

Semi-porous / Porous Material In some aspects of any of the pods described herein, the semi-porous or porous material retains or substantially retains the resin within the pod structure. In some examples of any of the pods described herein, the semi-porous or porous material does not allow (a) cells to flow from outside the pod into the inside of the pod, and (b). It is characterized by one or both of retaining or substantially retaining the resin inside the pod, (c) coating with a hydrophilic or hydrophobic coating. In some embodiments, the semi-porous or porous material does not allow cells to flow into the pod from outside the pod. In some embodiments, the semi-porous or porous material retains or substantially retains the resin inside the pod. In some aspects of any of the pods described herein, the semi-porous or porous material does not allow cells to flow from outside the pod into the pod, and retains the resin inside the pod. Or characterized by substantially retaining.

In some examples of any of the pods described herein, the semi-porous or porous material (a) does not allow mammalian cells to flow into the pod from outside the pod, and (b). ) Characterized by one or both of retaining or substantially retaining the resin inside the pod. In some examples of any of the pods described herein, the semi-porous or porous material does not allow mammalian cells to reversibly flow into the pod from outside the pod. In some examples of any of the pods described herein, the semi-porous or porous material (a) does not allow insect cells to flow into the pod from outside the pod, and (b). ) Characterized by one or both of retaining or substantially retaining the resin inside the pod. In some examples of any of the pods described herein, the semi-porous or porous material does not allow insect cells or yeast cells to reversibly flow into the pod from outside the pod. In some examples of any of the pods described herein, the semi-porous or porous material does not allow bacterial cells to reversibly flow into the pod from outside the pod.

In some examples, the material is a semipermeable or porous membrane. In some examples, the material is a semipermeable or porous mesh (eg nylon mesh).

In some examples, the materials are polyester, polyester with hydrophilic coating, cellulose acetate, cellulose nitrate, polyether sulfone (PES), polystyrene, polypropylene, acrylic acid copolymer, polyvinyl chloride (PVC), polyvinylidene fluoride. It may contain (at least in part) one or more of (PVDF), polytetrafluoroethylene (PTFE), and polycarbonate. In some embodiments, the material is a precision woven polymer (eg, any precision woven polymer). In some embodiments, the material is Sefar film (see, eg, Sefar's US website www.sefar.us). In some embodiments, the material is a Petex mesh sold by Sefar.

In some aspects of any of the pods described herein, the material is about 50 nm to about 50 μm, about 50 nm to about 45 μm, about 50 nm to about 40 μm, about 50 nm to about 35. μm, about 50 nm to about 30 μm, about 50 nm to about 25 μm, about 50 nm to about 20 μm, about 50 nm to about 18 μm, about 50 nm to about 16 μm, about 50 nm to about 14 μm, About 50 nm to about 12 μm, about 50 nm to about 10 μm, about 50 nm to about 8 μm, about 50 nm to about 6 μm, about 50 nm to about 5 μm, about 50 nm to about 4 μm, about 50 nm ~ about 3 μm, about 50 nm ~ about 2 μm, about 50 nm ~ about 1 μm, about 50 nm ~ about 500 nm, about 50 nm ~ about 100 nm, about 100 nm ~ about 50 μm, about 100 nm ~ About 45 μm, about 100 nm to about 40 μm, about 100 nm to about 35 μm, about 100 nm to about 30 μm, about 100 nm to about 25 μm, about 100 nm to about 20 μm, about 100 nm to about 18 μm, about 100 nm to about 16 μm, about 100 nm to about 14 μm, about 100 nm to about 12 μm, about 100 nm to about 10 μm, about 100 nm to about 8 μm, about 100 nm to about 6 μm, About 100 nm to about 5 μm, about 100 nm to about 4 μm, about 100 nm to about 3 μm, about 100 nm to about 2 μm, about 100 nm to about 1 μm, about 100 nm to about 500 nm, about 500 nm ~ about 50 μm, about 500 nm ~ about 45 μm, about 500 nm ~ about 40 μm, about 500 nm ~ about 35 μm, about 500 nm ~ about 30 μm, about 500 nm ~ about 25 μm, about 500 nm ~ About 20 μm, about 500 nm to about 18 μm, about 500 nm to about 16 μm, about 500 nm to about 14 μm, about 500 nm to about 12 μm, about 500 nm to about 10 μm, about 500 nm to about 8 μm, about 500 nm to about 6 μm, about 500 nm to about 5 μm, about 500 nm to about 4 μm, about 500 nm to about 3 μm, about 500 nm to about 2 μm, about 500 nm to about 1 μm, About 1 μm to about 50 μm, about 1 μm to about 45 μm, about 1 μm to about 40 μm, about 1 μm to about 35 μm, about 1 μm to about 30 μm, about 1 μm to about 25 μm, about 1 μm ~ about 20 μm, about 1 μm ~ about 18 μm, about 1 μm to about 16 μm, about 1 μm to about 14 μm, about 1 μm to about 12 μm, about 1 μm to about 10 μm, about 1 μm to about 8 μm, about 1 μm to about 6 μm, about 1 μm ~ About 5 μm, about 1 μm ~ about 4 μm, about 1 μm ~ about 3 μm, about 1 μm ~ about 2 μm, about 2 μm ~ about 50 μm, about 2 μm ~ about 45 μm, about 2 μm ~ 40 μm, about 2 μm to about 35 μm, about 2 μm to about 30 μm, about 2 μm to about 25 μm, about 2 μm to about 20 μm, about 2 μm to about 18 μm, about 2 μm to about 16 μm , About 2 μm to about 14 μm, about 2 μm to about 12 μm, about 2 μm to about 10 μm, about 2 μm to about 8 μm, about 2 μm to about 6 μm, about 2 μm to about 5 μm, about 2 μm to about 4 μm, about 2 μm to about 3 μm, about 3 μm to about 50 μm, about 3 μm to about 45 μm, about 3 μm to about 40 μm, about 3 μm to about 35 μm, about 3 μm ~ About 30 μm, about 3 μm ~ about 25 μm, about 3 μm ~ about 20 μm, about 3 μm ~ about 18 μm, about 3 μm ~ about 16 μm, about 3 μm ~ about 14 μm, about 3 μm ~ about 12 μm, about 3 μm to about 10 μm, about 3 μm to about 8 μm, about 3 μm to about 6 μm, about 3 μm to about 5 μm, about 3 μm to about 4 μm, about 4 μm to about 50 μm , About 4 μm to about 45 μm, about 4 μm to about 40 μm, about 4 μm to about 35 μm, about 4 μm to about 30 μm, about 4 μm to about 25 μm, about 4 μm to about 20 μm, about 4 μm to about 18 μm, about 4 μm to about 16 μm, about 4 μm to about 14 μm, about 4 μm to about 12 μm, about 4 μm to about 10 μm, about 4 μm to about 8 μm, about 4 μm ~ About 6 μm, about 4 μm ~ about 5 μm, about 5 μm ~ about 50 μm, about 5 μm ~ about 45 μm, about 5 μm ~ about 40 μm, about 5 μm ~ about 35 μm, about 5 μm ~ 30 μm, about 5 μm to about 25 μm, about 5 μm to about 20 μm, about 5 μm to about 18 μm, about 5 μm to about 16 μm, about 5 μm to about 14 μm, about 5 μm to about 12 μm , About 5 μm to about 10 μm, about 5 μm to about 8 μm, about 5 μm to about 6 μm, about 6 μm to about 50 μm, about 6 μm to about 45 μm, about 6 μm to about 40 μm, about 6 μm to about 35 μm, about 6 μm to about 30 μm, about 6 μm to about 25 μm, about 6 μm to about 20 μm, about 6 μm to about 18 μm, about 6 μm to about 16 μm, about 6 μm to about 14 μm, about 6 μm to about 12 μm, about 6 μm to about 10 μm, about 6 μm to about 8 μm , About 8 μm to about 50 μm, about 8 μm to about 45 μm, about 8 μm to about 40 μm, about 8 μm to about 35 μm, about 8 μm to about 30 μm, about 8 μm to about 25 μm, about 8 μm to about 20 μm, about 8 μm to about 18 μm, about 8 μm to about 16 μm, about 8 μm to about 14 μm, about 8 μm to about 12 μm, about 8 μm to about 10 μm, about 10 μm ~ About 50 μm, about 10 μm ~ about 45 μm, about 10 μm ~ about 40 μm, about 10 μm ~ about 35 μm, about 10 μm ~ about 30 μm, about 10 μm ~ about 25 μm, about 10 μm ~ 20 μm, about 10 μm to about 18 μm, about 10 μm to about 16 μm, about 10 μm to about 14 μm, about 10 μm to about 12 μm, about 12 μm to about 50 μm, about 12 μm to about 45 μm , About 12 μm to about 40 μm, about 12 μm to about 35 μm, about 12 μm to about 30 μm, about 12 μm to about 25 μm, about 12 μm to about 20 μm, about 12 μm to about 18 μm, about 12 μm to about 16 μm, about 12 μm to about 14 μm, about 14 μm to about 50 μm, about 14 μm to about 45 μm, about 14 μm to about 40 μm, about 14 μm to about 35 μm, about 14 μm ~ About 30 μm, about 14 μm ~ about 25 μm, about 14 μm ~ about 20 μm, about 14 μm ~ about 18 μm, about 14 μm ~ about 16 μm, about 16 μm ~ about 50 μm, about 16 μm ~ 45 μm, about 16 μm to about 40 μm, about 16 μm to about 35 μm, about 16 μm to about 30 μm, about 16 μm to about 25 μm, about 16 μm to about 20 μm, about 16 μm to about 18 μm , About 18 μm to about 50 μm, about 18 μm to about 45 μm, about 18 μm to about 40 μm, about 18 μm to about 35 μm, about 18 μm to about 30 μm, about 18 μm to about 25 μm, about 18 μm to about 20 μm, about 20 μm to about 50 μm, about 20 μm to about 45 μm, about 20 μm to about 40 μm, about 20 μm to about 35 μm, about 20 μm to about 30 μm, about 20 μm ~ About 25 μm, about 25 μm ~ about 50 μm, about 25 μm ~ about 45 μm, about 25 μm ~ about 40 μm, about 25 μm m ~ about 35 μm, about 25 μm ~ about 30 μm, about 30 μm ~ about 50 μm, about 30 μm ~ about 45 μm, about 30 μm ~ about 40 μm, about 30 μm ~ about 35 μm, about 35 μm ~ Pore diameter or average pore diameter of about 50 μm, about 35 μm to about 45 μm, about 35 μm to about 40 μm, about 40 μm to about 50 μm, about 40 μm to about 45 μm, or about 45 μm to about 50 μm Have. In some aspects of any of the pods described herein, the material is about 30 μm to about 70 μm, about 30 μm to about 68 μm, about 30 μm to about 66 μm, about 30 μm to about 64. μm, about 30 μm to about 62 μm, about 30 μm to about 60 μm, about 30 μm to about 58 μm, about 30 μm to about 56 μm, about 30 μm to about 54 μm, about 30 μm to about 52 μm, About 30 μm to about 50 μm, about 30 μm to about 48 μm, about 30 μm to about 46 μm, about 30 μm to about 44 μm, about 30 μm to about 42 μm, about 30 μm to about 40 μm, about 30 Μm ~ about 38 μm, about 30 μm ~ about 36 μm, about 30 μm ~ about 34 μm, about 30 μm ~ about 32 μm, about 32 μm ~ about 70 μm, about 32 μm ~ about 68 μm, about 32 μm ~ About 66 μm, about 32 μm to about 64 μm, about 32 μm to about 62 μm, about 32 μm to about 60 μm, about 32 μm to about 58 μm, about 32 μm to about 56 μm, about 32 μm to about 54 μm, about 32 μm to about 52 μm, about 32 μm to about 50 μm, about 32 μm to about 48 μm, about 32 μm to about 46 μm, about 32 μm to about 44 μm, about 32 μm to about 42 μm, About 32 μm to about 40 μm, about 32 μm to about 38 μm, about 32 μm to about 36 μm, about 32 μm to about 34 μm, about 34 μm to about 70 μm, about 34 μm to about 68 μm, about 34 μm to about 66 μm, about 34 μm to about 64 μm, about 34 μm to about 62 μm, about 34 μm to about 60 μm, about 34 μm to about 58 μm, about 34 μm to about 56 μm, about 34 μm About 54 μm, about 34 μm to about 52 μm, about 34 μm to about 50 μm, about 34 μm to about 48 μm, about 34 μm to about 46 μm, about 34 μm to about 44 μm, about 34 μm to about 42 μm, about 34 μm to about 40 μm, about 34 μm to about 38 μm, about 34 μm to about 36 μm, about 36 μm to about 70 μm, about 36 μm to about 68 μm, about 36 μm to about 66 μm, About 36 μm to about 64 μm, about 36 μm to about 62 μm, about 36 μm to about 60 μm, about 36 μm to about 58 μm, about 36 μm to about 56 μm, about 36 μm to about 54 μm, about 36 μm to about 52 μm, about 36 μm to about 50 μm, about 36 μm to about 48 μm, About 36 μm to about 46 μm, about 36 μm to about 44 μm, about 36 μm to about 42 μm, about 36 μm to about 40 μm, about 36 μm to about 38 μm, about 38 μm to about 70 μm, about 38 μm to about 68 μm, about 38 μm to about 66 μm, about 38 μm to about 64 μm, about 38 μm to about 62 μm, about 38 μm to about 60 μm, about 38 μm to about 58 μm, about 38 μm About 56 μm, about 38 μm to about 54 μm, about 38 μm to about 52 μm, about 38 μm to about 50 μm, about 38 μm to about 48 μm, about 38 μm to about 46 μm, about 38 μm to about 44 μm, about 38 μm to about 42 μm, about 38 μm to about 40 μm, about 40 μm to about 70 μm, about 40 μm to about 68 μm, about 40 μm to about 66 μm, about 40 μm to about 64 μm, About 40 μm to about 62 μm, about 40 μm to about 60 μm, about 40 μm to about 58 μm, about 40 μm to about 56 μm, about 40 μm to about 54 μm, about 40 μm to about 52 μm, about 40 μm to about 50 μm, about 40 μm to about 48 μm, about 40 μm to about 46 μm, about 40 μm to about 44 μm, about 40 μm to about 42 μm, about 42 μm to about 70 μm, about 42 μm About 68 μm, about 42 μm to about 66 μm, about 42 μm to about 64 μm, about 42 μm to about 62 μm, about 42 μm to about 60 μm, about 42 μm to about 58 μm, about 42 μm to about 56 μm, about 42 μm to about 54 μm, about 42 μm to about 52 μm, about 42 μm to about 50 μm, about 42 μm to about 48 μm, about 42 μm to about 46 μm, about 42 μm to about 44 μm, About 44 μm to about 70 μm, about 44 μm to about 68 μm, about 44 μm to about 66 μm, about 44 μm to about 64 μm, about 44 μm to about 62 μm, about 44 μm to about 60 μm, about 44 μm to about 58 μm, about 44 μm to about 56 μm, about 44 μm to about 54 μm, about 44 μm to about 52 μm, about 44 μm to about 50 μm, about 44 μm to about 48 μm, about 44 μm About 46 μm, about 46 μm to about 70 μm, about 46 μm to about 68 μm, about 46 μm to about 66 μm, about 46 μm to about 64 μm, about 46 μm to about 62 μm, about 46 μm to about 60 μm, about 46 μm to about 58 μm, about 46 μm to about 56 μm, about 46 μm ~ 54 μm, about 46 μm ~ about 52 μm, about 46 μm ~ about 50 μm, about 46 μm ~ about 48 μm, about 48 μm ~ about 70 μm, about 48 μm ~ about 68 μm, about 48 μm ~ 66 μm, about 48 μm to about 64 μm, about 48 μm to about 62 μm, about 48 μm to about 60 μm, about 48 μm to about 58 μm, about 48 μm to about 56 μm, about 48 μm to about 54 μm , About 48 μm to about 52 μm, about 48 μm to about 50 μm, about 50 μm to about 70 μm, about 50 μm to about 68 μm, about 50 μm to about 66 μm, about 50 μm to about 64 μm, about 50 μm to about 62 μm, about 50 μm to about 60 μm, about 50 μm to about 58 μm, about 50 μm to about 56 μm, about 50 μm to about 54 μm, about 50 μm to about 52 μm, about 52 μm ~ About 70 μm, about 52 μm ~ about 68 μm, about 52 μm ~ about 66 μm, about 52 μm ~ about 64 μm, about 52 μm ~ about 62 μm, about 52 μm ~ about 60 μm, about 52 μm ~ about 58 μm, about 52 μm to about 56 μm, about 52 μm to about 54 μm, about 54 μm to about 70 μm, about 54 μm to about 68 μm, about 54 μm to about 66 μm, about 54 μm to about 64 μm , About 54 μm to about 62 μm, about 54 μm to about 60 μm, about 54 μm to about 58 μm, about 54 μm to about 56 μm, about 56 μm to about 70 μm, about 56 μm to about 68 μm, about 56 μm to about 66 μm, about 56 μm to about 64 μm, about 56 μm to about 62 μm, about 56 μm to about 60 μm, about 56 μm to about 58 μm, about 58 μm to about 70 μm, about 58 μm ~ 68 μm, 58 μm ~ 66 μm, 58 μm ~ 64 μm, 58 μm ~ 62 μm, 58 μm ~ 60 μm, 60 μm ~ 70 μm, 60 μm ~ 68 μm, about 60 μm to about 66 μm, about 60 μm to about 64 μm, about 60 μm to about 62 μm, about 62 μm to about 70 μm, about 62 μm to about 68 μm, about 62 μm to about 66 μm , About 62 μm to about 64 μm, about 64 μm to about 70 μm, about 64 μm to about 68 μm, about 64 μm to about 66 μm, about 66 μm to about 70 μm, about 66 μm to about 68 μm, or It has a pore size of about 68 μm to about 70 μm or an average pore size.

Areas Covered by Semi-Porous / Porous Materials In some aspects of the pod, one or more of the walls, top, and bottom allow liquids to reversibly flow from outside the pod into the inside of the pod. At least one covered with a semi-porous or porous material (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, Includes (18, 19, or 20) areas. In some aspects of these pods, the pod is covered with a semi-porous or porous material (which allows liquid to reversibly flow into the pod from outside the pod), 1, 2, 3 in the wall. , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 areas; and / or (allowing liquid to reversibly flow into the pod from outside the pod ) 1,2,3,4,5,6,7,8,9,10,11,12,13,14, or 15 areas at the bottom, covered with a semi-porous or porous material; And / or 1, 2, 3, 4, 5, 6, 7 at the top, covered with a semi-porous or porous material (which allows liquid to reversibly flow into the pod from outside the pod) Includes 8, 9, 10, 11, 12, 13, 14, or 15 areas.

In some embodiments of the pods described herein, the wall comprises at least one area covered by the material. In some embodiments of the pods described herein, the bottom edge and the wall each include at least one area covered by the material. In some embodiments, the upper end comprises at least one area covered by the material. In some embodiments, the upper and lower ends each include at least one area covered by the material.

In some aspects of the pods described herein, semi-porous (allowing liquid to reversibly flow into the pod from outside the pod) at one or more of the walls, top, and bottom. Or the total number of areas covered by the porous material is 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some aspects of the pods described herein, semi-porous (allowing liquid to reversibly flow from outside the pod into the pod) at one or more of the walls, top, and bottom. Or the total number of areas covered by the porous material is 1-30, 1-28, 1-26, 1-24, 1-22, 1-20, 1-18, 1-16, 1-14, 1 ~ 12, 1 ~ 10, 1 ~ 8, 1 ~ 6, 1 ~ 4, 1 ~ 3, 1 or 2, 2 ~ 30, 2 ~ 28, 2 ~ 26, 2 ~ 24, 2 ~ 22, 2 ~ 20 , 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 2 or 3, 3-30, 3-28, 3-26, 3 ~ 24, 3 ~ 22, 3 ~ 20, 3 ~ 18, 3 ~ 16, 3 ~ 14, 3 ~ 12, 3 ~ 10, 3 ~ 8, 3 ~ 6, 3 or 4, 4 ~ 30, 4 ~ 28 , 4 to 26, 4 to 24, 4 to 22, 4 to 20, 4 to 18, 4 to 16, 4 to 14, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 6 to 30, 6 ~ 28, 6 ~ 26, 6 ~ 24, 6 ~ 22, 6 ~ 20, 6 ~ 18, 6 ~ 16, 6 ~ 14, 6 ~ 12, 6 ~ 10, 6 ~ 8, 8 ~ 30, 8 ~ 28 , 8 ~ 26, 8 ~ 24, 8 ~ 22, 8 ~ 20, 8 ~ 18, 8 ~ 16, 8 ~ 14, 8 ~ 12, 8 ~ 10, 10 ~ 30, 10 ~ 28, 10 ~ 26, 10 ~ 24, 10-22, 10-20, 10-18, 10-16, 10-14, 10-12, 12-30, 12-28, 12-26, 12-24, 12-22, 12-20 , 12-18, 12-16, 12-14, 14-30, 14-28, 14-26, 14-24, 14-22, 14-20, 14-18, 14-16, 16-30, 16 ~ 28, 16 ~ 26, 16 ~ 24, 16 ~ 22, 16 ~ 20, 16 ~ 18, 18 ~ 30, 18 ~ 28, 18 ~ 26, 18 ~ 24, 18 ~ 22, 18 ~ 20, 20 ~ 30 , 20-28, 20-26, 20-24, 22-22, 22-30, 22-28, 22-26, 22-24, 24-30, 24-28, 24-26, 26-30, 26 ~ 28, or 28-30.

In some aspects of any of the pods described herein, one or more of the at least one area covered by a semi-porous or porous material is circular, oval, square, rectangular, Elongated shape (eg, elongated line or rectangle), star, zigzag, or snake.

In some embodiments, one or more (eg, each) of the at least one area covered by a semi-porous or porous material is about 0.5 mm ² to about 60 cm ² , about 0.5 mm ² to about. 58 cm ² , about 0.5 mm ² to about 56 cm ² , about 0.5 mm ² to about 54 cm ² , about 0.5 mm ² to about 52 cm ² , about 0.5 mm ² to about 50 cm ² , about 0.5 mm ² to about 48 cm ² , about 0.5 mm ² to about 46 cm ² , about 0.5 mm ² to about 44 cm ² , about 0.5 mm ² to about 42 cm ² , about 0.5 mm ² to about 40 cm ² , about 0.5 mm ² to about 38 cm ² , about 0.5 mm ² to about 36 cm ² , about 0.5 mm ² to about 34 cm ² , about 0.5 mm ² to about 32 cm ² , 0.5 mm ² to about 30 cm ² , about 0.5 mm ² to about 28 cm ² , about 0.5 mm ² to about 26 cm ² , about 0.5 mm ² to about 24 cm ² , about 0.5 mm ² to about 22 cm ² , about 0.5 mm ² to about 20 cm ² , about 0.5 mm ² to about 18 cm ² , about 0.5 mm ² to about 16 cm ² , about 0.5 mm ² to about 14 cm ² , about 0.5 mm ² to about 12 cm ² , about 0.5 mm ² to about 10 cm ² , about 0.5 mm ² to about 8 cm ² , about 0.5 mm ² to about 6 cm ² , about 0.5 mm ² to about 5 cm ² , about 0.5 mm ² to about 4 cm ² , about 0.5 mm ² to about 3 cm ² , about 0.5 mm ² to about 2 cm ² , about 0.5 mm ² to about 1 cm ² , about 0.5 mm ² to about 5 mm ² , about 0.5 mm ² to about 2 mm ² , about 0.5 mm ² to about 1 mm ² , about 1 mm ² to about 60 cm ² , about 1 mm ² to about 58 cm ² , about 1 mm ² to about 56 cm ² , about 1 mm ² to about 54 cm ² , about 1 mm ² to about 52 cm ² , about 1 mm ² to about 50 cm ² , about 1 mm ² to about 48 cm ² , about 1 mm ² to about 46 cm ² , about 1 mm ² to about 44 cm ² , about 1 mm ² to about 42 cm ² , about 1 mm ² to about 40 cm ² , about 1 mm ² to about 38 cm ² , about 1 mm ² to about 36 cm ² , about 1 mm ² to about 34 cm ² , about 1 mm ² to about 32 cm ² , about 1 mm ² to about 30 cm ² , about 1 mm ² to about 28 cm ² , about 1 mm ² to about 26 cm ² , about 1 mm ² to about 24 cm ² , about 1 mm ² to about 22 cm ² , about 1 mm ² to about 20 cm ² , about 1 mm ² to about 18 cm ² , about 1 mm ² to about 16 cm ² , about 1 mm ² to about 14 cm ² , about 1 mm ² to about 12 cm ² , about 1 mm ² to about 10 cm ² , about 1 mm ² to about 8 cm ² , about 1 mm ² to about 6 cm ² , about 1 mm ² to about 5 cm ² , about 1 mm ² to about 4 cm ² , about 1 mm ² to about 3 cm ² , about 1 mm ² to about 2 cm ² , about 1 mm ² to about 1 cm ² , about 1 mm ² to about 5 mm ² , about 1 mm ² to about 2 mm ² , about 2 mm ² to about 60 cm ² , about 2 mm ² to about 58 cm ² , about 2 mm ² to about 56 cm ² , about 2 mm ² to about 54 cm ² , about 2 mm ² to about 52 cm ² , about 2 mm ² to about 50 cm ² , about 2 mm ² to about 48 cm ² , about 2 mm ² to about 46 cm ² , about 2 mm ² to about 44 cm ² , about 2 mm ² to about 42 cm ² , about 2 mm ² to about 40 cm ² , about 2 mm ² to about 38 cm ² , about 2 mm ² to about 36 cm ² , about 2 mm ² to about 34 cm ² , about 2 mm ² to about 32 cm ² , about 2 mm ² to about 30 cm ² , about 2 mm ² to about 28 cm ² , about 2 mm ² to about 26 cm ² , about 2 mm ² to about 24 cm ² , about 2 mm ² to about 22 cm ² , about 2 mm ² to about 20 cm ² , about 2 mm ² to about 18 cm ² , about 2 mm ² to about 16 cm ² , about 2 mm ² to about 14 cm ² , about 2 mm ² to about 12 cm ² , about 2 mm ² to about 10 cm ² , about 2 mm ² to about 8 cm ² , about 2 mm ² to about 6 cm ² , about 2 mm ² to about 5 cm ² , about 2 mm ² to about 4 cm ² , about 2 mm ² to about 3 cm ² , about 2 mm ² to about 2 cm ² , about 2 mm ² to about 1 cm ² , about 2 mm ² to about 5 mm ² , about 5 mm ² to about 60 cm ² , about 5 mm ² to about 58 cm ² , about 5 mm ² to about 56 cm ² , about 5 mm ² to about 54 cm ² , about 5 mm ² to about 52 cm ² , about 5 mm ² to about 50 cm ² , about 5 mm ² to about 48 cm ² , about 5 mm ² to about 46 cm ² , about 5 mm ² to about 44 cm ² , about 5 mm ² to about 42 cm ² , about 5 mm ² to about 40 cm ² , about 5 mm ² to about 38 cm ² , about 5 mm ² to about 36 cm ² , about 5 mm ² to about 34 cm ² , about 5 mm ² to about 32 cm ² , about 5 mm ² to about 30 cm ² , about 5 mm ² to about 28 cm ² , about 5 mm ² to about 26 cm ² , about 5 mm ² to about 24 cm ² , about 5 mm ² to about 22 cm ² , about 5 mm ² to about 20 cm ² , about 5 mm ² to about 18 cm ² , about 5 mm ² to about 16 cm ² , about 5 mm ² to about 14 cm ² , about 5 mm ² to about 12 cm ² , about 5 mm ² to about 10 cm ² , about 5 mm ² to about 8 cm ² , about 5 mm ² to about 6 cm ² , about 5 mm ² to about 5 cm ² , about 5 mm ² to about 4 cm ² , about 5 mm ² to about 3 cm ² , about 5 mm ² to about 2 cm ² , about 5 mm ² to about 1 cm ² , about 1 cm ² to about 60 cm ² , about 1 cm ² to about 58 cm ² , about 1 cm ² to about 56 cm ² , about 1 cm ² to about 54 cm ² , about 1 cm ² to about 52 cm ² , about 1 cm ² to about 50 cm ² , about 1 cm ² to about 48 cm ² , about 1 cm ² to about 46 cm ² , about 1 cm ² to about 44 cm ² , about 1 cm ² to about 42 cm ² , about 1 cm ² to about 40 cm ² , about 1 cm ² to about 38 cm ² , about 1 cm ² to about 36 cm ² , about 1 cm ² to about 34 cm ² , about 1 cm ² to about 3 2 cm ² , about 1 cm ² to about 30 cm ² , about 1 cm ² to about 28 cm ² , about 1 cm ² to about 26 cm ² , about 1 cm ² to about 24 cm ² , about 1 cm ² to about 1 cm ² to about 22 cm ² , about 1 cm ² to about 20 cm ² , about 1 cm ² to about 18 cm ² , about 1 cm ² to about 16 cm ² , about 1 cm ² to about 14 cm ² , about 1 cm ² to about 1 cm ² to about 12 cm ² , about 1 cm ² to about 10 cm ² , about 1 cm ² to about 8 cm ² , about 1 cm ² to about 6 cm ² , about 1 cm ² to about 5 cm ² , about 1 cm ² to about 1 cm ² 4 cm ² , about 1 cm ² to about 3 cm ² , about 1 cm ² to about 2 cm ² , about 2 cm ² to about 60 cm ² , about 2 cm ² to about 58 cm ² , about 2 cm ² to about 2 cm ² 56 cm ² , about 2 cm ² to about 54 cm ² , about 2 cm ² to about 52 cm ² , about 2 cm ² to about 50 cm ² , about 2 cm ² to about 48 cm ² , about 2 cm ² to about 2 cm ² to about 46 cm ² , about 2 cm ² to about 44 cm ² , about 2 cm ² to about 42 cm ² , about 2 cm ² to about 40 cm ² , about 2 cm ² to about 38 cm ² , about 2 cm ² to about 2 cm ² to about 36 cm ² , about 2 cm ² to about 34 cm ² , about 2 cm ² to about 32 cm ² , about 2 cm ² to about 30 cm ² , about 2 cm ² to about 28 cm ² , about 2 cm ² to about 2 cm ² to about 26 cm ² , about 2 cm ² to about 24 cm ² , about 2 cm ² to about 22 cm ² , about 2 cm ² to about 20 cm ² , about 2 cm ² to about 18 cm ² , about 2 cm ² to about 2 cm ² to about 16 cm ² , about 2 cm ² to about 14 cm ² , about 2 cm ² to about 12 cm ² , about 2 cm ² to about 10 cm ² , about 2 cm ² to about 8 cm ² , about 2 cm ² to about 2 cm ² to about 6 cm ² , about 2 cm ² to about 5 cm ² , about 2 cm ² to about 4 cm ² , about 2 cm ² to about 3 cm ² , about 3 cm ² to about 60 cm ² , about 3 cm ² to about 3 cm ² 58 cm ² , about 3 cm ² to about 56 cm ² , about 3 cm ² to about 54 cm ² , about 3 cm ² to about 52 cm ² , about 3 cm ² to about 50 cm ² , about 3 cm ² to about 48 cm ² , about 3 cm ² to about 46 cm ² , about 3 cm ² to about 44 cm ² , about 3 cm ² to about 42 cm ² , about 3 cm ² to about 40 cm ² , about 3 cm ² to about 38 cm ² , about 3 cm ² to about 36 cm ² , about 3 cm ² to about 34 cm ² , about 3 cm ² to about 32 cm ² , about 3 cm ² to about 30 cm ² , about 3 cm ² to about 28 cm ² , about 3 cm ² to about 26 cm ² , about 3 cm ² to about 24 cm ² , about 3 cm ² to about 22 cm ² , about 3 cm ² to about 20 cm ² , about 3 cm ² to about 18 cm ² , about 3 cm ² to about 16 cm ² , about 3 cm ² to about 14 cm ² , about 3 cm ² to about 12 cm ² , about 3 cm ² to about 10 cm ² , about 3 cm ² to about 8 cm ² , about 3 cm ² to about 6 cm ² , about 3 cm ² to about 5 cm ² , about 3 cm ² to about 4 cm ² , about 4 cm ² to about 60 cm ² , about 4 cm ² to about 58 cm ² , about 4 cm ² to about 56 cm ² , about 4 cm ² to about 54 cm ² , about 4 cm ² to about 52 cm ² , about 4 cm ² to about 50 cm ² , about 4 cm ² to about 48 cm ² , about 4 cm ² to about 46 cm ² , about 4 cm ² to about 44 cm ² , about 4 cm ² to about 42 cm ² , about 4 cm ² to about 40 cm ² , about 4 cm ² to about 38 cm ² , about 4 cm ² to about 36 cm ² , about 4 cm ² to about 34 cm ² , about 4 cm ² to about 32 cm ² , about 4 cm
² to about 30 cm ² , about 4 cm ² to about 28 cm ² , about 4 cm ² to about 26 cm ² , about 4 cm ² to about 24 cm ² , about 4 cm ² to about 22 cm ² , about 4 cm ² to about 20 cm ² , about 4 cm ² to about 18 cm ² , about 4 cm ² to about 16 cm ² , about 4 cm ² to about 14 cm ² , about 4 cm ² to about 12 cm ² , about 4 cm ² to about 10 cm ² , about 4 cm ² to about 8 cm ² , about 4 cm ² to about 6 cm ² , about 4 cm ² to about 5 cm ² , about 5 cm ² to about 60 cm ² , about 5 cm ² to about 58 cm ² , about 5 cm ² to about 56 cm ² , about 5 cm ² to about 54 cm ² , about 5 cm ² to about 52 cm ² , about 5 cm ² to about 50 cm ² , about 5 cm ² to about 48 cm ² , about 5 cm ² to about 46 cm ² , about 5 cm ² to about 44 cm ² , about 5 cm ² to about 42 cm ² , about 5 cm ² to about 40 cm ² , about 5 cm ² to about 38 cm ² , about 5 cm ² to about 36 cm ² , about 5 cm ² to about 34 cm ² , about 5 cm ² to about 32 cm ² , about 5 cm ² to about 30 cm ² , about 5 cm ² to about 28 cm ² , about 5 cm ² to about 26 cm ² , about 5 cm ² to about 24 cm ² , about 5 cm ² to about 22 cm ² , about 5 cm ² to about 20 cm ² , about 5 cm ² to about 18 cm ² , about 5 cm ² to about 16 cm ² , about 5 cm ² to about 14 cm ² , about 5 cm ² to about 12 cm ² , about 5 cm ² to about 10 cm ² , about 5 cm ² to about 8 cm ² , about 5 cm ² to about 6 cm ² , about 6 cm ² to about 60 cm ² , about 6 cm ² to about 58 cm ² , about 6 cm ² to about 56 cm ² , about 6 cm ² to about 54 cm ² , about 6 cm ² to about 52 cm ² , about 6 cm ² to about 50 cm ² , about 6 cm ² to about 48 cm ² , about 6 cm ² to about 46 cm ² , about 6 cm ² to about 44 cm ² , about 6 cm ² to about 42 cm ² , about 6 cm ² to about 40 cm ² , about 6 c m ² to about 38 cm ² , about 6 cm ² to about 36 cm ² , about 6 cm ² to about 34 cm ² , about 6 cm ² to about 32 cm ² , about 6 cm ² to about 30 cm ² , about 6 cm ² to about 28 cm ² , about 6 cm ² to about 26 cm ² , about 6 cm ² to about 24 cm ² , about 6 cm ² to about 22 cm ² , about 6 cm ² to about 20 cm ² , about 6 cm ² to about 18 cm ² , about 6 cm ² to about 16 cm ² , about 6 cm ² to about 14 cm ² , about 6 cm ² to about 12 cm ² , about 6 cm ² to about 10 cm ² , about 6 cm ² to about 8 cm ² , about 8 cm ² to about 60 cm ² , about 8 cm ² to about 58 cm ² , about 8 cm ² to about 56 cm ² , about 8 cm ² to about 54 cm ² , about 8 cm ² to about 52 cm ² , about 8 cm ² to about 50 cm ² , about 8 cm ² to about 48 cm ² , about 8 cm ² to about 46 cm ² , about 8 cm ² to about 44 cm ² , about 8 cm ² to about 42 cm ² , about 8 cm ² to about 40 cm ² , about 8 cm ² to about 38 cm ² , about 8 cm ² to about 36 cm ² , about 8 cm ² to about 34 cm ² , about 8 cm ² to about 32 cm ² , about 8 cm ² to about 30 cm ² , about 8 cm ² to about 28 cm ² , about 8 cm ² to about 26 cm ² , about 8 cm ² to about 24 cm ² , about 8 cm ² to about 22 cm ² , about 8 cm ² to about 20 cm ² , about 8 cm ² to about 18 cm ² , about 8 cm ² to about 16 cm ² , about 8 cm ² to about 14 cm ² , about 8 cm ² to about 12 cm ² , about 8 cm ² to about 10 cm ² , about 10 cm ² to about 60 cm ² , about 10 cm ² to about 58 cm ² , about 10 cm ² to about 56 cm ² , about 10 cm ² to about 54 cm ² , about 10 cm ² to about 52 cm ² , about 10 cm ² to about 50 cm ² , about 10 cm ² to about 48 cm ² , about 10 cm ² to about 46 cm ² , about 10 cm ² to about 44 cm ² , about 10 cm ² to about 42 cm ² , about 10 cm ² to about 40 cm ² , about 10 cm ² to about 38 cm ² , about 10 cm ² to about 36 cm ² , about 10 cm ² to about 34 cm ² , about 10 cm ² to about 32 cm ² , about 10 cm ² to about 30 cm ² , about 10 cm ² to about 28 cm ² , about 10 cm ² to about 26 cm ² , about 10 cm ² to about 24 cm ² , about 10 cm ² to about 22 cm ² , about 10 cm ² to about 20 cm ² , about 10 cm ² to about 18 cm ² , about 10 cm ² to about 16 cm ² , about 10 cm ² to about 14 cm ² , about 10 cm ² to about 12 cm ² , about 12 cm ² to about 60 cm ² , about 12 cm ² to about 58 cm ² , about 12 cm ² to about 56 cm ² , about 12 cm ² to about 54 cm ² , about 12 cm ² to about 52 cm ² , about 12 cm ² to about 50 cm ² , about 12 cm ² to about 48 cm ² , about 12 cm ² to about 46 cm ² , about 12 cm ² to about 44 cm ² , about 12 cm ² to about 42 cm ² , about 12 cm ² to about 40 cm ² , about 12 cm ² to about 38 cm ² , about 12 cm ² to about 36 cm ² , about 12 cm ² to about 34 cm ² , about 12 cm ² to about 32 cm ² , about 12 cm ² to about 30 cm ² , about 12 cm ² to about 28 cm ² , about 12 cm ² to about 26 cm ² , about 12 cm ² to about 24 cm ² , about 12 cm ² to about 22 cm ² , about 12 cm ² to about 20 cm ² , about 12 cm ² to about 18 cm ² , about 12 cm ² to about 16 cm ² , about 12 cm ² to about 14 cm ² , about 14 cm ² to about 60 cm ² , about 14 cm ² to about 58 cm ² , about 14 cm ² to about 56 cm ² , about 14 cm ² to about 54 cm ² , about 14 cm ² to about 52 cm ² , about 14 cm ² to about 50 cm ² , about 14 cm ² to about 48 cm ² , about 14 cm ² to about 46 cm ² , about 14 cm ² to about 44 cm ² , about 14 cm ² to about 42 cm ² , about 14 cm ² to about 40 cm ² , about 14 cm ² to about 38 cm ² , about 14 cm ² to about 36 cm ² , about 14 cm ² to about 34 cm ² , about 14 cm ² to about 32 cm ² , about 14 cm ² to about 30 cm ² , about 14 cm ² to about 28 cm ² , about 14 cm ² to about 26 cm ² , about 14 cm ² to about 24 cm ² , about 14 cm ² to about 22 cm ² , about 14 cm ² to about 20 cm ² , about 14 cm ² to about 18 cm ² , about 14 cm ² to about 16 cm ² , about 16 cm ² to about 60 cm ² , about 16 cm ² to about 58 cm ² , about 16 cm ² to about 56 cm ² , about 16 cm ² to about 54 cm ² , about 16 cm ² to about 52 cm ² , about 16 cm ² to about 50 cm ² , about 16 cm ² to about 48 cm ² , about 16 cm ² to about 46 cm ² , about 16 cm ² to about 44 cm ² , about 16 cm ² to about 42 cm ² , about 16 cm ² to about 40 cm ² , about 16 cm ² to about 38 cm ² , about 16 cm ² to about 36 cm ² , about 16 cm ² to about 34 cm ² , about 16 cm ² to about 32 cm ² , about 16 cm ² to about 30 cm ² , about 16 cm ² to about 28 cm ² , about 16 cm ² to about 26 cm ² , about 16 cm ² to about 24 cm ² , about 16 cm ² to about 22 cm ² , about 16 cm ² to about 20 cm ² , about 16 cm ² to about 18 cm ² , about 18 cm ² to about 60 cm ² , about 18 cm ² to about 58 cm ² , about 18 cm ² to about 56 cm ² , about 18 cm ² to about 54 cm ² , about 18 cm ² to about 52 cm ² , about 18 cm ² to about 50 cm ² , about 18 cm ² to about 48 cm ² , about 18 cm ² to about 46 cm ² , about 18 cm ² to about 44 cm ² , about 18 cm ² to about 42 cm ² , about 18 cm ² to about 40 cm ² , about 18 cm ² to about 38 cm ² , about 18 cm ² to about 36 cm ² , about 18 cm ² to about 34 cm ² , about 18 cm ² to about 32 cm ² , about 18 cm ² to about 30 cm ² , about 18 cm ² to about 28 cm ² , about 18 cm ² to about 26 cm ² , about 18 cm ² to about 24 cm ² , about 18 cm ² to about 22 cm ² , about 18 cm ² to about 20 cm ² , about 20 cm ² to about 60 cm ² , about 20 cm ² to about 58 cm ² , about 20 cm ² to about 56 cm ² , about 20 cm ² to about 54 cm ² , about 20 cm ² to about 52 cm ² , about 20 cm ² to about 50 cm ² , about 20 cm ² to about 48 cm ² , about 20 cm ² to about 46 cm ² , about 20 cm ² to about 44 cm ² , about 20 cm ² to about 42 cm ² , about 20 cm ² to about 40 cm ² , about 20 cm ² to about 38 cm ² , about 20 cm ² to about 36 cm ² , about 20 cm ² to about 34 cm ² , about 20 cm ² to about 32 cm ² , about 20 cm ² to about 30 cm ² , about 20 cm ² to about 28 cm ² , about 20 cm ² to about 26 cm ² , about 20 cm ² to about 24 cm ² , about 20 cm ² to about 22 cm ² , about 22 cm ² to about 60 cm ² , about 22 cm ² to about 58 cm ² , about 22 cm ² to about 56 cm ² , about 22 cm ² to about 54 cm ² , about 22 cm ² to about 52 cm ² , about 22 cm ² to about 50 cm ² , about 22 cm ² to about 48 cm ² , about 22 cm ² to about 46 cm ² , about 22 cm ² to about 44 cm ² , about 22 cm ² to about 42 cm ² , about 22 cm ² to about 40 cm ² , about 22 cm ² to about 38 cm ² , about 22 cm ² to about 36 cm ² , about 22 cm ² to about 34 cm ² , about 22 cm ² to about 32 cm ² , about 22 cm ² to about 30 cm ² , about 22 cm ²
~ About 28 cm ² , about 22 cm ² ~ about 26 cm ² , about 22 cm ² ~ about 24 cm ² , about 24 cm ² ~ about 60 cm ² , about 24 cm ² ~ about 58 cm ² , about 24 cm ² ~ About 56 cm ² , about 24 cm ² ~ about 54 cm ² , about 24 cm ² ~ about 52 cm ² , about 24 cm ² ~ about 50 cm ² , about 24 cm ² ~ about 48 cm ² , about 24 cm ² ~ About 46 cm ² , about 24 cm ² ~ about 44 cm ² , about 24 cm ² ~ about 42 cm ² , about 24 cm ² ~ about 40 cm ² , about 24 cm ² ~ about 38 cm ² , about 24 cm ² ~ About 36 cm ² , about 24 cm ² ~ about 34 cm ² , about 24 cm ² ~ about 32 cm ² , about 24 cm ² ~ about 30 cm ² , about 24 cm ² ~ about 28 cm ² , about 24 cm ² ~ About 26 cm ² , about 26 cm ² ~ about 60 cm ² , about 26 cm ² ~ about 58 cm ² , about 26 cm ² ~ about 56 cm ² , about 26 cm ² ~ about 54 cm ² , about 26 cm ² ~ About 52 cm ² , about 26 cm ² ~ about 50 cm ² , about 26 cm ² ~ about 48 cm ² , about 26 cm ² ~ about 46 cm ² , about 26 cm ² ~ about 44 cm ² , about 26 cm ² ~ About 42 cm ² , about 26 cm ² ~ about 40 cm ² , about 26 cm ² ~ about 38 cm ² , about 26 cm ² ~ about 36 cm ² , about 26 cm ² ~ about 34 cm ² , about 26 cm ² ~ About 32 cm ² , about 26 cm ² ~ about 30 cm ² , about 26 cm ² ~ about 28 cm ² , about 28 cm ² ~ about 60 cm ² , about 28 cm ² ~ about 58 cm ² , about 28 cm ² ~ About 56 cm ² , about 28 cm ² ~ about 54 cm ² , about 28 cm ² ~ about 52 cm ² , about 28 cm ² ~ about 50 cm ² , about 28 cm ² ~ about 48 cm ² , about 28 cm ² ~ About 46 cm ² , about 28 cm ² ~ about 44 cm ² , about 28 cm ² ~ about 42 cm ² , about 28 cm ² ~ about 40 cm ² , about 28 cm ² ~ about 38 cm ² , about 28 cm ² ~ About 36 cm ² , about 28 cm ² ~ about 34 cm ² , about 28 cm ² ~ about 32 cm ² , about 28 cm ² ~ about 30 cm ² , about 30 cm ² ~ about 60 cm ² , about 30 cm ² ~ About 58 cm ² , about 30 cm ² ~ about 56 cm ² , about 30 cm ² ~ about 54 cm ² , about 30 cm ² ~ about 52 cm ² , about 30 cm ² ~ about 50 cm ² , about 30 cm ² ~ About 48 cm ² , about 30 cm ² ~ about 46 cm ² , about 30 cm ² ~ about 44 cm ² , about 30 cm ² ~ about 42 cm ² , about 30 cm ² ~ about 40 cm ² , about 30 cm ² ~ About 38 cm ² , about 30 cm ² ~ about 36 cm ² , about 30 cm ² ~ about 34 cm ² , about 30 cm ² ~ about 32 cm ² , about 32 cm ² ~ about 60 cm ² , about 32 cm ² ~ About 58 cm ² , about 32 cm ² ~ about 56 cm ² , about 32 cm ² ~ about 54 cm ² , about 32 cm ² ~ about 52 cm ² , about 32 cm ² ~ about 50 cm ² , about 32 cm ² ~ About 48 cm ² , about 32 cm ² ~ about 46 cm ² , about 32 cm ² ~ about 44 cm ² , about 32 cm ² ~ about 42 cm ² , about 32 cm ² ~ about 40 cm ² , about 32 cm ² ~ About 38 cm ² , about 32 cm ² ~ about 36 cm ² , about 32 cm ² ~ about 34 cm ² , about 34 cm ² ~ about 60 cm ² , about 34 cm ² ~ about 58 cm ² , about 34 cm ² ~ About 56 cm ² , about 34 cm ² ~ about 54 cm ² , about 34 cm ² ~ about 52 cm ² , about 34 cm ² ~ about 50 cm ² , about 34 cm ² ~ about 48 cm ² , about 34 cm ² ~ About 46 cm ² , about 34 cm ² ~ about 44 cm ² , about 34 cm ² ~ about 42 cm ² , about 34 cm ² ~ about 40 cm ² , about 34 cm ² ~ about 38 cm ² , about 34 cm ² ~ About 36 cm ² , about 36 cm ² ~ about 60 cm ² , about 36 cm ² ~ about 58 cm ² , about 36 cm ² ~ about 56 cm ² , about 36 cm ² ~ about 54 cm ² , about 36 cm ² ~ About 52 cm ² , about 36 cm ² ~ about 50 cm ² , about 36 cm ² ~ about 48 cm ² , about 36 cm ² ~ about 46 cm ² , about 36 cm ² ~ about 44 cm ² , about 36 cm ² ~ About 42 cm ² , about 36 cm ² ~ about 40 cm ² , about 36 cm ² ~ about 38 cm ² , about 38 cm ² ~ about 60 cm ² , about 38 cm ² ~ about 58 cm ² , about 38 cm ² ~ About 56 cm ² , about 38 cm ² ~ about 54 cm ² , about 38 cm ² ~ about 52 cm ² , about 38 cm ² ~ about 50 cm ² , about 38 cm ² ~ about 48 cm ² , about 38 cm ² ~ About 46 cm ² , about 38 cm ² ~ about 44 cm ² , about 38 cm ² ~ about 42 cm ² , about 38 cm ² ~ about 40 cm ² , about 40 cm ² ~ about 60 cm ² , about 40 cm ² ~ About 58 cm ² , about 40 cm ² ~ about 56 cm ² , about 40 cm ² ~ about 54 cm ² , about 40 cm ² ~ about 52 cm ² , about 40 cm ² ~ about 50 cm ² , about 40 cm ² ~ About 48 cm ² , about 40 cm ² ~ about 46 cm ² , about 40 cm ² ~ about 44 cm ² , about 40 cm ² ~ about 42 cm ² , about 42 cm ² ~ about 60 cm ² , about 42 cm ² ~ About 58 cm ² , about 42 cm ² ~ about 56 cm ² , about 42 cm ² ~ about 54 cm ² , about 42 cm ² ~ about 52 cm ² , about 42 cm ² ~ about 50 cm ² , about 42 cm ² ~ About 48 cm ² , about 42 cm ² ~ about 46 cm ² , about 42 cm ² ~ about 44 cm ² , about 44 cm ² ~ about 60 cm ² , about 44 cm ² ~ about 58 cm ² , about 44 cm ² ~ About 56 cm ² , about 44 cm ² ~ about 54 cm ² , about 44 cm ² ~ about 52 cm ² , about 44 cm ² ~ about 50 cm ² , about 44 cm ² ~ about 48 cm ² , about 44 cm ² ~ About 46 cm ² , about 46 cm ² ~ about 60 cm ² , about 46 cm ² ~ about 58 cm ² , about 46 cm ² ~ about 56 cm ² , about 46 cm ² ~ about 54 cm ² , about 46 cm ² ~ About 52 cm ² , about 46 cm ² ~ about 50 cm ² , about 46 cm ² ~ about 48 cm ² , about 48 cm ² ~ about 60 cm ² , about 48 cm ² ~ about 58 cm ² , about 48 cm ² ~ About 56 cm ² , about 48 cm ² ~ about 54 cm ² , about 48 cm ² ~ about 52 cm ² , about 48 cm ² ~ about 50 cm ² , about 50 cm ² ~ about 60 cm ² , about 50 cm ² ~ About 58 cm ² , about 50 cm ² ~ about 56 cm ² , about 50 cm ² ~ about 54 cm ² , about 50 cm ² ~ about 52 cm ² , about 52 cm ² ~ about 60 cm ² , about 52 cm ² ~ About 58 cm ² , about 52 cm ² ~ about 56 cm ² , about 52 cm ² ~ about 54 cm ² , about 54 cm ² ~ about 60 cm ² , about 54 cm ² ~ about 58 cm ² , about 54 cm ² It has a surface area of ~ about 56 cm ² , about 56 cm ² ~ about 60 cm ² , about 56 cm ² ~ about 58 cm ² , or about 58 cm ² ~ about 60 cm ² .

In some aspects of any of the pods described herein, the total surface area of said at least one area covered by a semi-porous or porous material is about 0.5 mm ² to about 150 cm ² , about. 0.5 mm ² to about 140 cm ² , about 0.5 mm ² to about 130 cm ² , about 0.5 mm ² to about 120 cm ² , about 0.5 mm ² to about 110 cm ² , about 0.5 mm ² to about 100 cm ² , about 0.5 mm ² to about 90 cm ² , about 0.5 mm ² to about 80 cm ² , about 0.5 mm ² to about 70 cm ² , about 0.5 mm ² to about 60 cm ² , about 0.5 mm ² to about 50 cm ² , about 0.5 mm ² to about 40 cm ² , about 0.5 mm ² to about 30 cm ² , about 0.5 mm ² to about 20 cm ² , about 0.5 mm ² to about 18 cm ² , about 0.5 mm ² to about 16 cm ² , about 0.5 mm ² to about 14 cm ² , about 0.5 mm ² to about 12 cm ² , about 0.5 mm ² to about 10 cm ² , about 0.5 mm ² to about 8 cm ² , about 0.5 mm ² to about 6 cm ² , about 0.5 mm ² to about 5 cm ² , about 0.5 mm ² to about 4 cm ² , about 0.5 mm ² to about 3 cm ² , about 0.5 mm ² to about 2 cm ² , about 0.5 mm ² to about 1 cm ² , about 0.5 mm ² to about 5 mm ² , about 5 mm ² to about 150 cm ² , about 5 mm ² to about 140 cm ² , about 5 mm ² to about 130 cm ² , about 5 mm ² to about 120 cm ² , about 5 mm ² to about 110 cm ² , about 5 mm ² to about 100 cm ² , about 5 mm ² to about 90 cm ² , about 5 mm ² to about 80 cm ² , about 5 mm ² to about 70 cm ² , about 5 mm ² to about 60 cm ² , about 5 mm ² to about 50 cm ² , about 5 mm ² to about 40 cm ² , about 5 mm ² to about 30 cm ² , about 5 mm ² to about 20 cm ² , about 5 mm ² to about 18 cm ² , about 5 mm ² to about 16 cm ² , about 5 mm ² to about 14 cm ² , about 5 mm ² to about 12 cm ² , about 5 mm ² to about 10 cm ² , about 5 mm ² to about 8 cm ² , about 5 mm ² to about 6 cm ² , about 5 mm ² to about 5 cm ² , about 5 mm ² to about 4 cm ² , about 5 mm ² to about 3 cm ² , about 5 mm ² to about 2 cm ² , about 5 mm ² to about 1 cm ² , about 1 cm ² to about 150 cm ² , about 1 cm ² to about 140 cm ² , about 1 cm ² to about 130 cm ² , about 1 cm ² to about 120 cm ² , about 1 cm ² to about 110 cm ² , about 1 cm ² to about 100 cm ² , about 1 cm ² to about 90 cm ² , about 1 cm ² to about 80 cm ² , about 1 cm ² to about 70 cm ² , about 1 cm ² to about 60 cm ² , about 1 cm ² to about 50 cm ² , about 1 cm ² to about 40 cm ² , about 1 cm ² to about 30 cm ² , about 1 cm ² to about 20 cm ² , about 1 cm ² to about 18 cm ² , about 1 cm ² to about 16 cm ² , about 1 cm ² to about 14 cm ² , about 1 cm ² to about 12 cm ² , about 1 cm ² to about 10 cm ² , about 1 cm ² to about 8 cm ² , about 1 cm ² to about 6 cm ² , about 1 cm ² to about 5 cm ² , about 1 cm ² to about 4 cm ² , about 1 cm ² to about 3 cm ² , about 1 cm ² to about 2 cm ² , about 2 cm ² to about 150 cm ² , about 2 cm ² to about 140 cm ² , about 2 cm ² to about 130 cm ² , about 2 cm ² to about 120 cm ² , about 2 cm ² to about 110 cm ² , about 2 cm ² to about 100 cm ² , about 2 cm ² to about 90 cm ² , about 2 cm ² to about 80 cm ² , about 2 cm ² to about 70 cm ² , about 2 cm ² to about 60 cm ² , about 2 cm ² to about 50 cm ² , about 2 cm ² to about 40 cm ² , about 2 cm ² to about 30 cm ² , about 2 cm ² to about 20 cm ² , about 2 cm ² to about 18 cm ² , about 2 cm ² to about 16 cm ² , about 2 cm ² to about 14 cm ² , about 2 cm ² to about 12 cm ² , about 2 cm ² to about 10 cm ² , about 2 cm ² to about 8 cm ² , about 2 cm ² to about 6 cm ² , about 2 cm ² to about 5 cm ² , about 2 cm ² to about 4 cm ² , about 2 cm ² to about 3 cm ² , about 3 cm ² to about 150 cm ² , about 3 cm ² to about 3 cm ² 140 cm ² , about 3 cm ² to about 130 cm ² , about 3 cm ² to about 120 cm ² , about 3 cm ² to about 110 cm ² , about 3 cm ² to about 100 cm ² , about 3 cm ² to about 3 cm ² to about 90 cm ² , about 3 cm ² to about 80 cm ² , about 3 cm ² to about 70 cm ² , about 3 cm ² to about 60 cm ² , about 3 cm ² to about 50 cm ² , about 3 cm ² to about 3 cm ² to about 40 cm ² , about 3 cm ² to about 30 cm ² , about 3 cm ² to about 20 cm ² , about 3 cm ² to about 18 cm ² , about 3 cm ² to about 16 cm ² , about 3 cm ² to about 3 cm ² to about 14 cm ² , about 3 cm ² to about 12 cm ² , about 3 cm ² to about 10 cm ² , about 3 cm ² to about 8 cm ² , about 3 cm ² to about 6 cm ² , about 3 cm ² to about 3 cm ² to about 5 cm ² , about 3 cm ² to about 4 cm ² , about 4 cm ² to about 150 cm ² , about 4 cm ² to about 140 cm ² , about 4 cm ² to about 130 cm ² , about 4 cm ² to about 4 cm ² to about 120 cm ² , about 4 cm ² to about 110 cm ² , about 4 cm ² to about 100 cm ² , about 4 cm ² to about 90 cm ² , about 4 cm ² to about 80 cm ² , about 4 cm ² to about 4 cm ² to about 70 cm ² , about 4 cm ² to about 60 cm ² , about 4 cm ² to about 50 cm ² , about 4 cm ² to about 40 cm ² , about 4 cm ² to about 30 cm ² , about 4 cm ² to about 4 cm ² to about 20 cm ² , about 4 cm ² to about 18 cm ² , about 4 cm ² to about 16 cm ² , about 4 cm ² to about 14 cm ² , about 4 cm ² to about 12 cm ² , about 4 cm ² to about 4 cm ² to about 10 cm ² , about 4 cm ² to about 8 cm ² , about 4 cm ² to about 6 cm ² , about 4 cm ² to about 5 cm ² , about 5 cm ² to about 150 cm ² , about 5 cm ² to about 5 cm ² 140 cm ² , about 5 cm ² to about 130 cm ² , about 5 cm ² to about 120 cm ² , Approximately 5 cm ² to approximately 110 cm ² , Approximately 5 cm ² to approximately 100 cm ² , Approximately 5 cm ² to approximately 90 cm ² , Approximately 5 cm ² to approximately 80 cm ² , Approximately 5 cm ² to approximately 70 cm ² , Approximately 5 cm ² to approximately 60 cm ² , Approximately 5 cm ² to approximately 50 cm ² , Approximately 5 cm ² to approximately 40 cm ² , Approximately 5 cm ² to approximately 30 cm ² , Approximately 5 cm ² to approximately 20 cm ² , About 5 cm ² to about 18 cm ² , about 5 cm ² to about 16 cm ² , about 5 cm ² to about 14 cm ² , about 5 cm ² to about 12 cm ² , about 5 cm ² to about 10 cm ² , Approximately 5 cm ² to approximately 8 cm ² , Approximately 5 cm ² to approximately 6 cm ² , Approximately 6 cm ² to approximately 150 cm ² , Approximately 6 cm ² to approximately 140 cm ² , Approximately 6 cm ² to approximately 130 cm ² , Approximately 6 cm ² to approximately 120 cm ² , Approximately 6 cm ² to approximately 110 cm ² , Approximately 6 cm ² to approximately 100 cm ² , Approximately 6 cm ² to approximately 90 cm ² , Approximately 6 cm ² to approximately 80 cm ² , Approximately 6 cm ² to approximately 70 cm ² , Approximately 6 cm ² to approximately 60 cm ² , Approximately 6 cm ² to approximately 50 cm ² , Approximately 6 cm ² to approximately 40 cm ² , Approximately 6 cm ² to approximately 30 cm ² , About 6 cm ² to about 20 cm ² , about 6 cm ² to about 18 cm ² , about 6 cm ² to about 16 cm ² , about 6 cm ² to about 14 cm ² , about 6 cm ² to about 12 cm ² , Approximately 6 cm ² to approximately 10 cm ² , Approximately 6 cm ² to approximately 8 cm ² , Approximately 8 cm ² to approximately 150 cm ² , Approximately 8 cm ² to approximately 140 cm ² , Approximately 8 cm ² to approximately 130 cm ² , Approximately 8 cm ² to approximately 120 cm ² , Approximately 8 cm ² to approximately 110 cm ² , Approximately 8 cm ² to approximately 100 cm ² , Approximately 8 cm ² to approximately 90 cm ² , Approximately 8 cm ² to approximately 80 cm ² , Approximately 8 cm ² to approximately 70 cm ² , Approximately 8 cm ² to approximately 60 cm ² , Approximately 8 cm ² to approximately 50 cm ² , Approximately 8 cm ² to approximately 40 cm ² , Approximately 8 cm ² to approximately 30 cm ² , Approximately 8 cm ² to approximately 20 cm ² , Approximately 8 cm ² to approximately 18 cm ² , About 8 cm ² to about 16 cm ² , about 8 cm ² to about 14 cm ² , about 8 cm ² to about 12 cm ² , about 8 cm ² to about 10 cm ² , about 10 cm ² to about 150 cm ² , Approximately 10 cm ² to approximately 140 cm ² , Approximately 10 cm ² to approximately 130 cm ² , Approximately 10 cm ² to approximately 120 cm ² , Approximately 10 cm ² to approximately 110 cm ² , Approximately 10 cm ² to approximately 100 cm ² , About 10 cm ² to about 90 cm ² , about 10 cm ² to about 80 cm ² , about 10 cm ² to about 70 cm ² , about 10 cm ² to about 60 cm ² , about 10 cm ² to about 50 cm ² , About 10 cm ² to about 40 cm ² , about 10 cm ² to about 30 cm ² , about 10 cm ² to about 20 cm ² , about 10 cm ² to about 18 cm ² , about 10 cm ² to about 16 cm ² , Approximately 10 cm ² to approximately 14 cm ² , Approximately 10 cm ² to approximately 12 cm ² , Approximately 12 cm ² to approximately 150 cm ² , Approximately 12 cm ² to approximately 140 cm ² , Approximately 12 cm ² to approximately 130 cm ² , Approximately 12 cm ² to approximately 120 cm ² , Approximately 12 cm ² to approximately 110 cm ² , Approximately 12 cm ² to approximately 100 cm ² , Approximately 12 cm ² to approximately 90 cm ² , Approximately 12 cm ² to approximately 80 cm ² , Approximately 12 cm ² to approximately 70 cm ² , Approximately 12 cm ² to approximately 60 cm ² , Approximately 12 cm ² to approximately 50 cm ² , Approximately 12 cm ² to approximately 40 cm ² , Approximately 12 cm ² to approximately 30 cm ² , Approximately 12 cm ² to approximately 20 cm ² , Approximately 12 cm ² to approximately 18 cm ² , Approximately 12 cm ² to approximately 16 cm ² , Approximately 12 cm ² to approximately 14 cm ² , Approximately 14 cm ² to approximately 150 cm ² , About 14 cm
² to about 140 cm ² , about 14 cm ² to about 130 cm ² , about 14 cm ² to about 120 cm ² , about 14 cm ² to about 110 cm ² , about 14 cm ² to about 100 cm ² , about 14 cm ² to about 90 cm ² , about 14 cm ² to about 80 cm ² , about 14 cm ² to about 70 cm ² , about 14 cm ² to about 60 cm ² , about 14 cm ² to about 50 cm ² , about 14 cm ² to about 40 cm ² , about 14 cm ² to about 30 cm ² , about 14 cm ² to about 20 cm ² , about 14 cm ² to about 18 cm ² , about 14 cm ² to about 16 cm ² , about 16 cm ² to about 150 cm ² , about 16 cm ² to about 140 cm ² , about 16 cm ² to about 130 cm ² , about 16 cm ² to about 120 cm ² , about 16 cm ² to about 110 cm ² , about 16 cm ² to about 100 cm ² , about 16 cm ² to about 90 cm ² , about 16 cm ² to about 80 cm ² , about 16 cm ² to about 70 cm ² , about 16 cm ² to about 60 cm ² , about 16 cm ² to about 50 cm ² , about 16 cm ² to about 40 cm ² , about 16 cm ² to about 30 cm ² , about 16 cm ² to about 20 cm ² , about 16 cm ² to about 18 cm ² , about 18 cm ² to about 150 cm ² , about 18 cm ² to about 140 cm ² , about 18 cm ² to about 130 cm ² , about 18 cm ² to about 120 cm ² , about 18 cm ² to about 110 cm ² , about 18 cm ² to about 100 cm ² , about 18 cm ² to about 90 cm ² , about 18 cm ² to about 80 cm ² , about 18 cm ² to about 70 cm ² , about 18 cm ² to about 60 cm ² , about 18 cm ² to about 50 cm ² , about 18 cm ² to about 40 cm ² , about 18 cm ² to about 30 cm ² , about 18 cm ² to about 20 cm ² , about 20 cm ² to about 150 cm ² , about 20 cm ² to about 140 cm ² , about 20 cm ² to about 130 cm ² , about 20 cm ² to about 120 cm ² , about 20 cm ² to about 110 cm ² , about 20 c m ² to about 100 cm ² , about 20 cm ² to about 90 cm ² , about 20 cm ² to about 80 cm ² , about 20 cm ² to about 70 cm ² , about 20 cm ² to about 60 cm ² , about 20 cm ² to about 50 cm ² , about 20 cm ² to about 40 cm ² , about 20 cm ² to about 30 cm ² , about 30 cm ² to about 150 cm ² , about 30 cm ² to about 140 cm ² , about 30 cm ² to about 130 cm ² , about 30 cm ² to about 120 cm ² , about 30 cm ² to about 110 cm ² , about 30 cm ² to about 100 cm ² , about 30 cm ² to about 90 cm ² , about 30 cm ² to about 80 cm ² , about 30 cm ² to about 70 cm ² , about 30 cm ² to about 60 cm ² , about 30 cm ² to about 50 cm ² , about 30 cm ² to about 40 cm ² , about 40 cm ² to about 150 cm ² , about 40 cm ² to about 140 cm ² , about 40 cm ² to about 130 cm ² , about 40 cm ² to about 120 cm ² , about 40 cm ² to about 110 cm ² , about 40 cm ² to about 100 cm ² , about 40 cm ² to about 90 cm ² , about 40 cm ² to about 80 cm ² , about 40 cm ² to about 70 cm ² , about 40 cm ² to about 60 cm ² , about 40 cm ² to about 50 cm ² , about 50 cm ² to about 150 cm ² , about 50 cm ² to about 140 cm ² , about 50 cm ² to about 130 cm ² , about 50 cm ² to about 120 cm ² , about 50 cm ² to about 110 cm ² , about 50 cm ² to about 100 cm ² , about 50 cm ² to about 90 cm ² , about 50 cm ² to about 80 cm ² , about 50 cm ² to about 70 cm ² , about 50 cm ² to about 60 cm ² , about 60 cm ² to about 150 cm ² , about 60 cm ² to about 140 cm ² , about 60 cm ² to about 130 cm ² , about 60 cm ² to about 120 cm ² , about 60 cm ² to about 110 cm ² , about 60 cm ² to about 100 cm ² , about 60 cm ² to about 90 cm ² , about 60 cm ² to about 80 cm ² , About 60 cm ² to about 70 cm ² , about 70 cm ² to about 150 cm ² , about 70 cm ² to about 140 cm ² , about 70 cm ² to about 130 cm ² , about 70 cm ² to about 120 cm ² , About 70 cm ² to about 110 cm ² , about 70 cm ² to about 100 cm ² , about 70 cm ² to about 90 cm ² , about 70 cm ² to about 80 cm ² , about 80 cm ² to about 150 cm ² , About 80 cm ² to about 140 cm ² , about 80 cm ² to about 130 cm ² , about 80 cm ² to about 120 cm ² , about 80 cm ² to about 110 cm ² , about 80 cm ² to about 100 cm ² , About 80 cm ² to about 90 cm ² , about 90 cm ² to about 150 cm ² , about 90 cm ² to about 140 cm ² , about 90 cm ² to about 130 cm ² , about 90 cm ² to about 120 cm ² , About 90 cm ² to about 110 cm ² , about 90 cm ² to about 100 cm ² , about 100 cm ² to about 150 cm ² , about 100 cm ² to about 140 cm ² , about 100 cm ² to about 130 cm ² , About 100 cm ² to about 120 cm ² , about 100 cm ² to about 110 cm ² , about 110 cm ² to about 150 cm ² , about 110 cm ² to about 140 cm ² , about 110 cm ² to about 130 cm ² , About 110 cm ² to about 120 cm ² , about 120 cm ² to about 150 cm ² , about 120 cm ² to about 140 cm ² , about 120 cm ² to about 130 cm ² , about 130 cm ² to about 150 cm ² , It is about 130 cm ² to about 140 cm ² , or about 140 cm ² to about 150 cm ² .

How many of any of the pods described herein that the wall comprises at least one area covered with a semi-porous or porous material that allows liquid to reversibly flow from outside the pod into the pod. In that embodiment, the percentage of the total surface area of the wall occupied by said at least one area covered with a semi-porous or porous material that allows liquid to reversibly flow from the outside of the pod into the inside of the pod. About 0.1% to about 95%, about 0.1% to about 90%, about 0.1% to about 85%, about 0.1% to about 80%, about 0.1% to about 75%, about 0.1% to about 70%, about 0.1 % ~ About 65%, about 0.1% ~ about 60%, about 0.1% ~ about 55%, about 0.1% ~ about 50%, about 0.1% ~ about 45%, about 0.1% ~ about 40%, about 0.1% ~ About 35%, about 0.1% to about 30%, about 0.1% to about 25%, about 0.1% to about 20%, about 0.1% to about 18%, about 0.1% to about 16%, about 0.1% to about 14 %, About 0.1% to about 12%, About 0.1% to about 10%, About 0.1% to about 8%, About 0.1% to about 6%, About 0.1% to about 5%, About 0.1% to about 4%, About 0.1% to about 3%, about 0.1% to about 2%, about 0.1% to about 1%, about 0.1% to about 0.5%, about 0.1% to about 0.2%, about 0.2% to about 95%, about 0.2 % ~ About 90%, about 0.2% ~ about 85%, about 0.2% ~ about 80%, about 0.2% ~ about 75%, about 0.2% ~ about 70%, about 0.2% ~ about 65%, about 0.2% ~ About 60%, about 0.2% to about 55%, about 0.2% to about 50%, about 0.2% to about 45%, about 0.2% to about 40%, about 0.2% to about 35%, about 0.2% to about 30 %, About 0.2% to about 25%, About 0.2% to about 20%, About 0.2% to about 18%, About 0.2% to about 16%, About 0.2% to about 14%, About 0.2% to about 12%, About 0.2% to about 10%, about 0.2% to about 8%, about 0.2% to about 6%, about 0.2% to about 5%, about 0.2% to about 4%, about 0.2% to about 3%, about 0.2 % ~ About 2%, about 0.2% ~ about 1%, about 0.2% ~ about 0.5%, about 0.5% ~ about 95%, about 0.5% ~ about 90%, about 0.5% ~ about 85%, about 0.5% ~ About 80%, about 0.5% to about 75%, about 0.5% to about 70%, about 0.5% to about 65%, about 0.5% to about 60%, about 0.5% to about 55%, about 0.5% to about 50 %, About 0.5% to about 45%, about 0.5% to about 40%, about 0.5% to about 35%, about 0.5% to about 30%, about 0.5% to about 25%, about 0.5% to about 20%, about 0.5% to about 18%, about 0.5% to about 16%, about 0.5% to about 14%, about 0.5% to about 12%, about 0.5% to about 10%, about 0.5% to about 8% , About 0.5% to about 6%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2%, about 0.5% to about 1%, about 1% to about 95%, about 1% to about 90%, about 1% to about 85%, about 1% to about 80%, about 1% to about 75%, about 1% to about 70%, about 1% ~ About 65%, about 1% ~ about 60%, about 1% ~ about 55%, about 1% ~ about 50%, about 1% ~ about 45%, about 1% ~ about 40%, about 1% ~ about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 18%, about 1% to about 16%, about 1% to about 14% , About 1% to about 12%, about 1% to about 10%, about 1% to about 8%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 95%, about 2% to about 90%, about 2% to about 85%, about 2% to about 80%, about 2% ~ About 75%, about 2% ~ about 70%, about 2% ~ about 65%, about 2% ~ about 60%, about 2% ~ about 55%, about 2% ~ about 50%, about 2% ~ about 45%, about 2% to about 40%, about 2% to about 35%, about 2% to about 30%, about 2% to about 25%, about 2% to about 20%, about 2% to about 18% , About 2% to about 16%, about 2% to about 14%, about 2% to about 12%, about 2% to about 10%, about 2% to about 8%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 95%, about 3% to about 90%, about 3% to about 85%, about 3% ~ About 80%, about 3% ~ about 75%, about 3% ~ about 70%, about 3% ~ about 65%, about 3% ~ about 60%, about 3% ~ about 55%, about 3% ~ about 50%, about 3% to about 45%, about 3% to about 40%, about 3% to about 35%, about 3% to about 30%, about 3% to about 25%, about 3% to about 20% , About 3% to about 18%, about 3% to about 16%, about 3% to about 14%, about 3% to about 12%, about 3% to about 10%, about 3% to about 8%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4% to about 95%, about 4% to about 90%, about 4% to about 85%, about 4% ~ About 80%, about 4% ~ about 75%, about 4% ~ about 70%, about 4% ~ about 65%, about 4% ~ about 60%, about 4% ~ about 55%, about 4% ~ about 50%, about 4% to about 45%, about 4% to about 40%, about 4% to about 35%, about 4% to about 30%, about 4% to about 25%, about 4% to about 20% , About 4% ~ about 18%, about 4% ~ about 16%, about 4% ~ about 14%, about 4% ~ about 12%, about 4% ~ About 10%, about 4% to about 8%, about 4% to about 6%, about 4% to about 5%, about 5% to about 95%, about 5% to about 90%, about 5% to about 85 %, About 5% to about 80%, About 5% to about 75%, About 5% to about 70%, About 5% to about 65%, About 5% to about 60%, About 5% to about 55%, About 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5 % ~ About 20%, about 5% ~ about 18%, about 5% ~ about 16%, about 5% ~ about 14%, about 5% ~ about 12%, about 5% ~ about 10%, about 5% ~ About 8%, about 5% to about 6%, about 6% to about 95%, about 6% to about 90%, about 6% to about 85%, about 6% to about 80%, about 6% to about 75 %, About 6% to about 70%, About 6% to about 65%, About 6% to about 60%, About 6% to about 55%, About 6% to about 50%, About 6% to about 45%, About 6% to about 40%, about 6% to about 35%, about 6% to about 30%, about 6% to about 25%, about 6% to about 20%, about 6% to about 18%, about 6 % ~ About 16%, about 6% ~ about 14%, about 6% ~ about 12%, about 6% ~ about 10%, about 6% ~ about 8%, about 8% ~ about 95%, about 8% ~ About 90%, about 8% to about 85%, about 8% to about 80%, about 8% to about 75%, about 8% to about 70%, about 8% to about 65%, about 8% to about 60 %, About 8% to about 55%, About 8% to about 50%, About 8% to about 45%, About 8% to about 40%, About 8% to about 35%, About 8% to about 30%, About 8% to about 25%, about 8% to about 20%, about 8% to about 18%, about 8% to about 16%, about 8% to about 14%, about 8% to about 12%, about 8 % ~ About 10%, about 10% ~ about 95%, about 10% ~ about 90%, about 10% ~ about 85%, about 10% ~ about 80%, about 10% ~ about 75%, about 10% ~ About 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40 %, About 10% to about 35%, About 10% to about 30%, About 10% to about 25%, About 10% to about 20%, About 10% to about 18%, About 10% to about 16%, About 10% to about 14%, about 10% to about 12%, about 12% to about 95%, about 12% to about 90%, about 12% to about 85%, about 12% to about 80%, about 12 % ~ 75%, about 12% ~ 70%, about 12% ~ 65%, about 12% ~ 60%, about 12% ~ 55%, about 12% ~ about 50%, about 12% ~ About 45%, about 12% to about 40%, about 12% to about 35%, about 12% to about 30%, about 12% to about 25%, about 12% to about 20%, about 12% to about 18 %, About 12% to about 16%, About 12% to about 14%, about 14% to about 95%, about 14% to about 90%, about 14% to about 85%, about 14% to about 80%, about 14% to about 75%, about 14 % ~ About 70%, about 14% ~ about 65%, about 14% ~ about 60%, about 14% ~ about 55%, about 14% ~ about 50%, about 14% ~ about 45%, about 14% ~ About 40%, about 14% to about 35%, about 14% to about 30%, about 14% to about 25%, about 14% to about 20%, about 14% to about 18%, about 14% to about 16 %, About 16% to about 95%, About 16% to about 90%, About 16% to about 85%, About 16% to about 80%, About 16% to about 75%, About 16% to about 70%, About 16% to about 65%, about 16% to about 60%, about 16% to about 55%, about 16% to about 50%, about 16% to about 45%, about 16% to about 40%, about 16 % ~ About 35%, About 16% ~ About 30%, About 16% ~ About 25%, About 16% ~ About 20%, About 16% ~ About 18%, About 18% ~ About 95%, About 18% ~ About 90%, about 18% to about 85%, about 18% to about 80%, about 18% to about 75%, about 18% to about 70%, about 18% to about 65%, about 18% to about 60 %, About 18% to about 55%, About 18% to about 50%, About 18% to about 45%, About 18% to about 40%, About 18% to about 35%, About 18% to about 30%, About 18% to about 25%, about 18% to about 20%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20 % ~ 75%, about 20% ~ 70%, about 20% ~ 65%, about 20% ~ 60%, about 20% ~ 55%, about 20% ~ about 50%, about 20% ~ About 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 95%, about 25% to about 90 %, About 25% to about 85%, About 25% to about 80%, About 25% to about 75%, About 25% to about 70%, About 25% to about 65%, About 25% to about 60%, About 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30 % ~ 95%, about 30% ~ 90%, about 30% ~ about 85%, about 30% ~ about 80%, about 30% ~ about 75%, about 30% ~ about 70%, about 30% ~ About 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35 %, About 35% to about 95%, About 35% to about 90%, About 35% to about 85%, About 35% to about 80%, About 35% to about 75%, About 35% to about 70%, About 35% to about 65%, about 35% to about 60%, about 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40 % ~ 95%, 40% ~ 90%, 40% ~ 85%, 40% ~ 80%, 40% ~ 75%, 40% ~ 70%, 40% ~ About 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 45% to about 95%, about 45% to about 90 %, About 45% to about 85%, About 45% to about 80%, About 45% to about 75%, About 45% to about 70%, About 45% to about 65%, About 45% to about 60%, About 45% to about 55%, about 45% to about 50%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50 % ~ 75%, about 50% ~ 70%, about 50% ~ 65%, about 50% ~ 60%, about 50% ~ 55%, about 55% ~ about 95%, about 55% ~ About 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60 %, About 60% to about 95%, About 60% to about 90%, About 60% to about 85%, About 60% to about 80%, About 60% to about 75%, About 60% to about 70%, About 60% to about 65%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 65% to about 75%, about 65 % ~ About 70%, about 70% ~ about 95%, about 70% ~ about 90%, about 70% ~ about 85%, about 70% ~ about 80%, about 70% ~ about 75%, about 75% ~ About 95%, about 75% to about 90%, about 75% to about 85%, about 75% to about 80%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85 %, About 85% to about 95%, about 85% to about 90%, or about 90% to about 95%.

How many of any of the pods described herein, wherein the lower end comprises at least one area covered with a semi-porous or porous material that allows liquid to reversibly flow into the pod from outside the pod. In that embodiment, the percentage of total surface area at the bottom edge occupied by the at least one area covered with a semi-porous or porous material that allows the liquid to reversibly flow into the pod from outside the pod. About 1% to about 100%, about 1% to about 95%, about 1% to about 90%, about 1% to about 85%, about 1% to about 80%, about 1% to about 75%, about 1 % ~ About 70%, about 1% ~ about 65%, about 1% ~ about 60%, about 1% ~ about 55%, about 1% ~ about 50%, about 1% ~ about 45%, about 1% ~ About 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 18%, about 1% to about 16 %, About 1% to about 14%, About 1% to about 12%, About 1% to about 10%, About 1% to about 8%, About 1% to about 6%, About 1% to about 5%, About 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 100%, about 2% to about 95%, about 2% to about 90%, about 2 % ~ About 85%, about 2% ~ about 80%, about 2% ~ about 75%, about 2% ~ about 70%, about 2% ~ about 65%, about 2% ~ about 60%, about 2% ~ About 55%, about 2% to about 50%, about 2% to about 45%, about 2% to about 40%, about 2% to about 35%, about 2% to about 30%, about 2% to about 25 %, About 2% to about 20%, About 2% to about 18%, About 2% to about 16%, About 2% to about 14%, About 2% to about 12%, About 2% to about 10%, About 2% to about 8%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 100%, about 3 % ~ 95%, about 3% ~ 90%, about 3% ~ about 85%, about 3% ~ about 80%, about 3% ~ about 75%, about 3% ~ about 70%, about 3% ~ About 65%, about 3% to about 60%, about 3% to about 55%, about 3% to about 50%, about 3% to about 45%, about 3% to about 40%, about 3% to about 35 %, About 3% to about 30%, About 3% to about 25%, About 3% to about 20%, About 3% to about 18%, About 3% to about 16%, About 3% to about 14%, About 3% to about 12%, about 3% to about 10%, about 3% to about 8%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4 % ~ About 100%, about 4% ~ about 95%, about 4% ~ about 90%, about 4% ~ about 85%, about 4% ~ about 80%, about 4% ~ about 75%, about 4% ~ About 70%, about 4% to about 65%, about 4% to about 60%, about 4% to about 55%, About 4% to about 50%, about 4% to about 45%, about 4% to about 40%, about 4% to about 35%, about 4% to about 30%, about 4% to about 25%, about 4 % ~ About 20%, about 4% ~ about 18%, about 4% ~ about 16%, about 4% ~ about 14%, about 4% ~ about 12%, about 4% ~ about 10%, about 4% ~ About 8%, about 4% to about 6%, about 4% to about 5%, about 5% to about 100%, about 5% to about 95%, about 5% to about 90%, about 5% to about 85 %, About 5% to about 80%, About 5% to about 75%, About 5% to about 70%, About 5% to about 65%, About 5% to about 60%, About 5% to about 55%, About 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5 % ~ About 20%, about 5% ~ about 18%, about 5% ~ about 16%, about 5% ~ about 14%, about 5% ~ about 12%, about 5% ~ about 10%, about 5% ~ About 8%, about 5% to about 6%, about 6% to about 100%, about 6% to about 95%, about 6% to about 90%, about 6% to about 85%, about 6% to about 80 %, About 6% to about 75%, About 6% to about 70%, About 6% to about 65%, About 6% to about 60%, About 6% to about 55%, About 6% to about 50%, About 6% to about 45%, about 6% to about 40%, about 6% to about 35%, about 6% to about 30%, about 6% to about 25%, about 6% to about 20%, about 6 % ~ About 18%, about 6% ~ about 16%, about 6% ~ about 14%, about 6% ~ about 12%, about 6% ~ about 10%, about 6% ~ about 8%, about 8% ~ About 100%, about 8% to about 95%, about 8% to about 90%, about 8% to about 85%, about 8% to about 80%, about 8% to about 75%, about 8% to about 70 %, About 8% to about 65%, About 8% to about 60%, About 8% to about 55%, About 8% to about 50%, About 8% to about 45%, About 8% to about 40%, About 8% to about 35%, about 8% to about 30%, about 8% to about 25%, about 8% to about 20%, about 8% to about 18%, about 8% to about 16%, about 8 % ~ About 14%, about 8% ~ about 12%, about 8% ~ about 10%, about 10% ~ about 100%, about 10% ~ about 95%, about 10% ~ about 90%, about 10% ~ About 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55 %, About 10% to about 50%, About 10% to about 45%, About 10% to about 40%, About 10% to about 35%, About 10% to about 30%, About 10% to about 25%, About 10% to about 20%, about 10% to about 18%, about 10% to about 16%, about 10% to about 14%, about 10% to about 12%, about 12% to about 100%, about 12 % ~ 95%, about 12% ~ 90%, about 12% ~ About 85%, about 12% to about 80%, about 12% to about 75%, about 12% to about 70%, about 12% to about 65%, about 12% to about 60%, about 12% to about 55 %, About 12% to about 50%, About 12% to about 45%, About 12% to about 40%, About 12% to about 35%, About 12% to about 30%, About 12% to about 25%, About 12% to about 20%, about 12% to about 18%, about 12% to about 16%, about 12% to about 14%, about 14% to about 100%, about 14% to about 95%, about 14 % ~ 90%, about 14% ~ about 85%, about 14% ~ about 80%, about 14% ~ about 75%, about 14% ~ about 70%, about 14% ~ about 65%, about 14% ~ About 60%, about 14% to about 55%, about 14% to about 50%, about 14% to about 45%, about 14% to about 40%, about 14% to about 35%, about 14% to about 30 %, About 14% to about 25%, About 14% to about 20%, About 14% to about 18%, About 14% to about 16%, About 16% to about 100%, About 16% to about 95%, About 16% to about 90%, about 16% to about 85%, about 16% to about 80%, about 16% to about 75%, about 16% to about 70%, about 16% to about 65%, about 16 % ~ About 60%, about 16% ~ about 55%, about 16% ~ about 50%, about 16% ~ about 45%, about 16% ~ about 40%, about 16% ~ about 35%, about 16% ~ About 30%, about 16% to about 25%, about 16% to about 20%, about 16% to about 18%, about 18% to about 100%, about 18% to about 95%, about 18% to about 90 %, About 18% to about 85%, About 18% to about 80%, About 18% to about 75%, About 18% to about 70%, About 18% to about 65%, About 18% to about 60%, About 18% to about 55%, about 18% to about 50%, about 18% to about 45%, about 18% to about 40%, about 18% to about 35%, about 18% to about 30%, about 18 % ~ About 25%, About 18% ~ About 20%, About 20% ~ About 100%, About 20% ~ About 95%, About 20% ~ About 90%, About 20% ~ About 85%, About 20% ~ About 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50 %, About 20% to about 45%, About 20% to about 40%, About 20% to about 35%, About 20% to about 30%, About 20% to about 25%, About 25% to about 100%, About 25% to about 95%, about 25% to about 90%, about 25% to about 85%, about 25% to about 80%, about 25% to about 75%, about 25% to about 70%, about 25 % ~ About 65%, about 25% ~ about 60%, about 25% ~ about 55%, about 25% ~ about 50%, about 25% ~ about 45%, about 25% ~ about 40%, About 25% to about 35%, about 25% to about 30%, about 30% to about 100%, about 30% to about 95%, about 30% to about 90%, about 30% to about 85%, about 30 % ~ About 80%, about 30% ~ about 75%, about 30% ~ about 70%, about 30% ~ about 65%, about 30% ~ about 60%, about 30% ~ about 55%, about 30% ~ About 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 100%, about 35% to about 95%, about 35% to about 90 %, About 35% to about 85%, About 35% to about 80%, About 35% to about 75%, About 35% to about 70%, About 35% to about 65%, About 35% to about 60%, About 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 100%, about 40% to about 95%, about 40 % ~ 90%, about 40% ~ 85%, about 40% ~ about 80%, about 40% ~ about 75%, about 40% ~ about 70%, about 40% ~ about 65%, about 40% ~ About 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 45% to about 100%, about 45% to about 95%, about 45% to about 90 %, About 45% to about 85%, About 45% to about 80%, About 45% to about 75%, About 45% to about 70%, About 45% to about 65%, About 45% to about 60%, About 45% to about 55%, about 45% to about 50%, about 50% to about 100%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50 % ~ About 80%, about 50% ~ about 75%, about 50% ~ about 70%, about 50% ~ about 65%, about 50% ~ about 60%, about 50% ~ about 55%, about 55% ~ About 100%, about 55% to about 95%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70 %, About 55% to about 65%, about 55% to about 60%, about 60% to about 100%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, About 60% to about 80%, about 60% to about 75%, about 60% to about 70%, about 60% to about 65%, about 65% to about 100%, about 65% to about 95%, about 65 % ~ 90%, about 65% ~ about 85%, about 65% ~ about 80%, about 65% ~ about 75%, about 65% ~ about 70%; about 70% ~ about 100%, about 70% ~ About 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, about 70% to about 75%, about 75% to about 100%, about 75% to about 95 %, About 75% to about 90%, About 75% to about 85%, About 75% to about 80%, About 80% to about 100%, About 80% to about 95% , About 80% to about 90%, about 80% to about 85%, about 85% to about 100%, about 85% to about 95%, about 85% to about 90%, about 90% to about 100%, about 90% to about 95%, or about 95% to about 100%.

How many of any of the pods described herein, the top of which comprises at least one area covered with a semi-porous or porous material that allows liquid to reversibly flow into the pod from outside the pod. In that embodiment, the percentage of total surface area at the top edge, occupied by at least one area covered by a semi-porous or porous material that allows liquid to reversibly flow from outside the pod into the pod. About 1% to about 100%, about 1% to about 95%, about 1% to about 90%, about 1% to about 85%, about 1% to about 80%, about 1% to about 75%, about 1 % ~ About 70%, about 1% ~ about 65%, about 1% ~ about 60%, about 1% ~ about 55%, about 1% ~ about 50%, about 1% ~ about 45%, about 1% ~ About 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 18%, about 1% to about 16 %, About 1% to about 14%, About 1% to about 12%, About 1% to about 10%, About 1% to about 8%, About 1% to about 6%, About 1% to about 5%, About 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 100%, about 2% to about 95%, about 2% to about 90%, about 2 % ~ About 85%, about 2% ~ about 80%, about 2% ~ about 75%, about 2% ~ about 70%, about 2% ~ about 65%, about 2% ~ about 60%, about 2% ~ About 55%, about 2% to about 50%, about 2% to about 45%, about 2% to about 40%, about 2% to about 35%, about 2% to about 30%, about 2% to about 25 %, About 2% to about 20%, About 2% to about 18%, About 2% to about 16%, About 2% to about 14%, About 2% to about 12%, About 2% to about 10%, About 2% to about 8%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 100%, about 3 % ~ 95%, about 3% ~ 90%, about 3% ~ about 85%, about 3% ~ about 80%, about 3% ~ about 75%, about 3% ~ about 70%, about 3% ~ About 65%, about 3% to about 60%, about 3% to about 55%, about 3% to about 50%, about 3% to about 45%, about 3% to about 40%, about 3% to about 35 %, About 3% to about 30%, About 3% to about 25%, About 3% to about 20%, About 3% to about 18%, About 3% to about 16%, About 3% to about 14%, About 3% to about 12%, about 3% to about 10%, about 3% to about 8%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4 % ~ About 100%, about 4% ~ about 95%, about 4% ~ about 90%, about 4% ~ about 85%, about 4% ~ about 80%, about 4% ~ about 75%, about 4% ~ About 70%, about 4% to about 65%, about 4% to about 60%, about 4% to about 55%, About 4% to about 50%, about 4% to about 45%, about 4% to about 40%, about 4% to about 35%, about 4% to about 30%, about 4% to about 25%, about 4 % ~ About 20%, about 4% ~ about 18%, about 4% ~ about 16%, about 4% ~ about 14%, about 4% ~ about 12%, about 4% ~ about 10%, about 4% ~ About 8%, about 4% to about 6%, about 4% to about 5%, about 5% to about 100%, about 5% to about 95%, about 5% to about 90%, about 5% to about 85 %, About 5% to about 80%, About 5% to about 75%, About 5% to about 70%, About 5% to about 65%, About 5% to about 60%, About 5% to about 55%, About 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5 % ~ About 20%, about 5% ~ about 18%, about 5% ~ about 16%, about 5% ~ about 14%, about 5% ~ about 12%, about 5% ~ about 10%, about 5% ~ About 8%, about 5% to about 6%, about 6% to about 100%, about 6% to about 95%, about 6% to about 90%, about 6% to about 85%, about 6% to about 80 %, About 6% to about 75%, About 6% to about 70%, About 6% to about 65%, About 6% to about 60%, About 6% to about 55%, About 6% to about 50%, About 6% to about 45%, about 6% to about 40%, about 6% to about 35%, about 6% to about 30%, about 6% to about 25%, about 6% to about 20%, about 6 % ~ About 18%, about 6% ~ about 16%, about 6% ~ about 14%, about 6% ~ about 12%, about 6% ~ about 10%, about 6% ~ about 8%, about 8% ~ About 100%, about 8% to about 95%, about 8% to about 90%, about 8% to about 85%, about 8% to about 80%, about 8% to about 75%, about 8% to about 70 %, About 8% to about 65%, About 8% to about 60%, About 8% to about 55%, About 8% to about 50%, About 8% to about 45%, About 8% to about 40%, About 8% to about 35%, about 8% to about 30%, about 8% to about 25%, about 8% to about 20%, about 8% to about 18%, about 8% to about 16%, about 8 % ~ About 14%, about 8% ~ about 12%, about 8% ~ about 10%, about 10% ~ about 100%, about 10% ~ about 95%, about 10% ~ about 90%, about 10% ~ About 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55 %, About 10% to about 50%, About 10% to about 45%, About 10% to about 40%, About 10% to about 35%, About 10% to about 30%, About 10% to about 25%, About 10% to about 20%, about 10% to about 18%, about 10% to about 16%, about 10% to about 14%, about 10% to about 12%, about 12% to about 100%, about 12 % ~ 95%, about 12% ~ 90%, about 12% ~ About 85%, about 12% to about 80%, about 12% to about 75%, about 12% to about 70%, about 12% to about 65%, about 12% to about 60%, about 12% to about 55 %, About 12% to about 50%, About 12% to about 45%, About 12% to about 40%, About 12% to about 35%, About 12% to about 30%, About 12% to about 25%, About 12% to about 20%, about 12% to about 18%, about 12% to about 16%, about 12% to about 14%, about 14% to about 100%, about 14% to about 95%, about 14 % ~ 90%, about 14% ~ about 85%, about 14% ~ about 80%, about 14% ~ about 75%, about 14% ~ about 70%, about 14% ~ about 65%, about 14% ~ About 60%, about 14% to about 55%, about 14% to about 50%, about 14% to about 45%, about 14% to about 40%, about 14% to about 35%, about 14% to about 30 %, About 14% to about 25%, About 14% to about 20%, About 14% to about 18%, About 14% to about 16%, About 16% to about 100%, About 16% to about 95%, About 16% to about 90%, about 16% to about 85%, about 16% to about 80%, about 16% to about 75%, about 16% to about 70%, about 16% to about 65%, about 16 % ~ About 60%, about 16% ~ about 55%, about 16% ~ about 50%, about 16% ~ about 45%, about 16% ~ about 40%, about 16% ~ about 35%, about 16% ~ About 30%, about 16% to about 25%, about 16% to about 20%, about 16% to about 18%, about 18% to about 100%, about 18% to about 95%, about 18% to about 90 %, About 18% to about 85%, About 18% to about 80%, About 18% to about 75%, About 18% to about 70%, About 18% to about 65%, About 18% to about 60%, About 18% to about 55%, about 18% to about 50%, about 18% to about 45%, about 18% to about 40%, about 18% to about 35%, about 18% to about 30%, about 18 % ~ About 25%, About 18% ~ About 20%, About 20% ~ About 100%, About 20% ~ About 95%, About 20% ~ About 90%, About 20% ~ About 85%, About 20% ~ About 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50 %, About 20% to about 45%, About 20% to about 40%, About 20% to about 35%, About 20% to about 30%, About 20% to about 25%, About 25% to about 100%, About 25% to about 95%, about 25% to about 90%, about 25% to about 85%, about 25% to about 80%, about 25% to about 75%, about 25% to about 70%, about 25 % ~ About 65%, about 25% ~ about 60%, about 25% ~ about 55%, about 25% ~ about 50%, about 25% ~ about 45%, about 25% ~ about 40%, About 25% to about 35%, about 25% to about 30%, about 30% to about 100%, about 30% to about 95%, about 30% to about 90%, about 30% to about 85%, about 30 % ~ About 80%, about 30% ~ about 75%, about 30% ~ about 70%, about 30% ~ about 65%, about 30% ~ about 60%, about 30% ~ about 55%, about 30% ~ About 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 100%, about 35% to about 95%, about 35% to about 90 %, About 35% to about 85%, About 35% to about 80%, About 35% to about 75%, About 35% to about 70%, About 35% to about 65%, About 35% to about 60%, About 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 100%, about 40% to about 95%, about 40 % ~ 90%, about 40% ~ 85%, about 40% ~ about 80%, about 40% ~ about 75%, about 40% ~ about 70%, about 40% ~ about 65%, about 40% ~ About 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 45% to about 100%, about 45% to about 95%, about 45% to about 90 %, About 45% to about 85%, About 45% to about 80%, About 45% to about 75%, About 45% to about 70%, About 45% to about 65%, About 45% to about 60%, About 45% to about 55%, about 45% to about 50%, about 50% to about 100%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50 % ~ About 80%, about 50% ~ about 75%, about 50% ~ about 70%, about 50% ~ about 65%, about 50% ~ about 60%, about 50% ~ about 55%, about 55% ~ About 100%, about 55% to about 95%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70 %, About 55% to about 65%, about 55% to about 60%, about 60% to about 100%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, About 60% to about 80%, about 60% to about 75%, about 60% to about 70%, about 60% to about 65%, about 65% to about 100%, about 65% to about 95%, about 65 % ~ 90%, about 65% ~ about 85%, about 65% ~ about 80%, about 65% ~ about 75%, about 65% ~ about 70%; about 70% ~ about 100%, about 70% ~ About 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, about 70% to about 75%, about 75% to about 100%, about 75% to about 95 %, About 75% to about 90%, About 75% to about 85%, About 75% to about 80%, About 80% to about 100%, About 80% to about 95% , About 80% to about 90%, about 80% to about 85%, about 85% to about 100%, about 85% to about 95%, about 85% to about 90%, about 90% to about 100%, about 90% to about 95%, or about 95% to about 100%.

Container In some embodiments, the bottom edge of the container is flat, hemispherical, or conical.

In some examples of any of the pods described herein, the container is about 0.05 mL to about 1,000 mL, about 0.05 mL to about 950 mL, about 0.05 mL to about 900 mL, about 0.05 mL to about. 850 mL, about 0.05 mL to about 800 mL, about 0.05 mL to about 750 mL, about 0.05 mL to about 700 mL, about 0.05 mL to about 650 mL, about 0.05 mL to about 600 mL, about 0.05 mL to about 550 mL , About 0.05 mL to about 500 mL, about 0.05 mL to about 450 mL, about 0.05 mL to about 400 mL, about 0.05 mL to about 350 mL, about 0.05 mL to about 300 mL, about 0.05 mL to about 250 mL, about 0.05 mL to about 200 mL, about 0.05 mL to about 150 mL, about 0.05 mL to about 100 mL, about 0.05 mL to about 90 mL, about 0.05 mL to about 80 mL, about 0.05 mL to about 70 mL, about 0.05 mL ~ About 60 mL, about 0.05 mL ~ about 50 mL, about 0.05 mL ~ about 45 mL, about 0.05 mL ~ about 40 mL, about 0.05 mL ~ about 35 mL, about 0.05 mL ~ about 30 mL, about 0.05 mL ~ about 25 mL, about 0.05 mL to about 20 mL, about 0.05 mL to about 15 mL, about 0.05 mL to about 10 mL, about 0.05 mL to about 8 mL, about 0.05 mL to about 6 mL, about 0.05 mL to about 5 mL , About 0.05 mL ~ about 4 mL, about 0.05 mL ~ about 3 mL, about 0.05 mL ~ about 2 mL, about 0.05 mL ~ about 1 mL, about 0.05 mL ~ about 0.5 mL, about 0.05 mL ~ about 0.1 mL, about 0.1 mL to about 1,000 mL, about 0.1 mL to about 950 mL, about 0.1 mL to about 900 mL, about 0.1 mL to about 850 mL, about 0.1 mL to about 800 mL, about 0.1 mL to about 750 mL, about 0.1 mL ~ About 700 mL, about 0.1 mL ~ about 650 mL, about 0.1 mL ~ about 600 mL, about 0.1 mL ~ about 550 mL, about 0.1 mL ~ about 500 mL, about 0.1 mL ~ about 450 mL, about 0.1 mL ~ about 400 mL, about 0.1 mL to about 350 mL, about 0.1 mL to about 300 mL, about 0.1 mL to about 250 mL, about 0.1 mL to about 200 mL, about 0.1 mL to about 150 mL, about 0.1 mL to about 100 mL, about 0.1 mL to about 90 mL, about 0.1 mL to about 80 mL, about 0.1 mL to about 70 mL, about 0.1 mL to about 60 mL, about 0.1 mL to about 50 mL, about 0.1 mL ~ About 45 mL, about 0.1 mL ~ about 40 mL, about 0.1 mL ~ about 35 mL, about 0.1 mL ~ about 30 mL, about 0.1 mL ~ about 25 mL, about 0.1 mL ~ about 20 mL, about 0.1 mL ~ about 15 mL, about 0.1 mL to about 10 mL, about 0.1 mL to about 8 mL, about 0.1 mL to about 6 mL, about 0.1 mL to about 5 mL, about 0.1 mL to about 4 mL, about 0.1 mL to about 3 mL , About 0.1 mL to about 2 mL, about 0.1 mL to about 1 mL, about 0.1 mL to about 0.5 mL, about 0.5 mL to about 1,000 mL, about 0.5 mL to about 950 mL, about 0.5 mL to about 900 mL, about 0.5 mL to about 850 mL, about 0.5 mL to about 800 mL, about 0.5 mL to about 750 mL, about 0.5 mL to about 700 mL, about 0.5 mL to about 650 mL, about 0.5 mL to about 600 mL, about 0.5 mL ~ About 550 mL, about 0.5 mL ~ about 500 mL, about 0.5 mL ~ about 450 mL, about 0.5 mL ~ about 400 mL, about 0.5 mL ~ about 350 mL, about 0.5 mL ~ about 300 mL, about 0.5 mL ~ about 250 mL, about 0.5 mL to about 200 mL, about 0.5 mL to about 150 mL, about 0.5 mL to about 100 mL, about 0.5 mL to about 90 mL, about 0.5 mL to about 80 mL, about 0.5 mL to about 70 mL , About 0.5 mL to about 60 mL, about 0.5 mL to about 50 mL, about 0.5 mL to about 45 mL, about 0.5 mL to about 40 mL, about 0.5 mL to about 35 mL, about 0.5 mL to about 30 mL, about 0.5 mL to about 25 mL, about 0.5 mL to about 20 mL, about 0.5 mL to about 15 mL, about 0.5 mL to about 10 mL, about 0.5 mL to about 8 mL, about 0.5 mL to about 6 mL, about 0.5 mL ~ About 5 mL, about 0.5 mL ~ about 4 mL, about 0.5 mL ~ about 3 mL, about 0.5 mL ~ about 2 mL, about 0.5 mL ~ about 1 mL, about 1 mL ~ about 1,000 mL, about 1 mL ~ about 950 mL, about 1 mL to about 900 mL, about 1 mL to about 850 mL, about 1 mL to about 800 mL, about 1 mL ~ About 750 mL, about 1 mL ~ about 700 mL, about 1 mL ~ about 650 mL, about 1 mL ~ about 600 mL, about 1 mL ~ about 550 mL, about 1 mL ~ about 500 mL, about 1 mL ~ about 450 mL, about 1 mL to about 400 mL, about 1 mL to about 350 mL, about 1 mL to about 300 mL, about 1 mL to about 250 mL, about 1 mL to about 200 mL, about 1 mL to about 150 mL , About 1 mL to about 100 mL, about 1 mL to about 90 mL, about 1 mL to about 80 mL, about 1 mL to about 70 mL, about 1 mL to about 60 mL, about 1 mL to about 50 mL, about 1 mL to about 45 mL, about 1 mL to about 40 mL, about 1 mL to about 35 mL, about 1 mL to about 30 mL, about 1 mL to about 25 mL, about 1 mL to about 20 mL, about 1 mL ~ About 15 mL, about 1 mL ~ about 10 mL, about 1 mL ~ about 8 mL, about 1 mL ~ about 6 mL, about 1 mL ~ about 5 mL, about 1 mL ~ about 4 mL, about 1 mL ~ about 3 mL, about 1 mL to about 2 mL, about 2 mL to about 1,000 mL, about 2 mL to about 950 mL, about 2 mL to about 900 mL, about 2 mL to about 850 mL, about 2 mL to about 800 mL , About 2 mL to about 750 mL, about 2 mL to about 700 mL, about 2 mL to about 650 mL, about 2 mL to about 600 mL, about 2 mL to about 550 mL, about 2 mL to about 500 mL, about 2 mL to about 450 mL, about 2 mL to about 400 mL, about 2 mL to about 350 mL, about 2 mL to about 300 mL, about 2 mL to about 250 mL, about 2 mL to about 200 mL, about 2 mL ~ About 150 mL, about 2 mL ~ about 100 mL, about 2 mL ~ about 90 mL, about 2 mL ~ about 80 mL, about 2 mL ~ about 70 mL, about 2 mL ~ about 60 mL, about 2 mL ~ about 50 mL, about 2 mL to about 45 mL, about 2 mL to about 40 mL, about 2 mL to about 35 mL, about 2 mL to about 30 mL, about 2 mL to about 25 mL, about 2 mL to about 20 mL , About 2 mL to about 15 mL, about 2 mL to about 10 mL, about 2 mL to about 8 mL, about 2 mL to about 6 mL, about 2 mL to about 5 mL, about 2 mL to about 4 mL, about 2 mL to about 3 mL, about 3 mL to about 1,000 mL, about 3 mL to about 950 mL, about 3 mL to about 900 mL, about 3 mL to about 850 mL, about 3 mL to about 800 mL , About 3 mL to about 750 mL, about 3 mL to about 700 mL, about 3 mL to about 650 mL, about 3 mL to about 600 mL, about 3 mL to about 550 mL, about 3 mL to about 500 mL, about 3 mL to about 450 mL, about 3 mL to about 400 mL, about 3 mL to about 350 mL, about 3 mL to about 300 mL, about 3 mL to about 250 mL, about 3 mL to about 200 mL, about 3 mL ~ About 150 mL, about 3 mL ~ about 100 mL, about 3 mL ~ about 90 mL, about 3 mL ~ about 80 mL, about 3 mL ~ about 70 mL, about 3 mL ~ about 60 mL, about 3 mL ~ about 50 mL, about 3 mL to about 45 mL, about 3 mL to about 40 mL, about 3 mL to about 35 mL, about 3 mL to about 30 mL, about 3 mL to about 25 mL, about 3 mL to about 20 mL , About 3 mL to about 15 mL, about 3 mL to about 10 mL, about 3 mL to about 8 mL, about 3 mL to about 6 mL, about 3 mL to about 5 mL, about 3 mL to about 4 mL, about 4 mL to about 1,000 mL, about 4 mL to about 950 mL, about 4 mL to about 900 mL, about 4 mL to about 850 mL, about 4 mL to about 800 mL, about 4 mL to about 750 mL, about 4 mL ~ About 700 mL, about 4 mL ~ about 650 mL, about 4 mL ~ about 600 mL, about 4 mL ~ about 550 mL, about 4 mL ~ about 500 mL, about 4 mL ~ about 450 mL, about 4 mL ~ about 400 mL, about 4 mL to about 350 mL, about 4 mL to about 300 mL, about 4 mL to about 250 mL, about 4 mL to about 200 mL, about 4 mL to about 150 mL, about 4 mL to about 100 mL , About 4 mL to about 90 mL, about 4 mL to about 80 mL, about 4 mL to about 70 mL, about 4 mL to about 60 mL, about 4 mL to about 50 mL, about 4 mL to about 45 mL, about 4 mL to about 40 mL, about 4 mL to about 35 mL, about 4 mL to about 30 mL, about 4 mL to about 25 mL, about 4 mL to about 20 mL, about 4 mL to about 15 mL, about 4 mL ~ About 10 mL, about 4 mL ~ about 8 mL, about 4 mL ~ about 6 mL, about 4 mL ~ about 5 mL, about 5 mL ~ about 1,000 mL, about 5 mL ~ about 950 mL, about 5 mL ~ about 900 mL, about 5 mL to about 850 mL, about 5 mL to about 800 mL, about 5 mL to about 750 mL, about 5 mL to about 700 mL, about 5 mL to about 650 mL , About 5 mL to about 600 mL, about 5 mL to about 550 mL, about 5 mL to about 500 mL, about 5 mL to about 450 mL, about 5 mL to about 400 mL, about 5 mL to about 350 mL, about 5 mL to about 300 mL, about 5 mL to about 250 mL, about 5 mL to about 200 mL, about 5 mL to about 150 mL, about 5 mL to about 100 mL, about 5 mL to about 90 mL, about 5 mL ~ About 80 mL, about 5 mL ~ about 70 mL, about 5 mL ~ about 60 mL, about 5 mL ~ about 50 mL, about 5 mL ~ about 45 mL, about 5 mL ~ about 40 mL, about 5 mL ~ about 35 mL, about 5 mL to about 30 mL, about 5 mL to about 25 mL, about 5 mL to about 20 mL, about 5 mL to about 15 mL, about 5 mL to about 10 mL, about 5 mL to about 8 mL , About 5 mL to about 6 mL, about 6 mL to about 1,000 mL, about 6 mL to about 950 mL, about 6 mL to about 900 mL, about 6 mL to about 850 mL, about 6 mL to about 800 mL, about 6 mL to about 750 mL, about 6 mL to about 700 mL, about 6 mL to about 650 mL, about 6 mL to about 600 mL, about 6 mL to about 550 mL, about 6 mL to about 500 mL, about 6 mL ~ 450 mL, about 6 mL ~ about 400 mL, about 6 mL ~ about 350 mL, about 6 mL ~ about 300 mL, about 6 mL ~ about 250 mL, about 6 mL ~ about 200 mL, about 6 mL ~ about 150 mL, about 6 mL to about 100 mL, about 6 mL to about 90 mL, about 6 mL to about 80 mL, about 6 mL to about 70 mL, about 6 mL to about 60 mL, about 6 mL to about 50 mL , About 6 mL to about 45 mL, about 6 mL to about 40 mL, about 6 mL to about 35 mL, about 6 mL to about 30 mL, about 6 mL to about 25 mL, about 6 mL to about 20 mL, about 6 mL to about 15 mL, about 6 mL to about 10 mL, about 6 mL to about 8 mL, about 8 mL to about 1,000 mL, about 8 mL to about 950 mL, about 8 mL to about 900 mL, about 8 mL ~ About 850 mL, about 8 mL ~ about 800 mL, about 8 mL ~ about 750 mL, about 8 mL ~ about 700 mL, about 8 mL ~ about 650 mL, about 8 mL ~ about 600 mL, about 8 mL ~ about 550 mL, about 8 mL to about 500 mL, about 8 mL to about 450 mL, about 8 mL to about 400 mL, about 8 mL to about 350 mL, about 8 mL ~ About 300 mL, about 8 mL ~ about 250 mL, about 8 mL ~ about 200 mL, about 8 mL ~ about 150 mL, about 8 mL ~ about 100 mL, about 8 mL ~ about 90 mL, about 8 mL ~ about 80 mL, about 8 mL to about 70 mL, about 8 mL to about 60 mL, about 8 mL to about 50 mL, about 8 mL to about 45 mL, about 8 mL to about 40 mL, about 8 mL to about 35 mL , About 8 mL to about 30 mL, about 8 mL to about 25 mL, about 8 mL to about 20 mL, about 8 mL to about 15 mL, about 8 mL to about 10 mL, about 10 mL to about 1,000 mL, about 10 mL to about 950 mL, about 10 mL to about 900 mL, about 10 mL to about 850 mL, about 10 mL to about 800 mL, about 10 mL to about 750 mL, about 10 mL to about 700 mL, about 10 mL ~ About 650 mL, about 10 mL ~ about 600 mL, about 10 mL ~ about 550 mL, about 10 mL ~ about 500 mL, about 10 mL ~ about 450 mL, about 10 mL ~ about 400 mL, about 10 mL ~ about 350 mL, about 10 mL to about 300 mL, about 10 mL to about 250 mL, about 10 mL to about 200 mL, about 10 mL to about 150 mL, about 10 mL to about 100 mL, about 10 mL to about 90 mL , About 10 mL to about 80 mL, about 10 mL to about 70 mL, about 10 mL to about 60 mL, about 10 mL to about 50 mL, about 10 mL to about 45 mL, about 10 mL to about 40 mL, about 10 mL to about 35 mL, about 10 mL to about 30 mL, about 10 mL to about 25 mL, about 10 mL to about 20 mL, about 10 mL to about 15 mL, about 15 mL to about 1,000 mL, about 15 mL ~ About 950 mL, about 15 mL ~ about 900 mL, about 15 mL ~ about 850 mL, about 15 mL ~ about 800 mL, about 15 mL ~ about 750 mL, about 15 mL ~ about 700 mL, about 15 mL ~ about 650 mL, about 15 mL to about 600 mL, about 15 mL to about 550 mL, about 15 mL to about 500 mL, about 15 mL to about 450 mL, about 15 mL to about 400 mL, about 15 mL to about 350 mL , About 15 mL to about 300 mL, about 15 mL to about 250 mL, about 15 mL to about 200 mL, about 15 mL to about 150 mL, about 15 mL to about 100 mL, about 15 mL to about 90 mL, about 15 mL ~ about 80 mL, about 15 mL to about 70 mL, about 15 mL to about 60 mL, about 15 mL to about 50 mL, about 15 mL to about 45 mL, about 15 mL to about 40 mL, about 15 mL to about 35 mL, About 15 mL to about 30 mL, about 15 mL to about 25 mL, about 15 mL to about 20 mL, about 20 mL to about 1,000 mL, about 20 mL to about 950 mL, about 20 mL to about 900 mL, about 20 mL ~ about 850 mL, about 20 mL ~ about 800 mL, about 20 mL ~ about 750 mL, about 20 mL ~ about 700 mL, about 20 mL ~ about 650 mL, about 20 mL ~ about 600 mL, about 20 mL ~ About 550 mL, about 20 mL to about 500 mL, about 20 mL to about 450 mL, about 20 mL to about 400 mL, about 20 mL to about 350 mL, about 20 mL to about 300 mL, about 20 mL to about 250 mL, about 20 mL to about 200 mL, about 20 mL to about 150 mL, about 20 mL to about 100 mL, about 20 mL to about 90 mL, about 20 mL to about 80 mL, about 20 mL to about 70 mL, About 20 mL to about 60 mL, about 20 mL to about 50 mL, about 20 mL to about 45 mL, about 20 mL to about 40 mL, about 20 mL to about 35 mL, about 20 mL to about 30 mL, about 20 mL ~ about 25 mL, about 25 mL ~ about 1,000 mL, about 25 mL ~ about 950 mL, about 25 mL ~ about 900 mL, about 25 mL ~ about 850 mL, about 25 mL ~ about 800 mL, about 25 mL ~ About 750 mL, about 25 mL to about 700 mL, about 25 mL to about 650 mL, about 25 mL to about 600 mL, about 25 mL to about 550 mL, about 25 mL to about 500 mL, about 25 mL to about 450 mL, about 25 mL to about 400 mL, about 25 mL to about 350 mL, about 25 mL to about 300 mL, about 25 mL to about 250 mL, about 25 mL to about 200 mL, about 25 mL to about 150 mL, About 25 mL to about 100 mL, about 25 mL to about 90 mL, about 25 mL to about 80 mL, about 25 mL to about 70 mL, about 25 mL to about 60 mL, about 25 mL to about 50 mL, about 25 mL ~ about 45 mL, about 25 mL ~ about 40 mL, about 25 mL ~ about 35 mL, about 25 mL ~ about 30 mL, about 30 mL ~ about 1,000 mL, about 30 mL ~ about 950 m L, about 30 mL to about 900 mL, about 30 mL to about 850 mL, about 30 mL to about 800 mL, about 30 mL to about 750 mL, about 30 mL to about 700 mL, about 30 mL to about 650 mL, About 30 mL to about 600 mL, about 30 mL to about 550 mL, about 30 mL to about 500 mL, about 30 mL to about 450 mL, about 30 mL to about 400 mL, about 30 mL to about 350 mL, about 30 mL ~ about 300 mL, about 30 mL ~ about 250 mL, about 30 mL ~ about 200 mL, about 30 mL ~ about 150 mL, about 30 mL ~ about 100 mL, about 30 mL ~ about 90 mL, about 30 mL ~ About 80 mL, about 30 mL to about 70 mL, about 30 mL to about 60 mL, about 30 mL to about 50 mL, about 30 mL to about 45 mL, about 30 mL to about 40 mL, about 30 mL to about 35 mL, about 35 mL to about 1,000 mL, about 35 mL to about 950 mL, about 35 mL to about 900 mL, about 35 mL to about 850 mL, about 35 mL to about 800 mL, about 35 mL to about 750 mL, About 35 mL to about 700 mL, about 35 mL to about 650 mL, about 35 mL to about 600 mL, about 35 mL to about 550 mL, about 35 mL to about 500 mL, about 35 mL to about 450 mL, about 35 mL ~ about 400 mL, about 35 mL ~ about 350 mL, about 35 mL ~ about 300 mL, about 35 mL ~ about 250 mL, about 35 mL ~ about 200 mL, about 35 mL ~ about 150 mL, about 35 mL ~ About 100 mL, about 35 mL to about 90 mL, about 35 mL to about 80 mL, about 35 mL to about 70 mL, about 35 mL to about 60 mL, about 35 mL to about 50 mL, about 35 mL to about 45 mL, about 35 mL to about 40 mL, about 40 mL to about 1,000 mL, about 40 mL to about 950 mL, about 40 mL to about 900 mL, about 40 mL to about 850 mL, about 40 mL to about 800 mL, About 40 mL to about 750 mL, about 40 mL to about 700 mL, about 40 mL to about 650 mL, about 40 mL to about 600 mL, about 40 mL to about 550 mL, about 40 mL to about 500 mL, about 40 mL ~ about 450 mL, about 40 mL ~ about 400 mL, about 40 mL ~ about 350 mL, about 40 mL ~ about 300 mL, about 40 mL ~ about 250 mL
, About 40 mL to about 200 mL, about 40 mL to about 150 mL, about 40 mL to about 100 mL, about 40 mL to about 90 mL, about 40 mL to about 80 mL, about 40 mL to about 70 mL, about 40 mL to about 60 mL, about 40 mL to about 50 mL, about 40 mL to about 45 mL, about 45 mL to about 1,000 mL, about 45 mL to about 950 mL, about 45 mL to about 900 mL, about 45 mL ~ About 850 mL, about 45 mL ~ about 800 mL, about 45 mL ~ about 750 mL, about 45 mL ~ about 700 mL, about 45 mL ~ about 650 mL, about 45 mL ~ about 600 mL, about 45 mL ~ about 550 mL, about 45 mL to about 500 mL, about 45 mL to about 450 mL, about 45 mL to about 400 mL, about 45 mL to about 350 mL, about 45 mL to about 300 mL, about 45 mL to about 250 mL , About 45 mL to about 200 mL, about 45 mL to about 150 mL, about 45 mL to about 100 mL, about 45 mL to about 90 mL, about 45 mL to about 80 mL, about 45 mL to about 70 mL, about 45 mL to about 60 mL, about 45 mL to about 50 mL, about 50 mL to about 1,000 mL, about 50 mL to about 950 mL, about 50 mL to about 900 mL, about 50 mL to about 850 mL, about 50 mL ~ About 800 mL, about 50 mL ~ about 750 mL, about 50 mL ~ about 700 mL, about 50 mL ~ about 650 mL, about 50 mL ~ about 600 mL, about 50 mL ~ about 550 mL, about 50 mL ~ about 500 mL, about 50 mL to about 450 mL, about 50 mL to about 400 mL, about 50 mL to about 350 mL, about 50 mL to about 300 mL, about 50 mL to about 250 mL, about 50 mL to about 200 mL , About 50 mL to about 150 mL, about 50 mL to about 100 mL, about 50 mL to about 90 mL, about 50 mL to about 80 mL, about 50 mL to about 70 mL, about 50 mL to about 60 mL, about 60 mL to about 1,000 mL, about 60 mL to about 950 mL, about 60 mL to about 900 mL, about 60 mL to about 850 mL, about 60 mL to about 800 mL, about 60 mL to about 750 mL, about 60 mL ~ About 700 mL, about 60 mL ~ about 650 mL, about 60 mL ~ about 600 mL, about 60 mL ~ about 550 mL, about 60 mL ~ about 500 mL, about 60 mL ~ about 450 mL, about 60 mL ~ about 400 mL, about 60 mL ~ about 350 mL, about 60 mL ~ about 300 mL, about 60 mL ~ about 250 mL, about 60 mL ~ about 200 mL, about 60 mL ~ About 150 mL, about 60 mL to about 100 mL, about 60 mL to about 90 mL, about 60 mL to about 80 mL, about 60 mL to about 70 mL, about 70 mL to about 1,000 mL, about 70 mL to about 950 mL, about 70 mL to about 900 mL, about 70 mL to about 850 mL, about 70 mL to about 800 mL, about 70 mL to about 750 mL, about 70 mL to about 700 mL, about 70 mL to about 650 mL, About 70 mL to about 600 mL, about 70 mL to about 550 mL, about 70 mL to about 500 mL, about 70 mL to about 450 mL, about 70 mL to about 400 mL, about 70 mL to about 350 mL, about 70 mL ~ about 300 mL, about 70 mL ~ about 250 mL, about 70 mL ~ about 200 mL, about 70 mL ~ about 150 mL, about 70 mL ~ about 100 mL, about 70 mL ~ about 90 mL, about 70 mL ~ About 80 mL, about 80 mL to about 1,000 mL, about 80 mL to about 950 mL, about 80 mL to about 900 mL, about 80 mL to about 850 mL, about 80 mL to about 800 mL, about 80 mL to about 750 mL, about 80 mL to about 700 mL, about 80 mL to about 650 mL, about 80 mL to about 600 mL, about 80 mL to about 550 mL, about 80 mL to about 500 mL, about 80 mL to about 450 mL, About 80 mL to about 400 mL, about 80 mL to about 350 mL, about 80 mL to about 300 mL, about 80 mL to about 250 mL, about 80 mL to about 200 mL, about 80 mL to about 150 mL, about 80 mL ~ about 100 mL, about 80 mL ~ about 90 mL, about 90 mL ~ about 1,000 mL, about 90 mL ~ about 950 mL, about 90 mL ~ about 900 mL, about 90 mL ~ about 850 mL, about 90 mL ~ About 800 mL, about 90 mL to about 750 mL, about 90 mL to about 700 mL, about 90 mL to about 650 mL, about 90 mL to about 600 mL, about 90 mL to about 550 mL, about 90 mL to about 500 mL, about 90 mL to about 450 mL, about 90 mL to about 400 mL, about 90 mL to about 350 mL, about 90 mL to about 300 m L, about 90 mL to about 250 mL, about 90 mL to about 200 mL, about 90 mL to about 150 mL, about 90 mL to about 100 mL, about 100 mL to about 1,000 mL, about 100 mL to about 950 mL, About 100 mL to about 900 mL, about 100 mL to about 850 mL, about 100 mL to about 800 mL, about 100 mL to about 750 mL, about 100 mL to about 700 mL, about 100 mL to about 650 mL, about 100 mL ~ about 600 mL, about 100 mL ~ about 550 mL, about 100 mL ~ about 500 mL, about 100 mL ~ about 450 mL, about 100 mL ~ about 400 mL, about 100 mL ~ about 350 mL, about 100 mL ~ About 300 mL, about 100 mL to about 250 mL, about 100 mL to about 200 mL, about 100 mL to about 150 mL, about 150 mL to about 1,000 mL, about 150 mL to about 950 mL, about 150 mL to about 900 mL, about 150 mL to about 850 mL, about 150 mL to about 800 mL, about 150 mL to about 750 mL, about 150 mL to about 700 mL, about 150 mL to about 650 mL, about 150 mL to about 600 mL, About 150 mL to about 550 mL, about 150 mL to about 500 mL, about 150 mL to about 450 mL, about 150 mL to about 400 mL, about 150 mL to about 350 mL, about 150 mL to about 300 mL, about 150 mL-about 250 mL, about 150 mL-about 200 mL, about 200 mL-about 1,000 mL, about 200 mL-about 950 mL, about 200 mL-about 900 mL, about 200 mL-about 850 mL, about 200 mL- About 800 mL, about 200 mL to about 750 mL, about 200 mL to about 700 mL, about 200 mL to about 650 mL, about 200 mL to about 600 mL, about 200 mL to about 550 mL, about 200 mL to about 500 mL, about 200 mL to about 450 mL, about 200 mL to about 400 mL, about 200 mL to about 350 mL, about 200 mL to about 300 mL, about 200 mL to about 250 mL, about 250 mL to about 1,000 mL, About 250 mL to about 950 mL, about 250 mL to about 900 mL, about 250 mL to about 850 mL, about 250 mL to about 800 mL, about 250 mL to about 750 mL, about 250 mL to about 700 mL, about 2 50 mL to about 650 mL, about 250 mL to about 600 mL, about 250 mL to about 550 mL, about 250 mL to about 500 mL, about 250 mL to about 450 mL, about 250 mL to about 400 mL, about 250 mL ~ About 350 mL, about 250 mL ~ about 300 mL, about 300 mL ~ about 1,000 mL, about 300 mL ~ about 950 mL, about 300 mL ~ about 900 mL, about 300 mL ~ about 850 mL, about 300 mL ~ about 800 mL, about 300 mL to about 750 mL, about 300 mL to about 700 mL, about 300 mL to about 650 mL, about 300 mL to about 600 mL, about 300 mL to about 550 mL, about 300 mL to about 500 mL , About 300 mL to about 450 mL, about 300 mL to about 400 mL, about 300 mL to about 350 mL, about 350 mL to about 1,000 mL, about 350 mL to about 950 mL, about 350 mL to about 900 mL, about 350 mL to about 850 mL, about 350 mL to about 800 mL, about 350 mL to about 750 mL, about 350 mL to about 700 mL, about 350 mL to about 650 mL, about 350 mL to about 600 mL, about 350 mL ~ About 550 mL, about 350 mL ~ about 500 mL, about 350 mL ~ about 450 mL, about 350 mL ~ about 400 mL, about 400 mL ~ about 1,000 mL, about 400 mL ~ about 950 mL, about 400 mL ~ about 900 mL, about 400 mL to about 850 mL, about 400 mL to about 800 mL, about 400 mL to about 750 mL, about 400 mL to about 700 mL, about 400 mL to about 650 mL, about 400 mL to about 600 mL , About 400 mL to about 550 mL, about 400 mL to about 500 mL, about 400 mL to about 450 mL, about 450 mL to about 1,000 mL, about 450 mL to about 950 mL, about 450 mL to about 900 mL, about 450 mL to about 850 mL, about 450 mL to about 800 mL, about 450 mL to about 750 mL, about 450 mL to about 700 mL, about 450 mL to about 650 mL, about 450 mL to about 600 mL, about 450 mL ~ About 550 mL, about 450 mL ~ about 500 mL, about 500 mL ~ about 1,000 mL, about 500 mL ~ about 950 mL, about 500 mL ~ about 900 mL, about 500 mL ~ about 850 mL, About 500 mL to about 800 mL, about 500 mL to about 750 mL, about 500 mL to about 700 mL, about 500 mL to about 650 mL, about 500 mL to about 600 mL, about 500 mL to about 550 mL, about 550 mL ~ about 1,000 mL, about 550 mL ~ about 950 mL, about 550 mL ~ about 900 mL, about 550 mL ~ about 850 mL, about 550 mL ~ about 800 mL, about 550 mL ~ about 750 mL, about 550 mL ~ About 700 mL, about 550 mL to about 650 mL, about 550 mL to about 600 mL, about 600 mL to about 1,000 mL, about 600 mL to about 950 mL, about 600 mL to about 900 mL, about 600 mL to about 850 mL, about 600 mL to about 800 mL, about 600 mL to about 750 mL, about 600 mL to about 700 mL, about 600 mL to about 650 mL, about 650 mL to about 1,000 mL, about 650 mL to about 950 mL, About 650 mL to about 900 mL, about 650 mL to about 850 mL, about 650 mL to about 800 mL, about 650 mL to about 750 mL, about 650 mL to about 700 mL, about 700 mL to about 1,000 mL, about 700 mL ~ about 950 mL, about 700 mL ~ about 900 mL, about 700 mL ~ about 850 mL, about 700 mL ~ about 800 mL, about 700 mL ~ about 750 mL, about 750 mL ~ about 1,000 mL, about 750 mL ~ About 950 mL, about 750 mL to about 900 mL, about 750 mL to about 850 mL, about 750 mL to about 800 mL, about 800 mL to about 1,000 mL, about 800 mL to about 950 mL, about 800 mL to about 900 mL, about 800 mL to about 850 mL, about 850 mL to about 1,000 mL, about 850 mL to about 950 mL, about 850 mL to about 900 mL, about 900 mL to about 1,000 mL, about 900 mL to about 950 mL, Or it has an internal volume of about 950 mL to about 1,000 mL.

Walls Walls are polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), acrylic polymers, polyesters, silicone rubber, silicon, polyurethane, halogenated plastics (eg chlorinated polyethylene, chlorinated poly) Vinyl chloride, chlorosulfonated polyethylene, polychloroprene, ethylene fluorinated polypropylene), biaxially oriented polypropylene (BOPP), bioplastics, cellophane, ethane-derived plastics, nylon, polylactic acid (PLA), polyethylene terephthalate (PE or PETE plastic cord) 1), polypropylene, polystyrene, polycarbonate, polyeurothene, or poly (methylmethacrylate) may be constructed or partially constructed. Further exemplary compositions in which walls may be constructed include Bakelite (phenoloformaldehyde resin), Kevlar or Twaron (paraaramid), Kynar (polyfluorovinylidene), Mylar (polyethylene terephthalate film), Neoprene (polychloroprene), Nylon (polyamide 6,6), Orlon (polyacrylonitrile), Rilsan (polyamide 11 and 12), Technora (copolyamide), Teflon (polytetrafluoroethylene), Ultem (polyamide), Vectran (aromatic polyamide), Viton ( Includes polytetrafluoroethylene), or Zylon (poly-p-phenylene-2,6-benzobisoxazole (PBO). In some aspects of the pod, the walls are nylon, polypropylene, or polyvinyl alcohol (PVA). It may be constructed with or partially constructed with.

In some aspects of the pod, the wall has a cylindrical, conical, rectangular, square, spherical, hemispherical, hourglass, or oval shape.

Chromatographic Resin In some embodiments of any of the methods described herein, the pod further comprises a chromatographic resin placed inside the container. In some examples, the chromatographic resin is a commercially available chromatographic resin. In other examples, the chromatographic resin is not a commercially available chromatographic resin.

The chromatography resin may be any chromatography resin known in the art. For example, the chromatography resin is an affinity chromatography resin (for example, protein A resin, protein G resin, protein L resin, nickel resin, or magnetic bead resin), hydrophobic interaction chromatography resin, mixed mode chromatography resin, molecular sieving chromatography resin. You may. In some embodiments, the resin is a specific affinity resin that is specific for the recombinant protein of interest, such as a resin conjugated to an agent (eg, antibody, antibody fragment, peptide, and nucleic acid, etc.). is there. In some embodiments, the chromatographic resin is an alkali resistant resin. In some embodiments, the chromatographic resin is a sterile chromatographic resin.

The chromatography resin placed inside the container is about 0.1 mL to about 500 mL, about 0.1 mL to about 490 mL, about 0.1 mL to about 480 mL, about 0.1 mL to about 460 mL, about 0.1 mL to about 440 mL, About 0.1 mL to about 420 mL, about 0.1 mL to about 400 mL, about 0.1 mL to about 380 mL, about 0.1 mL to about 360 mL, about 0.1 mL to about 340 mL, about 0.1 mL to about 320 mL, about 0.1 mL ~ about 300 mL, about 0.1 mL ~ about 280 mL, about 0.1 mL ~ about 260 mL, about 0.1 mL ~ about 250 mL, about 0.1 mL ~ about 240 mL, about 0.1 mL ~ about 220 mL, about 0.1 mL ~ About 210 mL, about 0.1 mL to about 200 mL, about 0.1 mL to about 190 mL, about 0.1 mL to about 180 mL, about 0.1 mL to about 170 mL, about 0.1 mL to about 160 mL, about 0.1 mL to about 150 mL, about 0.1 mL to about 140 mL, about 0.1 mL to about 130 mL, about 0.1 mL to about 120 mL, about 0.1 mL to about 110 mL, about 0.1 mL to about 100 mL, about 0.1 mL to about 90 mL, About 0.1 mL to about 80 mL, about 0.1 mL to about 70 mL, about 0.1 mL to about 60 mL, about 0.1 mL to about 50 mL, about 0.1 mL to about 40 mL, about 0.1 mL to about 30 mL, about 0.1 mL ~ about 25 mL, about 0.1 mL ~ about 20 mL, about 0.1 mL ~ about 15 mL, about 0.1 mL ~ about 10 mL, about 0.1 mL ~ about 5 mL, about 0.1 mL ~ about 2 mL, about 0.1 mL ~ About 1 mL, about 0.2 mL to about 500 mL, about 0.2 mL to about 490 mL, about 0.2 mL to about 480 mL, about 0.2 mL to about 460 mL, about 0.2 mL to about 440 mL, about 0.2 mL to about 420 mL, about 0.2 mL to about 400 mL, about 0.2 mL to about 380 mL, about 0.2 mL to about 360 mL, about 0.2 mL to about 340 mL, about 0.2 mL to about 320 mL, about 0.2 mL to about 300 mL, About 0.2 mL to about 280 mL, about 0.2 mL to about 260 mL, about 0.2 mL to about 250 mL, about 0.2 mL to about 240 mL, about 0.2 mL to about 220 mL, about 0.2 mL to about 210 mL, about 0.2 mL ~ about 200 mL, about 0 .2 mL to about 190 mL, about 0.2 mL to about 180 mL, about 0.2 mL to about 170 mL, about 0.2 mL to about 160 mL, about 0.2 mL to about 150 mL, about 0.2 mL to about 140 mL, about 0.2 mL ~ about 130 mL, about 0.2 mL ~ about 120 mL, about 0.2 mL ~ about 110 mL, about 0.2 mL ~ about 100 mL, about 0.2 mL ~ about 90 mL, about 0.2 mL ~ about 80 mL, about 0.2 mL ~ About 70 mL, about 0.2 mL to about 60 mL, about 0.2 mL to about 50 mL, about 0.2 mL to about 40 mL, about 0.2 mL to about 30 mL, about 0.2 mL to about 25 mL, about 0.2 mL to about 20 mL, about 0.2 mL to about 15 mL, about 0.2 mL to about 10 mL, about 0.2 mL to about 5 mL, about 0.2 mL to about 2 mL, about 0.2 mL to about 1 mL, about 0.4 mL to about 500 mL, About 0.4 mL to about 490 mL, about 0.4 mL to about 480 mL, about 0.4 mL to about 460 mL, about 0.4 mL to about 440 mL, about 0.4 mL to about 420 mL, about 0.4 mL to about 400 mL, about 0.4 mL ~ about 380 mL, about 0.4 mL ~ about 360 mL, about 0.4 mL ~ about 340 mL, about 0.4 mL ~ about 320 mL, about 0.4 mL ~ about 300 mL, about 0.4 mL ~ about 280 mL, about 0.4 mL ~ About 260 mL, about 0.4 mL to about 250 mL, about 0.4 mL to about 240 mL, about 0.4 mL to about 220 mL, about 0.4 mL to about 210 mL, about 0.4 mL to about 200 mL, about 0.4 mL to about 190 mL, about 0.4 mL to about 180 mL, about 0.4 mL to about 170 mL, about 0.4 mL to about 160 mL, about 0.4 mL to about 150 mL, about 0.4 mL to about 140 mL, about 0.4 mL to about 130 mL, About 0.4 mL to about 120 mL, about 0.4 mL to about 110 mL, about 0.4 mL to about 100 mL, about 0.4 mL to about 90 mL, about 0.4 mL to about 80 mL, about 0.4 mL to about 70 mL, about 0.4 mL ~ about 60 mL, about 0.4 mL ~ about 50 mL, about 0.4 mL ~ about 40 mL, about 0.4 mL ~ about 30 mL, about 0.4 mL ~ about 25 mL, about 0.4 mL ~ about 20 mL, about 0.4 mL ~ About 15 mL, about 0.4 mL to about 10 mL, about 0.4 m L ~ about 5 mL, about 0.4 mL ~ about 2 mL, about 0.4 mL ~ about 1 mL, about 0.5 mL ~ about 500 mL, about 0.5 mL ~ about 490 mL, about 0.5 mL ~ about 480 mL, about 0.5 mL ~ About 460 mL, about 0.5 mL to about 440 mL, about 0.5 mL to about 420 mL, about 0.5 mL to about 400 mL, about 0.5 mL to about 380 mL, about 0.5 mL to about 360 mL, about 0.5 mL to about 340 mL, about 0.5 mL to about 320 mL, about 0.5 mL to about 300 mL, about 0.5 mL to about 280 mL, about 0.5 mL to about 260 mL, about 0.5 mL to about 250 mL, about 0.5 mL to about 240 mL, About 0.5 mL to about 220 mL, about 0.5 mL to about 210 mL, about 0.5 mL to about 200 mL, about 0.5 mL to about 190 mL, about 0.5 mL to about 180 mL, about 0.5 mL to about 170 mL, about 0.5 mL ~ about 160 mL, about 0.5 mL ~ about 150 mL, about 0.5 mL ~ about 140 mL, about 0.5 mL ~ about 130 mL, about 0.5 mL ~ about 120 mL, about 0.5 mL ~ about 110 mL, about 0.5 mL ~ About 100 mL, about 0.5 mL to about 90 mL, about 0.5 mL to about 80 mL, about 0.5 mL to about 70 mL, about 0.5 mL to about 60 mL, about 0.5 mL to about 50 mL, about 0.5 mL to about 40 mL, about 0.5 mL to about 30 mL, about 0.5 mL to about 25 mL, about 0.5 mL to about 20 mL, about 0.5 mL to about 15 mL, about 0.5 mL to about 10 mL, about 0.5 mL to about 5 mL, About 0.5 mL to about 2 mL, about 0.5 mL to about 1 mL, about 0.6 mL to about 500 mL, about 0.6 mL to about 490 mL, about 0.6 mL to about 480 mL, about 0.6 mL to about 460 mL, about 0.6 mL ~ about 440 mL, about 0.6 mL ~ about 420 mL, about 0.6 mL ~ about 400 mL, about 0.6 mL ~ about 380 mL, about 0.6 mL ~ about 360 mL, about 0.6 mL ~ about 340 mL, about 0.6 mL ~ About 320 mL, about 0.6 mL to about 300 mL, about 0.6 mL to about 280 mL, about 0.6 mL to about 260 mL, about 0.6 mL to about 250 mL, about 0.6 mL to about 240 mL, about 0.6 mL to about 220 mL, about 0.6 mL to about 210 mL, about 0.6 mL ~ about 200 mL, about 0.6 mL ~ about 190 mL, about 0.6 mL ~ about 180 mL, about 0.6 mL ~ about 170 mL, about 0.6 mL ~ about 160 mL, about 0.6 mL ~ about 150 mL, about 0.6 mL ~ About 140 mL, about 0.6 mL to about 130 mL, about 0.6 mL to about 120 mL, about 0.6 mL to about 110 mL, about 0.6 mL to about 100 mL, about 0.6 mL to about 90 mL, about 0.6 mL to about 80 mL, about 0.6 mL to about 70 mL, about 0.6 mL to about 60 mL, about 0.6 mL to about 50 mL, about 0.6 mL to about 40 mL, about 0.6 mL to about 30 mL, about 0.6 mL to about 25 mL, About 0.6 mL to about 20 mL, about 0.6 mL to about 15 mL, about 0.6 mL to about 10 mL, about 0.6 mL to about 5 mL, about 0.6 mL to about 2 mL, about 0.6 mL to about 1 mL, about 0.8 mL ~ about 500 mL, about 0.8 mL ~ about 490 mL, about 0.8 mL ~ about 480 mL, about 0.8 mL ~ about 460 mL, about 0.8 mL ~ about 440 mL, about 0.8 mL ~ about 420 mL, about 0.8 mL ~ About 400 mL, about 0.8 mL to about 380 mL, about 0.8 mL to about 360 mL, about 0.8 mL to about 340 mL, about 0.8 mL to about 320 mL, about 0.8 mL to about 300 mL, about 0.8 mL to about 280 mL, about 0.8 mL to about 260 mL, about 0.8 mL to about 250 mL, about 0.8 mL to about 240 mL, about 0.8 mL to about 220 mL, about 0.8 mL to about 210 mL, about 0.8 mL to about 200 mL, About 0.8 mL to about 190 mL, about 0.8 mL to about 180 mL, about 0.8 mL to about 170 mL, about 0.8 mL to about 160 mL, about 0.8 mL to about 150 mL, about 0.8 mL to about 140 mL, about 0.8 mL ~ about 130 mL, about 0.8 mL ~ about 120 mL, about 0.8 mL ~ about 110 mL, about 0.8 mL ~ about 100 mL, about 0.8 mL ~ about 90 mL, about 0.8 mL ~ about 80 mL, about 0.8 mL ~ About 70 mL, about 0.8 mL to about 60 mL, about 0.8 mL to about 50 mL, about 0.8 mL to about 40 mL, about 0.8 mL to about 30 mL, about 0.8 mL to about 25 mL, about 0.8 mL to about 20 mL, about 0.8 mL ~ about 15 mL, about 0.8 mL ~ About 10 mL, about 0.8 mL to about 5 mL, about 0.8 mL to about 2 mL, about 0.8 mL to about 1 mL, about 1 mL to about 500 mL, about 1 mL to about 490 mL, about 1 mL to about 480 mL, about 1 mL to about 460 mL, about 1 mL to about 440 mL, about 1 mL to about 420 mL, about 1 mL to about 400 mL, about 1 mL to about 380 mL, about 1 mL to about 360 mL, About 1 mL to about 340 mL, about 1 mL to about 320 mL, about 1 mL to about 300 mL, about 1 mL to about 280 mL, about 1 mL to about 260 mL, about 1 mL to about 250 mL, about 1 mL ~ about 240 mL, about 1 mL ~ about 220 mL, about 1 mL ~ about 210 mL, about 1 mL ~ about 200 mL, about 1 mL ~ about 190 mL, about 1 mL ~ about 180 mL, about 1 mL ~ About 170 mL, about 1 mL to about 160 mL, about 1 mL to about 150 mL, about 1 mL to about 140 mL, about 1 mL to about 130 mL, about 1 mL to about 120 mL, about 1 mL to about 110 mL, about 1 mL to about 100 mL, about 1 mL to about 90 mL, about 1 mL to about 80 mL, about 1 mL to about 70 mL, about 1 mL to about 60 mL, about 1 mL to about 50 mL, About 1 mL to about 40 mL, about 1 mL to about 30 mL, about 1 mL to about 25 mL, about 1 mL to about 20 mL, about 1 mL to about 15 mL, about 1 mL to about 10 mL, about 1 mL ~ about 5 mL, about 1 mL ~ about 2 mL, about 2 mL ~ about 500 mL, about 2 mL ~ about 490 mL, about 2 mL ~ about 480 mL, about 2 mL ~ about 460 mL, about 2 mL ~ About 440 mL, about 2 mL to about 420 mL, about 2 mL to about 400 mL, about 2 mL to about 380 mL, about 2 mL to about 360 mL, about 2 mL to about 340 mL, about 2 mL to about 320 mL, about 2 mL to about 300 mL, about 2 mL to about 280 mL, about 2 mL to about 260 mL, about 2 mL to about 250 mL, about 2 mL to about 240 mL, about 2 mL to about 220 mL, About 2 mL to about 210 mL, about 2 mL to about 200 mL, about 2 mL to about 190 mL, about 2 mL to about 180 mL, about 2 mL to about 170 mL, about 2 mL to about 160 mL, about 2 mL ~ about 150 mL, about 2 mL ~ about 140 mL, about 2 mL ~ about 130 mL, about 2 mL ~ about 120 mL, About 2 mL to about 110 mL, about 2 mL to about 100 mL, about 2 mL to about 90 mL, about 2 mL to about 80 mL, about 2 mL to about 70 mL, about 2 mL to about 60 mL, about 2 mL ~ about 50 mL, about 2 mL ~ about 40 mL, about 2 mL ~ about 30 mL, about 2 mL ~ about 25 mL, about 2 mL ~ about 20 mL, about 2 mL ~ about 15 mL, about 2 mL ~ About 10 mL, about 2 mL to about 5 mL, about 5 mL to about 500 mL, about 5 mL to about 490 mL, about 5 mL to about 480 mL, about 5 mL to about 460 mL, about 5 mL to about 440 mL, about 5 mL to about 420 mL, about 5 mL to about 400 mL, about 5 mL to about 380 mL, about 5 mL to about 360 mL, about 5 mL to about 340 mL, about 5 mL to about 320 mL, About 5 mL to about 300 mL, about 5 mL to about 280 mL, about 5 mL to about 260 mL, about 5 mL to about 250 mL, about 5 mL to about 240 mL, about 5 mL to about 220 mL, about 5 mL ~ about 210 mL, about 5 mL ~ about 200 mL, about 5 mL ~ about 190 mL, about 5 mL ~ about 180 mL, about 5 mL ~ about 170 mL, about 5 mL ~ about 160 mL, about 5 mL ~ About 150 mL, about 5 mL to about 140 mL, about 5 mL to about 130 mL, about 5 mL to about 120 mL, about 5 mL to about 110 mL, about 5 mL to about 100 mL, about 5 mL to about 90 mL, about 5 mL to about 80 mL, about 5 mL to about 70 mL, about 5 mL to about 60 mL, about 5 mL to about 50 mL, about 5 mL to about 40 mL, about 5 mL to about 30 mL, About 5 mL to about 25 mL, about 5 mL to about 20 mL, about 5 mL to about 15 mL, about 5 mL to about 10 mL, about 10 mL to about 500 mL, about 10 mL to about 490 mL, about 10 mL ~ about 480 mL, about 10 mL ~ about 460 mL, about 10 mL ~ about 440 mL, about 10 mL ~ about 420 mL, about 10 mL ~ about 400 mL, about 10 mL ~ about 380 mL, about 10 mL ~ About 360 mL, about 10 mL to about 340 mL, about 10 mL to about 320 mL, about 10 mL to about 300 mL, about 10 mL to about 280 mL, about 10 mL to about 260 mL, about 10 mL to about 250 mL, about 10 mL to about 240 mL, about 10 mL to about 220 mL, about 10 mL to about 210 m L, about 10 mL to about 200 mL, about 10 mL to about 190 mL, about 10 mL to about 180 mL, about 10 mL to about 170 mL, about 10 mL to about 160 mL, about 10 mL to about 150 mL, About 10 mL to about 140 mL, about 10 mL to about 130 mL, about 10 mL to about 120 mL, about 10 mL to about 110 mL, about 10 mL to about 100 mL, about 10 mL to about 90 mL, about 10 mL ~ about 80 mL, about 10 mL ~ about 70 mL, about 10 mL ~ about 60 mL, about 10 mL ~ about 50 mL, about 10 mL ~ about 40 mL, about 10 mL ~ about 30 mL, about 10 mL ~ About 25 mL, about 10 mL to about 20 mL, about 10 mL to about 15 mL, about 15 mL to about 500 mL, about 15 mL to about 490 mL, about 15 mL to about 480 mL, about 15 mL to about 460 mL, about 15 mL to about 440 mL, about 15 mL to about 420 mL, about 15 mL to about 400 mL, about 15 mL to about 380 mL, about 15 mL to about 360 mL, about 15 mL to about 340 mL, About 15 mL to about 320 mL, about 15 mL to about 300 mL, about 15 mL to about 280 mL, about 15 mL to about 260 mL, about 15 mL to about 250 mL, about 15 mL to about 240 mL, about 15 mL ~ 220 mL, about 15 mL ~ 210 mL, about 15 mL ~ about 200 mL, about 15 mL ~ about 190 mL, about 15 mL ~ about 180 mL, about 15 mL ~ about 170 mL, about 15 mL ~ About 160 mL, about 15 mL to about 150 mL, about 15 mL to about 140 mL, about 15 mL to about 130 mL, about 15 mL to about 120 mL, about 15 mL to about 110 mL, about 15 mL to about 100 mL, about 15 mL to about 90 mL, about 15 mL to about 80 mL, about 15 mL to about 70 mL, about 15 mL to about 60 mL, about 15 mL to about 50 mL, about 15 mL to about 40 mL, About 15 mL to about 30 mL, about 15 mL to about 25 mL, about 15 mL to about 20 mL, about 20 mL to about 500 mL, about 20 mL to about 490 mL, about 20 mL to about 480 mL, about 20 mL ~ about 460 mL, about 20 mL ~ about 440 mL, about 20 mL ~ about 420 mL, about 20 mL ~ about 400 mL, about 20 mL ~ about 380 mL, about 20 mL ~ about 360 mL, about 20 mL to about 340 mL, about 20 mL to about 320 mL, about 20 mL to about 300 mL, about 20 mL to about 280 mL, about 20 mL to about 260 mL, about 20 mL to about 250 mL, About 20 mL to about 240 mL, about 20 mL to about 220 mL, about 20 mL to about 210 mL, about 20 mL to about 200 mL, about 20 mL to about 190 mL, about 20 mL to about 180 mL, about 20 mL ~ about 170 mL, about 20 mL ~ about 160 mL, about 20 mL ~ about 150 mL, about 20 mL ~ about 140 mL, about 20 mL ~ about 130 mL, about 20 mL ~ about 120 mL, about 20 mL ~ About 110 mL, about 20 mL to about 100 mL, about 20 mL to about 90 mL, about 20 mL to about 80 mL, about 20 mL to about 70 mL, about 20 mL to about 60 mL, about 20 mL to about 50 mL, about 20 mL to about 40 mL, about 20 mL to about 30 mL, about 20 mL to about 25 mL, about 25 mL to about 500 mL, about 25 mL to about 490 mL, about 25 mL to about 480 mL, About 25 mL to about 460 mL, about 25 mL to about 440 mL, about 25 mL to about 420 mL, about 25 mL to about 400 mL, about 25 mL to about 380 mL, about 25 mL to about 360 mL, about 25 mL ~ about 340 mL, about 25 mL ~ about 320 mL, about 25 mL ~ about 300 mL, about 25 mL ~ about 280 mL, about 25 mL ~ about 260 mL, about 25 mL ~ about 250 mL, about 25 mL ~ About 240 mL, about 25 mL to about 220 mL, about 25 mL to about 210 mL, about 25 mL to about 200 mL, about 25 mL to about 190 mL, about 25 mL to about 180 mL, about 25 mL to about 170 mL, about 25 mL to about 160 mL, about 25 mL to about 150 mL, about 25 mL to about 140 mL, about 25 mL to about 130 mL, about 25 mL to about 120 mL, about 25 mL to about 110 mL, About 25 mL to about 100 mL, about 25 mL to about 90 mL, about 25 mL to about 80 mL, about 25 mL to about 70 mL, about 25 mL to about 60 mL, about 25 mL to about 50 mL, about 25 mL ~ about 40 mL, about 25 mL ~ about 30 mL, about 30 mL ~ about 500 mL, about 30 mL ~ about 490 mL, about 30 mL ~ about 480 mL, about 30 mL ~ About 460 mL, about 30 mL to about 440 mL, about 30 mL to about 420 mL, about 30 mL to about 400 mL, about 30 mL to about
380 mL, about 30 mL to about 360 mL, about 30 mL to about 340 mL, about 30 mL to about 320 mL, about 30 mL to about 300 mL, about 30 mL to about 280 mL, about 30 mL to about 260 mL , About 30 mL to about 250 mL, about 30 mL to about 240 mL, about 30 mL to about 220 mL, about 30 mL to about 210 mL, about 30 mL to about 200 mL, about 30 mL to about 190 mL, about 30 mL to about 180 mL, about 30 mL to about 170 mL, about 30 mL to about 160 mL, about 30 mL to about 150 mL, about 30 mL to about 140 mL, about 30 mL to about 130 mL, about 30 mL ~ About 120 mL, about 30 mL ~ about 110 mL, about 30 mL ~ about 100 mL, about 30 mL ~ about 90 mL, about 30 mL ~ about 80 mL, about 30 mL ~ about 70 mL, about 30 mL ~ about 60 mL, about 30 mL to about 50 mL, about 30 mL to about 40 mL, about 40 mL to about 500 mL, about 40 mL to about 490 mL, about 40 mL to about 480 mL, about 40 mL to about 460 mL , About 40 mL to about 440 mL, about 40 mL to about 420 mL, about 40 mL to about 400 mL, about 40 mL to about 380 mL, about 40 mL to about 360 mL, about 40 mL to about 340 mL, about 40 mL to about 320 mL, about 40 mL to about 300 mL, about 40 mL to about 280 mL, about 40 mL to about 260 mL, about 40 mL to about 250 mL, about 40 mL to about 240 mL, about 40 mL ~ About 220 mL, about 40 mL ~ about 210 mL, about 40 mL ~ about 200 mL, about 40 mL ~ about 190 mL, about 40 mL ~ about 180 mL, about 40 mL ~ about 170 mL, about 40 mL ~ about 160 mL, about 40 mL to about 150 mL, about 40 mL to about 140 mL, about 40 mL to about 130 mL, about 40 mL to about 120 mL, about 40 mL to about 110 mL, about 40 mL to about 100 mL , About 40 mL to about 90 mL, about 40 mL to about 80 mL, about 40 mL to about 70 mL, about 40 mL to about 60 mL, about 40 mL to about 50 mL, about 50 mL to about 500 mL, about 50 mL to about 490 mL, about 50 mL to about 480 mL, about 50 mL to about 460 mL, about 50 mL to about 440 mL, about 50 mL to about 420 mL , About 50 mL to about 400 mL, about 50 mL to about 380 mL, about 50 mL to about 360 mL, about 50 mL to about 340 mL, about 50 mL to about 320 mL, about 50 mL to about 300 mL, about 50 mL to about 280 mL, about 50 mL to about 260 mL, about 50 mL to about 250 mL, about 50 mL to about 240 mL, about 50 mL to about 220 mL, about 50 mL to about 210 mL, about 50 mL ~ About 200 mL, about 50 mL ~ about 190 mL, about 50 mL ~ about 180 mL, about 50 mL ~ about 170 mL, about 50 mL ~ about 160 mL, about 50 mL ~ about 150 mL, about 50 mL ~ about 140 mL, about 50 mL to about 130 mL, about 50 mL to about 120 mL, about 50 mL to about 110 mL, about 50 mL to about 100 mL, about 50 mL to about 90 mL, about 50 mL to about 80 mL , About 50 mL to about 70 mL, about 50 mL to about 60 mL, about 60 mL to about 500 mL, about 60 mL to about 490 mL, about 60 mL to about 480 mL, about 60 mL to about 460 mL, about 60 mL to about 440 mL, about 60 mL to about 420 mL, about 60 mL to about 400 mL, about 60 mL to about 380 mL, about 60 mL to about 360 mL, about 60 mL to about 340 mL, about 60 mL ~ About 320 mL, about 60 mL ~ about 300 mL, about 60 mL ~ about 280 mL, about 60 mL ~ about 260 mL, about 60 mL ~ about 250 mL, about 60 mL ~ about 240 mL, about 60 mL ~ about 220 mL, about 60 mL to about 210 mL, about 60 mL to about 200 mL, about 60 mL to about 190 mL, about 60 mL to about 180 mL, about 60 mL to about 170 mL, about 60 mL to about 160 mL , About 60 mL to about 150 mL, about 60 mL to about 140 mL, about 60 mL to about 130 mL, about 60 mL to about 120 mL, about 60 mL to about 110 mL, about 60 mL to about 100 mL, about 60 mL to about 90 mL, about 60 mL to about 80 mL, about 60 mL to about 70 mL, about 70 mL to about 500 mL, about 70 mL to about 490 mL, about 70 mL to about 480 mL, about 70 mL ~ About 460 mL, about 70 mL ~ about 440 mL, about 70 mL ~ about 420 mL, about 70 mL ~ about 400 mL, about 70 mL ~ about 380 mL, about 70 mL to about 360 mL, about 70 mL to about 340 mL, about 70 mL to about 320 mL, about 70 mL to about 300 mL, about 70 mL to about 280 mL, about 70 mL to about 260 mL, about 70 mL ~ About 250 mL, about 70 mL ~ about 240 mL, about 70 mL ~ about 220 mL, about 70 mL ~ about 210 mL, about 70 mL ~ about 200 mL, about 70 mL ~ about 190 mL, about 70 mL ~ about 180 mL, about 70 mL to about 170 mL, about 70 mL to about 160 mL, about 70 mL to about 150 mL, about 70 mL to about 140 mL, about 70 mL to about 130 mL, about 70 mL to about 120 mL , About 70 mL to about 110 mL, about 70 mL to about 100 mL, about 70 mL to about 90 mL, about 70 mL to about 80 mL, about 80 mL to about 500 mL, about 80 mL to about 490 mL, about 80 mL to about 480 mL, about 80 mL to about 460 mL, about 80 mL to about 440 mL, about 80 mL to about 420 mL, about 80 mL to about 400 mL, about 80 mL to about 380 mL, about 80 mL ~ About 360 mL, about 80 mL ~ about 340 mL, about 80 mL ~ about 320 mL, about 80 mL ~ about 300 mL, about 80 mL ~ about 280 mL, about 80 mL ~ about 260 mL, about 80 mL ~ about 250 mL, about 80 mL to about 240 mL, about 80 mL to about 220 mL, about 80 mL to about 210 mL, about 80 mL to about 200 mL, about 80 mL to about 190 mL, about 80 mL to about 180 mL , About 80 mL to about 170 mL, about 80 mL to about 160 mL, about 80 mL to about 150 mL, about 80 mL to about 140 mL, about 80 mL to about 130 mL, about 80 mL to about 120 mL, about 80 mL to about 110 mL, about 80 mL to about 100 mL, about 80 mL to about 90 mL, about 90 mL to about 500 mL, about 90 mL to about 490 mL, about 90 mL to about 480 mL, about 90 mL ~ About 460 mL, about 90 mL ~ about 440 mL, about 90 mL ~ about 420 mL, about 90 mL ~ about 400 mL, about 90 mL ~ about 380 mL, about 90 mL ~ about 360 mL, about 90 mL ~ about 340 mL, about 90 mL to about 320 mL, about 90 mL to about 300 mL, about 90 mL to about 280 mL, about 90 mL to about 260 mL , About 90 mL to about 250 mL, about 90 mL to about 240 mL, about 90 mL to about 220 mL, about 90 mL to about 210 mL, about 90 mL to about 200 mL, about 90 mL to about 190 mL, about 90 mL to about 180 mL, about 90 mL to about 170 mL, about 90 mL to about 160 mL, about 90 mL to about 150 mL, about 90 mL to about 140 mL, about 90 mL to about 130 mL, about 90 mL ~ About 120 mL, about 90 mL ~ about 110 mL, about 90 mL ~ about 100 mL, about 100 mL ~ about 500 mL, about 100 mL ~ about 490 mL, about 100 mL ~ about 480 mL, about 100 mL ~ about 460 mL, about 100 mL to about 440 mL, about 100 mL to about 420 mL, about 100 mL to about 400 mL, about 100 mL to about 380 mL, about 100 mL to about 360 mL, about 100 mL to about 340 mL , About 100 mL to about 320 mL, about 100 mL to about 300 mL, about 100 mL to about 280 mL, about 100 mL to about 260 mL, about 100 mL to about 250 mL, about 100 mL to about 240 mL, about 100 mL to about 220 mL, about 100 mL to about 210 mL, about 100 mL to about 200 mL, about 100 mL to about 190 mL, about 100 mL to about 180 mL, about 100 mL to about 170 mL, about 100 mL ~ About 160 mL, about 100 mL ~ about 150 mL, about 100 mL ~ about 140 mL, about 100 mL ~ about 130 mL, about 100 mL ~ about 120 mL, about 100 mL ~ about 110 mL, about 110 mL ~ about 500 mL, about 110 mL to about 490 mL, about 110 mL to about 480 mL, about 110 mL to about 460 mL, about 110 mL to about 440 mL, about 110 mL to about 420 mL, about 110 mL to about 400 mL , About 110 mL to about 380 mL, about 110 mL to about 360 mL, about 110 mL to about 340 mL, about 110 mL to about 320 mL, about 110 mL to about 300 mL, about 110 mL to about 280 mL, about 110 mL to about 260 mL, about 110 mL to about 250 mL, about 110 mL to about 240 mL, about 110 mL to about 220 mL, about 110 mL to about 210 mL, about 110 mL to about 200 mL, about 110 mL ~ About 190 mL, about 110 m L ~ about 180 mL, about 110 mL ~ about 170 mL, about 110 mL ~ about 160 mL, about 110 mL ~ about 150 mL, about 110 mL ~ about 140 mL, about 110 mL ~ about 130 mL, about 110 mL ~ About 120 mL, about 120 mL to about 500 mL, about 120 mL to about 490 mL, about 120 mL to about 480 mL, about 120 mL to about 460 mL, about 120 mL to about 440 mL, about 120 mL to about 420 mL, about 120 mL to about 400 mL, about 120 mL to about 380 mL, about 120 mL to about 360 mL, about 120 mL to about 340 mL, about 120 mL to about 320 mL, about 120 mL to about 300 mL, About 120 mL to about 280 mL, about 120 mL to about 260 mL, about 120 mL to about 250 mL, about 120 mL to about 240 mL, about 120 mL to about 220 mL, about 120 mL to about 210 mL, about 120 mL ~ about 200 mL, about 120 mL ~ about 190 mL, about 120 mL ~ about 180 mL, about 120 mL ~ about 170 mL, about 120 mL ~ about 160 mL, about 120 mL ~ about 150 mL, about 120 mL ~ About 140 mL, about 120 mL to about 130 mL, about 130 mL to about 500 mL, about 130 mL to about 490 mL, about 130 mL to about 480 mL, about 130 mL to about 460 mL, about 130 mL to about 440 mL, about 130 mL to about 420 mL, about 130 mL to about 400 mL, about 130 mL to about 380 mL, about 130 mL to about 360 mL, about 130 mL to about 340 mL, about 130 mL to about 320 mL, About 130 mL to about 300 mL, about 130 mL to about 280 mL, about 130 mL to about 260 mL, about 130 mL to about 250 mL, about 130 mL to about 240 mL, about 130 mL to about 220 mL, about 130 mL ~ about 210 mL, about 130 mL ~ about 200 mL, about 130 mL ~ about 190 mL, about 130 mL ~ about 180 mL, about 130 mL ~ about 170 mL, about 130 mL ~ about 160 mL, about 130 mL ~ About 150 mL, about 130 mL to about 140 mL, about 140 mL to about 500 mL, about 140 mL to about 490 mL, about 140 mL to about 480 mL, about 140 mL to about 460 mL, about 140 mL to about 440 m L, about 140 mL to about 420 mL, about 140 mL to about 400 mL, about 140 mL to about 380 mL, about 140 mL to about 360 mL, about 140 mL to about 340 mL, about 140 mL to about 320 mL, About 140 mL to about 300 mL, about 140 mL to about 280 mL, about 140 mL to about 260 mL, about 140 mL to about 250 mL, about 140 mL to about 240 mL, about 140 mL to about 220 mL, about 140 mL ~ about 210 mL, about 140 mL ~ about 200 mL, about 140 mL ~ about 190 mL, about 140 mL ~ about 180 mL, about 140 mL ~ about 170 mL, about 140 mL ~ about 160 mL, about 140 mL ~ About 150 mL, about 150 mL to about 500 mL, about 150 mL to about 490 mL, about 150 mL to about 480 mL, about 150 mL to about 460 mL, about 150 mL to about 440 mL, about 150 mL to about 420 mL, about 150 mL to about 400 mL, about 150 mL to about 380 mL, about 150 mL to about 360 mL, about 150 mL to about 340 mL, about 150 mL to about 320 mL, about 150 mL to about 300 mL, About 150 mL to about 280 mL, about 150 mL to about 260 mL, about 150 mL to about 250 mL, about 150 mL to about 240 mL, about 150 mL to about 220 mL, about 150 mL to about 210 mL, about 150 mL ~ about 200 mL, about 150 mL ~ about 190 mL, about 150 mL ~ about 180 mL, about 150 mL ~ about 170 mL, about 150 mL ~ about 160 mL, about 160 mL ~ about 500 mL, about 160 mL ~ About 490 mL, about 160 mL to about 480 mL, about 160 mL to about 460 mL, about 160 mL to about 440 mL, about 160 mL to about 420 mL, about 160 mL to about 400 mL, about 160 mL to about 380 mL, about 160 mL to about 360 mL, about 160 mL to about 340 mL, about 160 mL to about 320 mL, about 160 mL to about 300 mL, about 160 mL to about 280 mL, about 160 mL to about 260 mL, About 160 mL to about 250 mL, about 160 mL to about 240 mL, about 160 mL to about 220 mL, about 160 mL to about 210 mL, about 160 mL to about 200 mL, about 160 mL to about 190 mL, about 160 m L ~ about 180 mL, about 160 mL ~ about 170 mL, about 170 mL ~ about 500 mL, about 170 mL ~ about 490 mL, about 170 mL ~ about 480 mL, about 170 mL ~ about 460 mL, about 170 mL ~ About 440 mL, about 170 mL to about 420 mL, about 170 mL to about 400 mL, about 170 mL to about 380 mL, about 170 mL to about 360 mL, about 170 mL to about 340 mL, about 170 mL to about 320 mL, about 170 mL to about 300 mL, about 170 mL to about 280 mL, about 170 mL to about 260 mL, about 170 mL to about 250 mL, about 170 mL to about 240 mL, about 170 mL to about 220 mL, About 170 mL-about 210 mL, about 170 mL-about 200 mL, about 170 mL-about 190 mL, about 170 mL-about 180 mL, about 180 mL-about 500 mL, about 180 mL-about 490 mL, about 180 mL ~ about 480 mL, about 180 mL ~ about 460 mL, about 180 mL ~ about 440 mL, about 180 mL ~ about 420 mL, about 180 mL ~ about 400 mL, about 180 mL ~ about 380 mL, about 180 mL ~ About 360 mL, about 180 mL to about 340 mL, about 180 mL to about 320 mL, about 180 mL to about 300 mL, about 180 mL to about 280 mL, about 180 mL to about 260 mL, about 180 mL to about 250 mL, about 180 mL to about 240 mL, about 180 mL to about 220 mL, about 180 mL to about 210 mL, about 180 mL to about 200 mL, about 180 mL to about 190 mL, about 190 mL to about 500 mL, About 190 mL to about 490 mL, about 190 mL to about 480 mL, about 190 mL to about 460 mL, about 190 mL to about 440 mL, about 190 mL to about 420 mL, about 190 mL to about 400 mL, about 190 mL ~ about 380 mL, about 190 mL ~ about 360 mL, about 190 mL ~ about 340 mL, about 190 mL ~ about 320 mL, about 190 mL ~ about 300 mL, about 190 mL ~ about 280 mL, about 190 mL ~ About 260 mL, about 190 mL to about 250 mL, about 190 mL to about 240 mL, about 190 mL to about 220 mL, about 190 mL to about 210 mL, about 190 mL to about 200 mL, about 200 mL to about 500 m L, about 200 mL to about 490 mL, about 200 mL to about 480 mL, about 200 mL to about 460 mL, about 200 mL to about 440 mL, about 200 mL to about 420 mL, about 200 mL to about 400 mL, About 200 mL to about 380 mL, about 200 mL to about 360 mL, about 200 mL to about 340 mL, about 200 mL to about 320 mL, about 200 mL to about 300 mL, about 200 mL to about 280 mL, about 200 mL ~ about 260 mL, about 200 mL ~ about 250 mL, about 200 mL ~ about 240 mL, about 200 mL ~ about 220 mL, about 200 mL ~ about 210 mL, about 210 mL ~ about 500 mL, about 210 mL ~ About 490 mL, about 210 mL to about 480 mL, about 210 mL to about 460 mL, about 210 mL to about 440 mL, about 210 mL to about 420 mL, about 210 mL to about 400 mL, about 210 mL to about 380 mL, about 210 mL to about 360 mL, about 210 mL to about 340 mL, about 210 mL to about 320 mL, about 210 mL to about 300 mL, about 210 mL to about 280 mL, about 210 mL to about 260 mL, About 210 mL to about 250 mL, about 210 mL to about 240 mL, about 210 mL to about 220 mL, about 220 mL to about 500 mL, about 220 mL to about 490 mL, about 220 mL to about 480 mL, about 220 mL ~ about 460 mL, about 220 mL ~ about 440 mL, about 220 mL ~ about 420 mL, about 220 mL ~ about 400 mL, about 220 mL ~ about 380 mL, about 220 mL ~ about 360 mL, about 220 mL ~ About 340 mL, about 220 mL to about 320 mL, about 220 mL to about 300 mL, about 220 mL to about 280 mL, about 220 mL to about 260 mL, about 220 mL to about 250 mL, about 220 mL to about 240 mL, about 240 mL to about 500 mL, about 240 mL to about 490 mL, about 240 mL to about 480 mL, about 240 mL to about 460 mL, about 240 mL to about 440 mL, about 240 mL to about 420 mL, About 240 mL to about 400 mL, about 240 mL to about 380 mL, about 240 mL to about 360 mL, about 240 mL to about 340 mL, about 240 mL to about 320 mL, about 240 mL to about 300 mL, about 240 m L ~ about 280 mL, about 240 mL ~ about 260 mL, about 240 mL ~ about 250 mL, about 250 mL ~ about 500 mL, about 250 mL ~ about 490 mL, about 250 mL ~ about 480 mL, about 250 mL ~ Approximately 460 mL, approximately 250 mL to approximately 440 mL, approximately 250 mL to approximately 420 mL, approximately 250 mL to
About 400 mL, about 250 mL to about 380 mL, about 250 mL to about 360 mL, about 250 mL to about 340 mL, about 250 mL to about 320 mL, about 250 mL to about 300 mL, about 250 mL to about 280 mL, about 250 mL to about 260 mL, about 260 mL to about 500 mL, about 260 mL to about 490 mL, about 260 mL to about 480 mL, about 260 mL to about 460 mL, about 260 mL to about 440 mL, About 260 mL to about 420 mL, about 260 mL to about 400 mL, about 260 mL to about 380 mL, about 260 mL to about 360 mL, about 260 mL to about 340 mL, about 260 mL to about 320 mL, about 260 mL ~ about 300 mL, about 260 mL ~ about 280 mL, about 280 mL ~ about 500 mL, about 280 mL ~ about 490 mL, about 280 mL ~ about 480 mL, about 280 mL ~ about 460 mL, about 280 mL ~ About 440 mL, about 280 mL-about 420 mL, about 280 mL-about 400 mL, about 280 mL-about 380 mL, about 280 mL-about 360 mL, about 280 mL-about 340 mL, about 280 mL-about 320 mL, about 280 mL to about 300 mL, about 300 mL to about 500 mL, about 300 mL to about 490 mL, about 300 mL to about 480 mL, about 300 mL to about 460 mL, about 300 mL to about 440 mL, About 300 mL to about 420 mL, about 300 mL to about 400 mL, about 300 mL to about 380 mL, about 300 mL to about 360 mL, about 300 mL to about 340 mL, about 300 mL to about 320 mL, about 320 mL ~ about 500 mL, about 320 mL ~ about 490 mL, about 320 mL ~ about 480 mL, about 320 mL ~ about 460 mL, about 320 mL ~ about 440 mL, about 320 mL ~ about 420 mL, about 320 mL ~ About 400 mL, about 320 mL to about 380 mL, about 320 mL to about 360 mL, about 320 mL to about 340 mL, about 340 mL to about 500 mL, about 340 mL to about 490 mL, about 340 mL to about 480 mL, about 340 mL to about 460 mL, about 340 mL to about 440 mL, about 340 mL to about 420 mL, about 340 mL to about 400 mL, about 340 mL to about 380 mL, about 340 mL to about 360 mL, About 360 mL to about 500 mL, about 360 mL to about 490 mL, about 360 mL to about 480 mL, about 360 mL to about 460 mL, about 360 mL to about 440 mL, about 360 mL to about 420 mL, about 360 mL ~ about 400 mL, about 360 mL ~ about 380 mL, about 380 mL ~ about 500 mL, about 380 mL ~ about 490 mL, about 380 mL ~ about 480 mL, about 380 mL ~ about 460 mL, about 380 mL ~ About 440 mL, about 380 mL to about 420 mL, about 380 mL to about 400 mL, about 400 mL to about 500 mL, about 400 mL to about 490 mL, about 400 mL to about 480 mL, about 400 mL to about 460 mL, about 400 mL to about 440 mL, about 400 mL to about 420 mL, about 420 mL to about 500 mL, about 420 mL to about 490 mL, about 420 mL to about 480 mL, about 420 mL to about 460 mL, About 420 mL to about 440 mL, about 440 mL to about 500 mL, about 440 mL to about 490 mL, about 440 mL to about 480 mL, about 440 mL to about 460 mL, about 460 mL to about 500 mL, about 460 It may have a volume of mL to about 490 mL, about 460 mL to about 480 mL, about 480 mL to about 500 mL, about 480 mL to about 490 mL, or about 490 mL to about 500 mL.

In some embodiments, the chromatographic resin placed in the pod is about 1 x 10 ^-6 to about 1 x 10 ^-10 , about 1 x 10 ^-6 to about 0.5 x 10 ^-10 , about 1 x 10 ^-6. ~ About 1 x 10 ^-9 , About 1 x 10 ^-6 ~ About 0.5 x 10 ^-9 , About 1 x 10 ^-6 ~ About 1 x 10 ^-8 , About 1 x 10 ^-6 ~ About 0.5 x 10 ^-8 , Approximately 1 x 10 ^-6 to approximately 1 x 10 ^-7 , approximately 1 x 10 ^-6 to approximately 0.5 x 10 ^-7 , approximately 0.5 x 10 ^-7 to approximately 1 x 10 ^-10 , approximately 0.5 x 10 ^-7 to approximately 0.5 x 10 ^-10 , about 0.5 x 10 ^-7 ~ about 1 x 10 ^-9 , about 0.5 x 10 ^-7 ~ about 0.5 x 10 ^-9 , about 0.5 x 10 ^-7 ~ about 1 x 10 ^-8 , about 0.5 x 10 ^-7 to about 0.5 x 10 ^-8 , about 0.5 x 10 ^-7 to about 1 x 10 ^-7 , about 1 x 10 ^-7 to about 1 x 10 ^-10 , about 1 x 10 ^-7 to about 0.5 x 10 ^-10 , about 0.5 x 10 ^-7 ~ about 1 x 10 ^-9 , about 1 x 10 ^-7 ~ about 0.5 x 10 ^-9 , about 1 x 10 ^-7 ~ about 1 x 10 ^-8 , about 1 x 10 ^-7 to about 0.5 x 10 ^-8, about 0.5 x 10 ^-8 to about 1 x 10 ^-10, about 0.5 x 10 ^-8 to about 0.5 x 10 ^-10, about 0.5 x 10 ^-8 to about 1 x 10 ^{- 9} , about 0.5 x 10 ^-8 to about 0.5 x 10 ^-9 , about 0.5 x 10 ^-8 to about 1 x 10 ^-8 , about 1 x 10 ^-8 to about 1 x 10 ^-10 , about 1 x 10 ^-8 ~ About 0.5 x 10 ^-10 , about 1 x 10 ^-8 ~ about 1 x 10 ^-9 , about 1 x 10 ^-8 ~ about 0.5 x 10 ^-9 , about 0.5 x 10 ^-9 ~ about 1 x 10 ^-10 , About 0.5 x 10 ^-9 to about 0.5 x 10 ^-10 , about 0.5 x 10 ^-9 to about 1 x 10 ^-9 , about 1 x 10 ^-9 to about 1 x 10 ^-10 , about 1 x 10 ^-9 to about It has a sterility assurance level of 0.5 x 10 ^-10 , or about 0.5 x 10 ^-10 to about 1 x 10 ^-10 .

Kits Kits containing any of the pods described herein are also provided herein. (A) Any pod described herein, one or more areas covered with a semi-porous or porous material that allows liquid to reversibly flow from outside the pod into the interior of the pod. , Containing pods on the wall of the container) and; (b) securely secured on each of the one or more areas on the wall to prevent fluid from leaking from inside the pod to outside the pod. Kits, including, one or more panels, are also provided herein. (A) Any pod described herein, one or more areas covered with a semi-porous or porous material that allows liquid to reversibly flow from outside the pod into the interior of the pod. Cover the one or more areas on the wall to prevent fluid from leaking from inside the pod to outside the pod through the at least one area on the wall. A kit is also provided herein that includes an outer sleeve, which can be securely secured onto the pod.

One or both of (a) pods of any of the pods described herein; (b) elution outlets that can be stably anchored on the bottom edge, and buffer reservoirs that can be stably anchored on the top edge. Kits containing, and are also provided herein. (A) Any pod described herein, one or more areas covered with a semi-porous or porous material that allows liquid to reversibly flow from outside the pod into the interior of the pod. , And; (b) the one on the wall as the movable sleeve rotates to prevent fluid from leaking from inside the pod to the outside of the pod through the at least one area on the wall. Alternatively, a kit including a movable outer sleeve, which is connected to a wall so that multiple areas can be covered, is also provided herein. A kit comprising (a) any of the pods described herein; (b) means for stably positioning the long axis of the pod in a horizontal position within the culture vessel is also described herein. provide.

Any kit described herein may include means for stably positioning the pod within the culture vessel. An exemplary means for stably positioning the pod within a culture vessel (eg, any culture vessel described herein) is with brackets; with braces; on the openings in the culture vessel and. It may include an adapter that allows the pod to be inserted into the culture vessel (without dropping the pod into the culture vessel). In some embodiments, the means for stably positioning the pod within a culture vessel (eg, any culture vessel described herein) is flexible and the pod is within the culture vessel. Allows it to move with the movement of the liquid culture without falling. In some embodiments, the means for stably positioning the pod within the culture vessel (eg, any of the culture vessels described herein) is rigid and the pod falls into the culture vessel. Allows to withstand the movement of liquid cultures. Non-limiting examples of means for stably positioning the pod within a culture vessel (eg, any culture vessel described herein) are shown in FIGS. 10-19, 36, and 37.

In some embodiments, the pod is stably secured by tape the pod inside the culture vessel. In some embodiments, the pod is stably secured by snapping the pod into the culture vessel.

In some embodiments, the kit includes an adapter manifold that allows multiple pods to be edited into a plate array, for example to further facilitate an automated high throughput purification approach.

Non-limiting examples of culture vessels include multi-well plates (eg 6-well plates, 12-well plates, 18-well plates, 24-well plates, 48-well plates, 96-well plates, or 384-well plates), shaking flasks (eg, 6-well plates). Includes Erlenmeyer flask or T-flask), shaking tube (eg Eppendorf tube), wave bag, bioreactor (eg benchtop bioreactor, perfusion bioreactor, fed batch bioreactor), rotating tube bioreactor .. Further examples of bioreactors are known in the art.

Some aspects of any of the kits described herein include the one or more (eg, 1, 2) of said on the wall to prevent fluid from leaking through the wall from inside the pod to outside the pod. One or more (eg 1, 2, 3, 4, 5, 6, 7) that can be securely fixed on each of the, 3, 4, 5, 6, 7, 8, 9, or 10 areas. , 8, 9, or 10) panels may be further included. The one or more panels may be made of one or more of any of the exemplary materials described herein, which may be used to make a wall. The one or more panels are designed to cover any of the areas described herein. The one or more panels may be designed to be clicked, snapped, or screwed into the wall to cover the one or more areas of the wall as described herein.

Some aspects of the kits described herein are to cover the at least one area of the wall so that fluid is prevented from leaking from inside the pod to the outside of the pod through the at least one area of the wall. An outer sleeve that can be securely secured on the pod may be further included. As will be appreciated in the art, the sleeve may be constructed of at least partially flexible rubber or silicon so that it can be slid onto the container to cover at least one area of the wall. In some embodiments, the sleeve may be an empty chromatographic column that fits onto the container.

Some aspects of the kit described herein are such that when the movable outer sleeve rotates, the movable outer sleeve is prevented from leaking from the inside of the pod to the outside of the pod through the at least one area of the wall. A movable outer sleeve, which is connected to the wall so that at least one area can be covered, may further be included. In some embodiments, the rotation of the movable outer sleeve required to cover at least one area of the wall is clockwise. In some embodiments, the rotation of the movable outer sleeve required to cover at least one area of the wall is counterclockwise. In an alternative embodiment, the movable outer sleeve slides up or down to prevent the fluid from leaking from the inside of the pod to the outside of the pod through the at least one area of the wall. A movable outer sleeve is connected to the wall so that it can be covered.

Some aspects of the kit further include an elution outlet that can be stably anchored on the bottom edge. The elution outlet may be a funnel or a flat surface with ports. In some embodiments, the elution outlet may include means for controlling the flow of fluid exiting the container. Non-limiting examples of means for controlling the flow of fluid out of a container include a screw valve or a stopcock. Further exemplary means for controlling the flow of fluid exiting the container are known in the art. In other embodiments, the elution outlet does not include a means for controlling the flow of fluid out of the container. The elution outlet may be designed to snap, click, or screw onto the bottom edge of the container. In some embodiments of any of the kits provided herein, the buffer reservoir has one or more (eg, two or three) O-rings (eg, fixed to a pod or buffer reservoir). It may be used to seal the pod.

Some aspects of any of the kits described herein further include a buffer reservoir that can be stably anchored on top. The buffer reservoir may be snap-fastened, click-fastened, or screwed onto the top edge of the container. The buffer reservoir may have any shape to hold a volume of fluid that may flow into the container. For example, the buffer reservoir may be cylindrical, rectangular, hemispherical, spherical, or oval. In some embodiments, the buffer reservoir may include means for controlling the flow of fluid from the buffer reservoir into the container. Non-limiting examples of means for controlling the flow of fluid out of the buffer reservoir and into the container include screw valves or stopcocks. Further exemplary means for controlling the flow of fluid out of the buffer reservoir and into the container are known in the art. In another embodiment, the buffer reservoir does not include means for controlling the flow of fluid out of the buffer reservoir and into the container. The buffer reservoir is 0.1 mL to about 500 mL, about 0.1 mL to about 450 mL, about 0.1 mL to about 400 mL, about 0.1 mL to about 350 mL, about 0.1 mL to about 300 mL, about 0.1 mL to about 250. mL, about 0.1 mL to about 200 mL, about 0.1 mL to about 150 mL, about 0.1 mL to about 100 mL, about 0.1 mL to about 80 mL, about 0.1 mL to about 60 mL, about 0.1 mL to about 50 mL, About 0.1 mL to about 40 mL, about 0.1 mL to about 30 mL, about 0.1 mL to about 20 mL, about 0.1 mL to about 10 mL, about 0.1 mL to about 5 mL, about 0.1 mL to about 2 mL, about 0.1 mL ~ about 1 mL, about 0.2 mL ~ about 500 mL, about 0.2 mL ~ about 450 mL, about 0.2 mL ~ about 400 mL, about 0.2 mL ~ about 350 mL, about 0.2 mL ~ about 300 mL, about 0.2 mL ~ About 250 mL, about 0.2 mL to about 200 mL, about 0.2 mL to about 150 mL, about 0.2 mL to about 100 mL, about 0.2 mL to about 80 mL, about 0.2 mL to about 60 mL, about 0.2 mL to about 50 mL, about 0.2 mL to about 40 mL, about 0.2 mL to about 30 mL, about 0.2 mL to about 20 mL, about 0.2 mL to about 10 mL, about 0.2 mL to about 5 mL, about 0.2 mL to about 2 mL, About 0.2 mL to about 1 mL, 0.5 mL to about 500 mL, about 0.5 mL to about 450 mL, about 0.5 mL to about 400 mL, about 0.5 mL to about 350 mL, about 0.5 mL to about 300 mL, about 0.5 mL ~ About 250 mL, about 0.5 mL ~ about 200 mL, about 0.5 mL ~ about 150 mL, about 0.5 mL ~ about 100 mL, about 0.5 mL ~ about 80 mL, about 0.5 mL ~ about 60 mL, about 0.5 mL ~ about 50 mL, about 0.5 mL to about 40 mL, about 0.5 mL to about 30 mL, about 0.5 mL to about 20 mL, about 0.5 mL to about 10 mL, about 0.5 mL to about 5 mL, about 0.5 mL to about 2 mL , About 2 mL to about 500 mL, about 2 mL to about 450 mL, about 2 mL to about 400 mL, about 2 mL to about 350 mL, about 2 mL to about 300 mL, about 2 mL to about 250 mL, about 2 mL to about 200 mL, about 2 mL to about 150 mL, about 2 mL to about 100 mL, about 2 mL to about 8 0 mL, about 2 mL to about 60 mL, about 2 mL to about 50 mL, about 2 mL to about 40 mL, about 2 mL to about 30 mL, about 2 mL to about 20 mL, about 2 mL to about 10 mL , About 2 mL to about 5 mL, about 5 mL to about 500 mL, about 5 mL to about 450 mL, about 5 mL to about 400 mL, about 5 mL to about 350 mL, about 5 mL to about 300 mL, about 5 mL to about 250 mL, about 5 mL to about 200 mL, about 5 mL to about 150 mL, about 5 mL to about 100 mL, about 5 mL to about 80 mL, about 5 mL to about 60 mL, about 5 mL ~ About 50 mL, about 5 mL ~ about 40 mL, about 5 mL ~ about 30 mL, about 5 mL ~ about 20 mL, about 5 mL ~ about 10 mL, about 10 mL ~ about 500 mL, about 10 mL ~ about 450 mL, about 10 mL to about 400 mL, about 10 mL to about 350 mL, about 10 mL to about 300 mL, about 10 mL to about 250 mL, about 10 mL to about 200 mL, about 10 mL to about 150 mL , About 10 mL to about 100 mL, about 10 mL to about 80 mL, about 10 mL to about 60 mL, about 10 mL to about 50 mL, about 10 mL to about 40 mL, about 10 mL to about 30 mL, about 10 mL to about 20 mL, about 20 mL to about 500 mL, about 20 mL to about 450 mL, about 20 mL to about 400 mL, about 20 mL to about 350 mL, about 20 mL to about 300 mL, about 20 mL ~ About 250 mL, about 20 mL ~ about 200 mL, about 20 mL ~ about 150 mL, about 20 mL ~ about 100 mL, about 20 mL ~ about 80 mL, about 20 mL ~ about 60 mL, about 20 mL ~ about 50 mL, about 20 mL to about 40 mL, about 20 mL to about 30 mL, about 30 mL to about 500 mL, about 30 mL to about 450 mL, about 30 mL to about 400 mL, about 30 mL to about 350 mL , About 30 mL to about 300 mL, about 30 mL to about 250 mL, about 30 mL to about 200 mL, about 30 mL to about 150 mL, about 30 mL to about 100 mL, about 30 mL to about 80 mL, about 30 mL to about 60 mL, about 30 mL to about 50 mL, about 30 mL to about 40 mL, about 50 mL to about 500 mL, about 50 mL to about 450 mL, about 50 mL to about 400 mL, about 50 mL ~ About 350 mL, about 50 mL ~ about 300 m L, about 50 mL to about 250 mL, about 50 mL to about 200 mL, about 50 mL to about 150 mL, about 50 mL to about 100 mL, about 50 mL to about 80 mL, or about 50 mL to about 60 mL It may be designed to hold a volume of fluid.

(A) with any of the pods described herein (eg, pods having any of the properties or aspects described herein in any combination); (b) an oblong with an upper end and a fluid conduit. An inlet system with a cylindrical body and a lower end, the upper end of the inlet system having a fluid inlet and configured to be stably attached to the upper end of the pod; and stably connected to the pod. Occasionally allows the flow of fluid through the fluid inlet and fluid conduit and out of the bottom edge of the inlet system into the pod; and the elongated cylindrical body of the inlet system is configured to fit inside the wall of the pod. An outlet system with an inlet system; (c) an outlet system with a fluid outlet, configured to be stably attached to the lower end of the pod, and a fluid outlet when stably connected to the lower end of the pod. A kit including an outlet system, which allows the flow of fluid through the pod, is also provided herein. In some aspects of these kits, the top of the inlet system and the top of the pod include threading that allows the top of the pod to be stably attached to the top of the inlet system. In some aspects of these kits, when the inlet system and pod are stably attached, the top of the inlet system and the top of the pod form a seal (eg, a fluid-tight seal). In some aspects of these kits, threading at the top of the inlet system, or threading at the top of the pod, comprises an O-ring. Other means for providing a stable connection between the top of the inlet system and the top of the pod are known in the art. In some aspects of these kits, the pod contains a chromatographic resin; and the inlet system and pod are such that the lower end of the inlet system is close to the chromatographic resin when the inlet system and pod are stably attached. It is composed of. In some aspects of these kits, the inner surface of the pod and the lower end of the inlet system are configured to allow stable attachment of the pod to the lower end of the inlet system. In some aspects of these kits, when the inlet system and the pod are stably attached, the inner surface of the pod and the lower end of the inlet system form a seal (eg, a fluid-tight seal). In some aspects of these kits, the inner surface of the pod and the lower edge of the inlet system include threading that allows stable attachment of the pod to the lower edge of the inlet system. In some aspects of these kits, threading on the inner surface of the pod, or threading on the lower edge of the inlet system, comprises an O-ring. Other means for providing a stable connection between the inner surface of the pod and the lower end of the inlet system are known in the art. In some aspects of these kits, the outlet system and the bottom edge of the pod include threading that allows the outlet system to be stably attached to the bottom edge of the pod. In some aspects of these kits, threading in the outlet system, or threading at the bottom edge of the pod, comprises an O-ring. In some aspects of these kits, the fluid inlet can be attached to a tubing (eg, a tubing connected to a chromatography system). In some aspects of these kits, the fluid outlet can be attached to a tubing (eg, a tubing connected to a chromatography system). Some aspects of these kits further include (d) means for stably positioning the long axis of the pod in a horizontal position within the culture vessel. Some aspects of these kits further include (d) means for stably positioning the long axis of the pod in a vertical position within the culture vessel. In some aspects of these kits, the elongated cylindrical body of the inlet system is about 0.1 cm to about 50 cm, about 0.1 cm to about 45 cm, about 0.1 cm to about 40 cm, about 0.1 cm to about 35 cm. , About 0.1 cm to about 30 cm, about 0.1 cm to about 25 cm, about 0.1 cm to about 20 cm, about 0.1 cm to about 18 cm, about 0.1 cm to about 16 cm, about 0.1 cm to about 14 cm, about 0.1 cm to about 12 cm, about 0.1 cm to about 10 cm, about 0.1 cm to about 8 cm, about 0.1 cm to about 6 cm, about 0.1 cm to about 5 cm, about 0.1 cm to about 4.5 cm, about 0.1 cm ~ About 4.0 cm, about 0.1 cm ~ about 3.5 cm, about 0.1 cm ~ about 3.0 cm, about 0.1 cm ~ about 2.5 cm, about 0.1 cm ~ about 2.0 cm, about 0.1 cm ~ about 1.5 cm, about 0.1 cm ~ about 1.0 cm, about 0.1 cm to about 0.5 cm, about 0.5 cm to about 50 cm, about 0.5 cm to about 45 cm, about 0.5 cm to about 40 cm, about 0.5 cm to about 35 cm, about 0.5 cm to about 30 cm , About 0.5 cm to about 25 cm, about 0.5 cm to about 20 cm, about 0.5 cm to about 18 cm, about 0.5 cm to about 16 cm, about 0.5 cm to about 14 cm, about 0.5 cm to about 12 cm, about 0.5 cm to about 10 cm, about 0.5 cm to about 8 cm, about 0.5 cm to about 6 cm, about 0.5 cm to about 5 cm, about 0.5 cm to about 4.5 cm, about 0.5 cm to about 4.0 cm, about 0.5 cm ~ About 3.5 cm, about 0.5 cm ~ about 3.0 cm, about 0.5 cm ~ about 2.5 cm, about 0.5 cm ~ about 2.0 cm, about 0.5 cm ~ about 1.5 cm, about 0.5 cm ~ about 1.0 cm, about 1.0 cm ~ about 50 cm, about 1.0 cm to about 45 cm, about 1.0 cm to about 40 cm, about 1.0 cm to about 35 cm, about 1.0 cm to about 30 cm, about 1.0 cm to about 25 cm, about 1.0 cm to about 20 cm , About 1.0 cm to about 18 cm, about 1.0 cm to about 16 cm, about 1.0 cm to about 14 cm, about 1.0 cm to about 12 cm, about 1.0 cm to about 10 cm, about 1.0 cm to about 8 cm, about 1.0 cm to about 6 cm, about 1.0 cm to about 5 cm, about 1.0 cm to about 4.5 cm, about 1.0 c m ~ about 4.0 cm, about 1.0 cm ~ about 3.5 cm, about 1.0 cm ~ about 3.0 cm, about 1.0 cm ~ about 2.5 cm, about 1.0 cm ~ about 2.0 cm, about 1.0 cm ~ about 1.5 cm, about 1.5 cm ~ About 50 cm, about 1.5 cm to about 45 cm, about 1.5 cm to about 40 cm, about 1.5 cm to about 35 cm, about 1.5 cm to about 30 cm, about 1.5 cm to about 25 cm, about 1.5 cm to about 20 cm, about 1.5 cm to about 18 cm, about 1.5 cm to about 16 cm, about 1.5 cm to about 14 cm, about 1.5 cm to about 12 cm, about 1.5 cm to about 10 cm, about 1.5 cm to about 8 cm, About 1.5 cm to about 6 cm, about 1.5 cm to about 5 cm, about 1.5 cm to about 4.5 cm, about 1.5 cm to about 4.0 cm, about 1.5 cm to about 3.5 cm, about 1.5 cm to about 3.0 cm, about 1.5 cm to about 2.5 cm, about 1.5 cm to about 2.0 cm, about 2.0 cm to about 50 cm, about 2.0 cm to about 45 cm, about 2.0 cm to about 40 cm, about 2.0 cm to about 35 cm, about 2.0 cm About 30 cm, about 2.0 cm to about 25 cm, about 2.0 cm to about 20 cm, about 2.0 cm to about 18 cm, about 2.0 cm to about 16 cm, about 2.0 cm to about 14 cm, about 2.0 cm to about 12 cm, about 2.0 cm to about 10 cm, about 2.0 cm to about 8 cm, about 2.0 cm to about 6 cm, about 2.0 cm to about 5 cm, about 2.0 cm to about 4.5 cm, about 2.0 cm to about 4.0 cm, About 2.0 cm to about 3.5 cm, about 2.0 cm to about 3.0 cm, about 2.0 cm to about 2.5 cm, about 2.5 cm to about 50 cm, about 2.5 cm to about 45 cm, about 2.5 cm to about 40 cm, about 2.5 cm to about 35 cm, about 2.5 cm to about 30 cm, about 2.5 cm to about 25 cm, about 2.5 cm to about 20 cm, about 2.5 cm to about 18 cm, about 2.5 cm to about 16 cm, about 2.5 cm About 14 cm, about 2.5 cm to about 12 cm, about 2.5 cm to about 10 cm, about 2.5 cm to about 8 cm, about 2.5 cm to about 6 cm, about 2.5 cm to about 5 cm, about 2.5 cm to about 4.5 cm, about 2.5 cm to about 4.0 cm, about 2.5 cm to about 3.5 cm, about 2.5 cm to about 3.0 cm, about 3.0 cm to about 50 cm, about 3.0 cm to about 45 cm, about 3.0 cm to about 40 cm, about 3.0 cm to about 35 cm, about 3.0 cm to about 30 cm, about 3.0 cm to about 25 cm, about 3.0 cm to About 20 cm, about 3.0 cm to about 18 cm, about 3.0 cm to about 16 cm, about 3.0 cm to about 14 cm, about 3.0 cm to about 12 cm, about 3.0 cm to about 10 cm, about 3.0 cm to about 8 cm, about 3.0 cm to about 6 cm, about 3.0 cm to about 5 cm, about 3.0 cm to about 4.5 cm, about 3.0 cm to about 4.0 cm, about 3.0 cm to about 3.5 cm, about 3.5 cm to about 50 cm, About 3.5 cm to about 45 cm, about 3.5 cm to about 40 cm, about 3.5 cm to about 35 cm, about 3.5 cm to about 30 cm, about 3.5 cm to about 25 cm, about 3.5 cm to about 20 cm, about 3.5 cm ~ about 18 cm, about 3.5 cm ~ about 16 cm, about 3.5 cm ~ about 14 cm, about 3.5 cm ~ about 12 cm, about 3.5 cm ~ about 10 cm, about 3.5 cm ~ about 8 cm, about 3.5 cm ~ About 6 cm, about 3.5 cm to about 5 cm, about 3.5 cm to about 4.5 cm, about 3.5 cm to about 4.0 cm, about 4.0 cm to about 50 cm, about 4.0 cm to about 45 cm, about 4.0 cm to about 40 cm, about 4.0 cm to about 35 cm, about 4.0 cm to about 30 cm, about 4.0 cm to about 25 cm, about 4.0 cm to about 20 cm, about 4.0 cm to about 18 cm, about 4.0 cm to about 16 cm, About 4.0 cm to about 14 cm, about 4.0 cm to about 12 cm, about 4.0 cm to about 10 cm, about 4.0 cm to about 8 cm, about 4.0 cm to about 6 cm, about 4.0 cm to about 5 cm, about 4.0 cm ~ about 4.5 cm, about 4.5 cm ~ about 50 cm, about 4.5 cm ~ about 45 cm, about 4.5 cm ~ about 40 cm, about 4.5 cm ~ about 35 cm, about 4.5 cm ~ about 30 cm, about 4.5 cm ~ About 25 cm, about 4.5 cm to about 20 cm, about 4.5 cm to about 18 cm, about 4.5 cm to about 16 cm, about 4.5 cm to about 14 cm, about 4.5 cm to about 12 cm, about 4.5 cm to about 10 cm, about 4.5 cm to about 8 cm, about 4.5 cm to about 6 cm, about 4.5 cm to about 5 cm, about 5 cm to about 50 cm, about 5 cm to about 45 cm, about 5 cm to about 40 cm, about 5 cm to about 35 cm, about 5 cm to about 30 cm, about 5 cm to about 25 cm, about 5 cm to about 20 cm, about 5 cm to about 18 cm , About 5 cm to about 16 cm, about 5 cm to about 14 cm, about 5 cm to about 12 cm, about 5 cm to about 10 cm, about 5 cm to about 8 cm, about 5 cm to about 6 cm, about 6 cm to about 50 cm, about 6 cm to about 45 cm, about 6 cm to about 40 cm, about 6 cm to about 35 cm, about 6 cm to about 30 cm, about 6 cm to about 25 cm, about 6 cm ~ About 20 cm, about 6 cm ~ about 18 cm, about 6 cm ~ about 16 cm, about 6 cm ~ about 14 cm, about 6 cm ~ about 12 cm, about 6 cm ~ about 10 cm, about 6 cm ~ about 8 cm, about 8 cm to about 50 cm, about 8 cm to about 45 cm, about 8 cm to about 40 cm, about 8 cm to about 35 cm, about 8 cm to about 30 cm, about 8 cm to about 25 cm , About 8 cm to about 20 cm, about 8 cm to about 18 cm, about 8 cm to about 16 cm, about 8 cm to about 14 cm, about 8 cm to about 12 cm, about 8 cm to about 10 cm, about 10 cm to about 50 cm, about 10 cm to about 45 cm, about 10 cm to about 40 cm, about 10 cm to about 35 cm, about 10 cm to about 30 cm, about 10 cm to about 25 cm, about 10 cm ~ About 20 cm, about 10 cm ~ about 18 cm, about 10 cm ~ about 16 cm, about 10 cm ~ about 14 cm, about 10 cm ~ about 12 cm, about 12 cm ~ about 50 cm, about 12 cm ~ about 45 cm, about 12 cm to about 40 cm, about 12 cm to about 35 cm, about 12 cm to about 30 cm, about 12 cm to about 25 cm, about 12 cm to about 20 cm, about 12 cm to about 18 cm , About 12 cm to about 16 cm, about 12 cm to about 14 cm, about 14 cm to about 50 cm, about 14 cm to about 45 cm, about 14 cm to about 40 cm, about 14 cm to about 35 cm, about 14 cm to about 30 cm, about 14 cm to about 25 cm, about 14 cm to about 20 cm, about 14 cm to about 18 cm, about 14 cm to about 16 cm, about 16 cm to about 50 cm, about 16 cm ~ About 45 cm, about 16 cm ~ about 40 cm, about 16 cm ~ about 35 cm, about 16 cm ~ about 30 cm, about 16 cm ~ about 25 cm, about 16 cm ~ about 20 c m, about 16 cm to about 18 cm, about 18 cm to about 50 cm, about 18 cm to about 45 cm, about 18 cm to about 40 cm, about 18 cm to about 35 cm, about 18 cm to about 30 cm, About 18 cm to about 25 cm, about 18 cm to about 20 cm, about 20 cm to about 50 cm, about 20 cm to about 45 cm, about 20 cm to about 40 cm, about 20 cm to about 35 cm, about 20 cm ~ about 30 cm, about 20 cm ~ about 25 cm, about 25 cm ~ about 50 cm, about 25 cm ~ about 45 cm, about 25 cm ~ about 40 cm, about 25 cm ~ about 35 cm, about 25 cm ~ About 30 cm, about 30 cm to about 50 cm, about 30 cm to about 45 cm, about 30 cm to about 40 cm, about 30 cm to about 35 cm, about 35 cm to about 50 cm, about 35 cm to about 45 cm, about 35 cm to about 40 cm, about 40 cm to about 50 cm, about 40 cm to about 45 cm, or about 45 cm to about 50 cm; and / or about 0.1 cm to about 30 cm, About 0.1 cm to about 28 cm, about 0.1 cm to about 26 cm, about 0.1 cm to about 24 cm, about 0.1 cm to about 22 cm, about 0.1 cm to about 20 cm, about 0.1 cm to about 18 cm, about 0.1 cm to about 16 cm, about 0.1 cm to about 14 cm, about 0.1 cm to about 12 cm, about 0.1 cm to about 10 cm, about 0.1 cm to about 9 cm, about 0.1 cm to about 8 cm, about 0.1 cm About 7 cm, about 0.1 cm to about 6 cm, about 0.1 cm to about 5 cm, about 0.1 cm to about 4 cm, about 0.1 cm to about 3 cm, about 0.1 cm to about 2 cm, about 0.1 cm to about 1 cm, about 0.1 cm to about 0.5 cm, about 0.5 cm to about 30 cm, about 0.5 cm to about 28 cm, about 0.5 cm to about 26 cm, about 0.5 cm to about 24 cm, about 0.5 cm to about 22 cm, About 0.5 cm to about 20 cm, about 0.5 cm to about 18 cm, about 0.5 cm to about 16 cm, about 0.5 cm to about 14 cm, about 0.5 cm to about 12 cm, about 0.5 cm to about 10 cm, about 0.5 cm to about 9 cm, about 0.5 cm to about 8 cm, about 0.5 cm to about 7 cm, about 0.5 cm to about 6 cm, about 0.5 cm to about 5 cm, about 0.5 cm to about 4 cm, about 0.5 cm About 3 cm, about 0.5 cm ~ About 2 cm, about 0.5 cm ~ about 1 cm, about 1 cm ~ about 30 cm, about 1 cm ~ about 28 cm, about 1 cm ~ about 26 cm, about 1 cm ~ about 24 cm, about 1 cm ~ about 22 cm, about 1 cm to about 20 cm, about 1 cm to about 18 cm, about 1 cm to about 16 cm, about 1 cm to about 14 cm, about 1 cm to about 12 cm, about 1 cm to about 10 cm , About 1 cm to about 9 cm, about 1 cm to about 8 cm, about 1 cm to about 7 cm, about 1 cm to about 6 cm, about 1 cm to about 5 cm, about 1 cm to about 4 cm, about 1 cm to about 3 cm, about 1 cm to about 2 cm, about 2 cm to about 30 cm, about 2 cm to about 28 cm, about 2 cm to about 26 cm, about 2 cm to about 24 cm, about 2 cm ~ About 22 cm, about 2 cm ~ about 20 cm, about 2 cm ~ about 18 cm, about 2 cm ~ about 16 cm, about 2 cm ~ about 14 cm, about
2 cm to about 12 cm, about 2 cm to about 10 cm, about 2 cm to about 9 cm, about 2 cm to about 8 cm, about 2 cm to about 7 cm, about 2 cm to about 6 cm, about 2 cm ~ About 5 cm, about 2 cm ~ about 4 cm, about 2 cm ~ about 3 cm, about 3 cm ~ about 30 cm, about 3 cm ~ about 28 cm, about 3 cm ~ about 26 cm, about 3 cm ~ about 24 cm, about 3 cm to about 22 cm, about 3 cm to about 20 cm, about 3 cm to about 18 cm, about 3 cm to about 16 cm, about 3 cm to about 14 cm, about 3 cm to about 12 cm , About 3 cm to about 10 cm, about 3 cm to about 9 cm, about 3 cm to about 8 cm, about 3 cm to about 7 cm, about 3 cm to about 6 cm, about 3 cm to about 5 cm, about 3 cm to about 4 cm, about 4 cm to about 30 cm, about 4 cm to about 28 cm, about 4 cm to about 26 cm, about 4 cm to about 24 cm, about 4 cm to about 22 cm, about 4 cm ~ About 20 cm, about 4 cm ~ about 18 cm, about 4 cm ~ about 16 cm, about 4 cm ~ about 14 cm, about 4 cm ~ about 12 cm, about 4 cm ~ about 10 cm, about 4 cm ~ about 9 cm, about 4 cm to about 8 cm, about 4 cm to about 7 cm, about 4 cm to about 6 cm, about 4 cm to about 5 cm, about 5 cm to about 30 cm, about 5 cm to about 28 cm , About 5 cm to about 26 cm, about 5 cm to about 24 cm, about 5 cm to about 22 cm, about 5 cm to about 20 cm, about 5 cm to about 18 cm, about 5 cm to about 16 cm, about 5 cm to about 14 cm, about 5 cm to about 12 cm, about 5 cm to about 10 cm, about 5 cm to about 9 cm, about 5 cm to about 8 cm, about 5 cm to about 7 cm, about 5 cm ~ About 6 cm, about 6 cm ~ about 30 cm, about 6 cm ~ about 28 cm, about 6 cm ~ about 26 cm, about 6 cm ~ about 24 cm, about 6 cm ~ about 22 cm, about 6 cm ~ about 20 cm, about 6 cm to about 18 cm, about 6 cm to about 16 cm, about 6 cm to about 14 cm, about 6 cm to about 12 cm, about 6 cm to about 10 cm, about 6 cm to about 9 cm , About 6 cm to about 8 cm, about 6 cm to about 7 cm, about 7 cm to about 30 cm, about 7 cm to about 28 cm, about 7 cm to about 26 cm, about 7 cm to about 24 cm, about 7 cm to about 22 cm, about 7 cm to about 20 cm, about 7 cm to about 18 cm, about 7 cm to about 16 cm , About 7 cm to about 14 cm, about 7 cm to about 12 cm, about 7 cm to about 10 cm, about 7 cm to about 9 cm, about 7 cm to about 8 cm, about 8 cm to about 30 cm, about 8 cm to about 28 cm, about 8 cm to about 26 cm, about 8 cm to about 24 cm, about 8 cm to about 22 cm, about 8 cm to about 20 cm, about 8 cm to about 18 cm, about 8 cm ~ About 16 cm, about 8 cm ~ about 14 cm, about 8 cm ~ about 12 cm, about 8 cm ~ about 10 cm, about 8 cm ~ about 9 cm, about 9 cm ~ about 30 cm, about 9 cm ~ about 28 cm, about 9 cm to about 26 cm, about 9 cm to about 24 cm, about 9 cm to about 22 cm, about 9 cm to about 20 cm, about 9 cm to about 18 cm, about 9 cm to about 16 cm , About 9 cm to about 14 cm, about 9 cm to about 12 cm, about 9 cm to about 10 cm, about 10 cm to about 30 cm, about 10 cm to about 28 cm, about 10 cm to about 26 cm, about 10 cm to about 24 cm, about 10 cm to about 22 cm, about 10 cm to about 20 cm, about 10 cm to about 18 cm, about 10 cm to about 16 cm, about 10 cm to about 14 cm, about 10 cm ~ About 12 cm, about 12 cm ~ about 30 cm, about 12 cm ~ about 28 cm, about 12 cm ~ about 26 cm, about 12 cm ~ about 24 cm, about 12 cm ~ about 22 cm, about 12 cm ~ about 20 cm, about 12 cm to about 18 cm, about 12 cm to about 16 cm, about 12 cm to about 14 cm, about 14 cm to about 30 cm, about 14 cm to about 28 cm, about 14 cm to about 26 cm , About 14 cm to about 24 cm, about 14 cm to about 22 cm, about 14 cm to about 20 cm, about 14 cm to about 18 cm, about 14 cm to about 16 cm, about 16 cm to about 30 cm, about 16 cm to about 28 cm, about 16 cm to about 26 cm, about 16 cm to about 24 cm, about 16 cm to about 22 cm, about 16 cm to about 20 cm, about 16 cm to about 18 cm, about 18 cm ~ About 30 cm, about 18 cm ~ about 28 cm, about 18 cm ~ about 26 cm, about 18 cm ~ about 24 cm, about 18 cm ~ about 22 cm, about 18 cm ~ about 20 cm, about 20 cm ~ about 30 cm, about 20 cm to about 28 cm, about 20 cm to about 26 cm, about 20 cm to about 24 cm, about 20 cm to about 22 cm, about 22 cm to about 30 cm, about 22 cm to about 28 cm, about 22 cm to about 26 cm, about 22 cm to about 24 cm, about 24 cm to about 30 cm, about 24 cm to about 28 cm, about 24 cm to about 26 cm It has a diameter or average diameter of about 26 cm to about 30 cm, about 26 cm to about 28 cm, or about 28 cm to about 30 cm.

In some embodiments, the fluid conduit in the inlet system is about 0.1 mm to about 10 mm, about 0.1 mm to about 9.5 mm, about 0.1 mm to about 9.0 mm, about 0.1 mm to about 9.0 mm, about 0.1 mm to. About 8.5 mm, about 0.1 mm to about 8.0 mm, about 0.1 mm to about 7.5 mm, about 0.1 mm to about 7.0 mm, about 0.1 mm to about 6.5 mm, about 0.1 mm to about 6.0 mm, about 0.1 mm to about 5.5 mm, about 0.1 mm to about 5.0 mm, about 0.1 mm to about 4.5 mm, about 0.1 mm to about 4.0 mm, about 0.1 mm to about 3.5 mm, about 0.1 mm to about 3.0 mm, about 0.1 mm to about 2.5 mm, About 0.1 mm to about 2.0 mm, about 0.1 mm to about 1.5 mm, about 0.1 mm to about 1.0 mm, about 0.1 mm to about 0.5 mm, about 0.5 mm to about 10 mm, about 0.5 mm to about 9.5 mm, about 0.5 mm ~ about 9.0 mm, about 0.5 mm ~ about 9.0 mm, about 0.5 mm ~ about 8.5 mm, about 0.5 mm ~ about 8.0 mm, about 0.5 mm ~ about 7.5 mm, about 0.5 mm ~ about 7.0 mm, about 0.5 mm ~ About 6.5 mm, about 0.5 mm to about 6.0 mm, about 0.5 mm to about 5.5 mm, about 0.5 mm to about 5.0 mm, about 0.5 mm to about 4.5 mm, about 0.5 mm to about 4.0 mm, about 0.5 mm to about 3.5 mm, about 0.5 mm to about 3.0 mm, about 0.5 mm to about 2.5 mm, about 0.5 mm to about 2.0 mm, about 0.5 mm to about 1.5 mm, about 0.5 mm to about 1.0 mm, about 1.0 mm to about 10 mm, About 1.0 mm to about 9.5 mm, about 1.0 mm to about 9.0 mm, about 1.0 mm to about 9.0 mm, about 1.0 mm to about 8.5 mm, about 1.0 mm to about 8.0 mm, about 1.0 mm to about 7.5 mm, about 1.0 mm ~ about 7.0 mm, about 1.0 mm ~ about 6.5 mm, about 1.0 mm ~ about 6.0 mm, about 1.0 mm ~ about 5.5 mm, about 1.0 mm ~ about 5.0 mm, about 1.0 mm ~ about 4.5 mm, about 1.0 mm ~ It has an inner diameter of about 4.0 mm, about 1.0 mm to about 3.5 mm, about 1.0 mm to about 3.0 mm, about 1.0 mm to about 2.5 mm, about 1.0 mm to about 2.0 mm, or about 1.0 mm to about 1.5 mm.

In some aspects of these kits, the pod contains a chromatography resin (eg, any type of chromatography resin described herein), and the inlet system and pod are stable with the inlet system and pod. When installed, the bottom edge of the inlet system is approximately 1 mm to approximately 30 mm from the chromatography resin (eg, approximately 1 mm to approximately 25 mm, approximately 1 mm to approximately 20 mm, approximately 1 mm to approximately 15 mm, approximately 1 mm). ~ About 10 mm, about 1 mm ~ about 5 mm). In some aspects of these kits, the pod may contain any volume of chromatographic resin described herein.

In some aspects of these kits, the inlet system has a generally conical shape. In some aspects of these kits, the outlet system has a generally conical shape. Non-limiting examples of a kit containing a pod, an inlet system, and an outlet system are shown in FIGS. 27 (unassembled) and 28 (assembled). Non-limiting examples of other kits described herein are shown in FIGS. 1A-1E, 3, 23-26, and 35.

Some aspects of these kits are (d) adapters with upper and lower ends, the upper end of the adapter being configured to be stably attached to the lower end of the pod, and one or more lower ends of the adapter. When the upper end of the adapter has blades and is stably attached to the lower end of the pod, the positioning of the one or more blades (eg, liquid reversibly flows from the outside of the pod into the inside of the pod). It may further include an adapter that facilitates the flow of fluid entering the pod (through a material that allows it). A non-limiting example of such an adapter is shown in Figure 34. A diagram showing an exemplary adapter attached to an exemplary pod is shown in FIG.

In some aspects of these kits, the inner surface of the pod and the top edge of the adapter are configured to allow stable attachment of the pod to the top edge of the adapter. In some embodiments of these kits, the inner surface of the pod and the top edge of the adapter form a seal (eg, a fluid-tight seal) when the adapter and pod are stably attached. In some aspects of these kits, the inner surface of the pod and the top edge of the adapter include threading that allows stable attachment of the pod to the top edge of the adapter. In some aspects of these kits, threading on the inner surface of the pod, or threading on the top edge of the adapter, comprises an O-ring. Other means for providing a stable connection between the inner surface of the pod and the upper end of the adapter are known in the art.

Some aspects of any of the kits described herein may further include instructions for performing any of the methods described herein.

Method A method of capturing recombinant protein produced by liquid cell culture, comprising contacting any of the pods provided herein with liquid cell culture for a period of time. The method is described herein under conditions where at least a portion of the at least one area results in contact with a liquid cell culture medium, and the contact results in the capture of recombinant protein by a chromatographic resin. Provide to the book.

A method of purifying a recombinant protein produced by liquid cell culture, wherein (a) any of the pods provided herein is in contact with the liquid cell culture for a period of time, at least one of the above. The steps are carried out under conditions that result in contact of at least a portion of one area with liquid cell culture, and that contact results in the capture of recombinant protein by a chromatographic resin; and (b) said. Also provided herein is a method comprising eluting the recombinant protein from the chromatographic resin after a period of time, thereby purifying the recombinant protein. In some embodiments, elution (and optional wash steps) may be performed using a chromatographic system (eg, AKTA system).

In some embodiments of any of the methods described herein, the pod is a chromatographic resin placed inside the container (eg, any chromatographic resin described herein or known in the art). ), Whichever pod described herein. In some embodiments, the pods are arranged such that the vertical axis of the pods is placed approximately horizontally (eg, horizontally) in the liquid cell culture medium. In some embodiments, the pods are arranged such that the vertical axis of the pods is placed approximately vertically (eg, vertically) in the liquid cell culture medium. In some embodiments, the pod comprises a chromatographic resin that is a commercially available chromatographic resin. In some embodiments, the chromatography resin is an affinity chromatography resin (eg, protein A resin, protein G resin, protein L resin, nickel resin, or magnetic bead resin). In some embodiments, the pod or chromatographic resin may be frozen or stored for a period of time after the contact step and before the recombinant protein is eluted from the chromatographic resin in the pod. In some embodiments, the pod or chromatographic resin is frozen or stored for a period of time, the pod or chromatographic resin is used for a period of time after the contact step and after the pod or chromatographic resin has been contacted with the wash buffer. It may be frozen or stored.

In some embodiments, the stage of contact is at least a portion of the at least one area (eg, at least 10% of the at least one area, at least 15% of the at least one area, at least 20 of the at least one area. %, At least 25% of the at least one area, at least 30% of the at least one area, at least 35% of the at least one area, at least 40% of the at least one area, at least 45 of the at least one area %, At least 50% of the at least one area, at least 55% of the at least one area, at least 60% of the at least one area, at least 65% of the at least one area, at least 70 of the at least one area %, At least 75% of the at least one area, at least 80% of the at least one area, at least 85% of the at least one area, at least 90% of the at least one area, at least 95 of the at least one area %, At least 97% of the at least one area, at least 98% of the at least one area, at least 99% of the at least one area) results in contact with the liquid cell culture medium. In some embodiments, at least 20% of the total surface area of the at least one area (eg, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88% , At least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) Contact.

In some embodiments, the contact is performed under conditions that result in the capture of the recombinant protein by the chromatographic resin. In some embodiments, capture is up to 60% to 100% of the binding capacity of the chromatographic resin (eg, up to 60% to 70%, up to 60% to 75%, up to 60% to 80%, up to 60% to 85). %, Maximum 60% -90%, Maximum 60% -95%, Maximum 65% -70%, Maximum 65% -75%, Maximum 65% -80%, Maximum 65% -85%, Maximum 65% -90% , Maximum 65% -95%, Maximum 65% -100%, Maximum 70% -75%, Maximum 70% -80%, Maximum 70% -85%, Maximum 70% -90%, Maximum 70% -95%, Maximum 70% -100%, Maximum 75% -80%, Maximum 75% -85%, Maximum 75% -90%, Maximum 75% -95%, Maximum 75% -100%, Maximum 80% -85%, Maximum 80% -90%, up to 80% -95%, up to 80% -100%, up to 85% -90%, up to 85% -95%, up to 85% -100%, up to 90% -95%, up to 95 % To 100%, up to 50% to 55%, up to 50% to 60%, up to 50% to 65%, up to 50% to 70%, up to 50% to 75%, up to 50% to 80%, up to 50% ~ 85%, up to 50% ~ 90%, up to 50% ~ 95%, up to 50% ~ 100%, up to 55% -60%, up to 55% ~ 65%, up to 55% ~ 70%, up to 55% ~ 75%, up to 55% -80%, up to 55% -85%, up to 55% -90%, up to 55% -95%, up to 55% -100%, up to 50%, up to 51%, up to 52%, Up to 53%, up to 54%, up to 55%, up to 56%, up to 57%, up to 58%, up to 59%, up to 60%, up to 61%, up to 62%, up to 63%, up to 64%, up to 65 %, Max 66%, Max 67%, Max 68%, Max 69%, Max 70%, Max 71%, Max 72%, Max 73%, Max 74%, Max 75%, Max 76%, Max 77%, Up to 78%, up to 79%, up to 80%, up to 81%, up to 82%, up to 83%, up to 84%, up to 85%, up to 86%, up to 87%, up to 88%, up to 89%, up to 90 Recombinant protein binds to levels of%, up to 91%, up to 92%, up to 93%, up to 94%, up to 95%, up to 96%, up to 97%, up to 98%, up to 99%, or 100%) It may result in being done.

In some embodiments, capture is about 0.05 mg to about 80 mg (eg, about 0.05 mg to about 0.1 mg, about 0.05 mg to about 0.2 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.6 mg). , About 0.05 mg to about 0.8 mg, about 0.05 mg to about 1 mg, about 0.05 mg to about 2 mg, about 0.05 mg to about 5 mg, about 0.05 mg to about 10 mg, about 0.05 mg to about 15 mg, about 0.05 mg to about 20 mg, about 0.05 mg to about 25 mg, about 0.05 mg to about 30 mg, about 0.05 mg to about 35 mg, about 0.05 mg to about 40 mg, about 0.05 mg to about 45 mg, about 0.05 mg ~ About 50 mg, about 0.05 mg ~ about 55 mg, about 0.05 mg ~ about 60 mg, about 0.05 mg ~ about 65 mg, about 0.05 mg ~ about 70 mg, about 0.05 mg ~ about 75 mg, about 0.05 mg ~ about 80 mg, about 0.1 mg to about 0.2 mg, about 0.1 mg to about 0.5 mg, about 0.1 mg to about 0.6 mg, about 0.1 mg to about 0.8 mg, about 0.1 mg to about 1 mg, about 0.1 mg to about 2 mg , About 0.1 mg to about 5 mg, about 0.1 mg to about 10 mg, about 0.1 mg to about 15 mg, about 0.1 mg to about 20 mg, about 0.1 mg to about 25 mg, about 0.1 mg to about 30 mg, about 0.1 mg to about 35 mg, about 0.1 mg to about 40 mg, about 0.1 mg to about 45 mg, about 0.1 mg to about 50 mg, about 0.1 mg to about 55 mg, about 0.1 mg to about 60 mg, about 0.1 mg ~ About 65 mg, about 0.1 mg ~ about 70 mg, about 0.1 mg ~ about 75 mg, about 0.1 mg ~ about 80 mg, about 0.2 mg ~ about 0.5 mg, about 0.2 mg ~ about 0.6 mg, about 0.2 mg ~ about 0.8 mg, about 0.2 mg to about 1 mg, about 0.2 mg to about 2 mg, about 0.2 mg to about 5 mg, about 0.2 mg to about 10 mg, about 0.2 mg to about 15 mg, about 0.2 mg to about 20 mg , About 0.2 mg to about 25 mg, about 0.2 mg to about 30 mg, about 0.2 mg to about 35 mg, about 0.2 mg to about 40 mg, about 0.2 mg to about 45 mg, about 0.2 mg to about 50 mg, about 0.2 mg ~ about 55 mg, about 0.2 mg ~ about 60 mg, about 0.2 mg ~ About 65 mg, about 0.2 mg to about 70 mg, about 0.2 mg to about 75 mg, about 0.2 mg to about 80 mg, about 0.5 mg to about 0.6 mg, about 0.5 mg to about 0.8 mg, about 0.5 mg to about 1 mg, about 0.5 mg to about 2 mg, about 0.5 mg to about 5 mg, about 0.5 mg to about 10 mg, about 0.5 mg to about 15 mg, about 0.5 mg to about 20 mg, about 0.5 mg to about 25 mg, About 0.5 mg to about 30 mg, about 0.5 mg to about 35 mg, about 0.5 mg to about 40 mg, about 0.5 mg to about 45 mg, about 0.5 mg to about 50 mg, about 0.5 mg to about 55 mg, about 0.5 mg ~ about 60 mg, about 0.5 mg ~ about 65 mg, about 0.5 mg ~ about 70 mg, about 0.5 mg ~ about 75 mg, about 0.5 mg ~ about 80 mg, about 0.6 mg ~ about 0.8 mg, about 0.6 mg ~ About 1 mg, about 0.6 mg to about 2 mg, about 0.6 mg to about 5 mg, about 0.6 mg to about 10 mg, about 0.6 mg to about 15 mg, about 0.6 mg to about 20 mg, about 0.6 mg to about 25 mg, about 0.6 mg to about 30 mg, about 0.6 mg to about 35 mg, about 0.6 mg to about 40 mg, about 0.6 mg to about 45 mg, about 0.6 mg to about 50 mg, about 0.6 mg to about 55 mg, About 0.6 mg to about 60 mg, about 0.6 mg to about 65 mg, about 0.6 mg to about 70 mg, about 0.6 mg to about 75 mg, about 0.6 mg to about 80 mg, about 0.8 mg to about 1 mg, about 0.8 mg ~ about 2 mg, about 0.8 mg ~ about 5 mg, about 0.8 mg ~ about 10 mg, about 0.8 mg ~ about 15 mg, about 0.8 mg ~ about 20 mg, about 0.8 mg ~ about 25 mg, about 0.8 mg ~ About 30 mg, about 0.8 mg to about 35 mg, about 0.8 mg to about 40 mg, about 0.8 mg to about 45 mg, about 0.8 mg to about 50 mg, about 0.8 mg to about 55 mg, about 0.8 mg to about 60 mg, about 0.8 mg to about 65 mg, about 0.8 mg to about 70 mg, about 0.8 mg to about 75 mg, about 0.8 mg to about 80 mg, about 1 mg to about 2 mg, about 1 mg to about 5 mg, About 1 mg to about 10 mg, about 1 mg to about 15 mg, about 1 mg to about 20 mg, about 1 mg to about 25 mg, about 1 mg to about 30 mg, about 1 mg to about 35 mg, about 1 mg to about 40 mg, about 1 mg to about 45 mg, about 1 mg to about 50 mg, about 1 mg to about 55 mg, about 1 mg to about 60 mg, about 1 mg to about 65 mg, About 1 mg to about 70 mg, about 1 mg to about 75 mg, about 1 mg to about 80 mg, about 2 mg to about 5 mg, about 2 mg to about 10 mg, about 2 mg to about 15 mg, about 2 mg ~ about 20 mg, about 2 mg ~ about 25 mg, about 2 mg ~ about 30 mg, about 2 mg ~ about 35 mg, about 2 mg ~ about 40 mg, about 2 mg ~ about 45 mg, about 2 mg ~ About 50 mg, about 2 mg to about 55 mg, about 2 mg to about 60 mg, about 2 mg to about 65 mg, about 2 mg to about 70 mg, about 2 mg to about 75 mg, about 2 mg to about 80 mg, about 5 mg to about 10 mg, about 5 mg to about 15 mg, about 5 mg to about 20 mg, about 5 mg to about 25 mg, about 5 mg to about 30 mg, about 5 mg to about 35 mg, About 5 mg to about 40 mg, about 5 mg to about 45 mg, about 5 mg to about 50 mg, about 5 mg to about 55 mg, about 5 mg to about 60 mg, about 5 mg to about 65 mg, about 5 mg ~ about 70 mg, about 5 mg ~ about 75 mg, about 5 mg ~ about 80 mg, about 10 mg ~ about 15 mg, about 10 mg ~ about 20 mg, about 10 mg ~ about 25 mg, about 10 mg ~ About 30 mg, about 10 mg to about 35 mg, about 10 mg to about 40 mg, about 10 mg to about 45 mg, about 10 mg to about 50 mg, about 10 mg to about 55 mg, about 10 mg to about 60 mg, about 10 mg to about 65 mg, about 10 mg to about 70 mg, about 10 mg to about 75 mg, about 10 mg to about 80 mg, about 15 mg to about 20 mg, about 15 mg to about 20 mg, About 15 mg to about 25 mg, about 15 mg to about 30 mg, about 15 mg to about 35 mg, about 15 mg to about 40 mg, about 15 mg to about 45 mg, about 15 mg to about 50 mg, about 15 mg ~ about 55 mg, about 15 mg ~ about 60 mg, about 15 mg ~ about 65 mg, about 15 mg ~ about 70 mg, about 15 mg ~ about 75 mg, about 15 mg ~ about 80 mg, about 20 mg ~ About 25 mg, about 20 mg to about 30 mg, about 20 mg to about 35 mg, about 20 mg to about 40 mg, about 20 mg to about 45 mg, about 20 mg to about 50 mg, about 2 0 mg to about 55 mg, about 20 mg to about 60 mg, about 20 mg to about 65 mg, about 20 mg to about 70 mg, about 20 mg to about 75 mg, about 20 mg to about 80 mg, about 25 mg ~ About 30 mg, about 25 mg ~ about 35 mg, about 25 mg ~ about 40 mg, about 25 mg ~ about 45 mg, about 25 mg ~ about 50 mg, about 25 mg ~ about 55 mg, about 25 mg ~ about 60 mg, about 25 mg to about 65 mg, about 25 mg to about 70 mg, about 25 mg to about 75 mg, about 25 mg to about 80 mg, about 30 mg to about 35 mg, about 30 mg to about 40 mg , About 30 mg to about 45 mg, about 30 mg to about 50 mg, about 30 mg to about 55 mg, about 30 mg to about 60 mg, about 30 mg to about 65 mg, about 30 mg to about 70 mg, about 30 mg to about 75 mg, about 30 mg to about 80 mg, about 35 mg to about 40 mg, about 35 mg to about 45 mg, about 35 mg to about 50 mg, about 35 mg to about 55 mg, about 35 mg ~ About 60 mg, about 35 mg ~ about 65 mg, about 35 mg ~ about 70 mg, about 35 mg ~ about 75 mg, about 35 mg ~ about 80 mg, about 40 mg ~ about 45 mg, about 40 mg ~ about 50 mg, about 45 mg to about 55 mg, about 45 mg to about 60 mg, about 45 mg to about 65 mg, about 45 mg to about 70 mg, about 45 mg to about 75 mg, about 45 mg to about 80 mg , About 50 mg to about 55 mg, about 50 mg to about 60 mg, about 50 mg to about 65 mg, about 50 mg to about 70 mg, about 50 mg to about 75 mg, about 50 mg to about 80 mg, about 55 mg to about 60 mg, about 55 mg to about 65 mg, about 55 mg to about 70 mg, about 55 mg to about 75 mg, about 55 mg to about 80 mg, about 60 mg to about 65 mg, about 60 mg ~ About 70 mg, about 60 mg ~ about 75 mg, about 60 mg ~ about 80 mg, about 65 mg ~ about 70 mg, about 65 mg ~ about 75 mg, about 65 mg ~ about 80 mg, about 70 mg ~ about 75 mg, about 70 mg to about 80 mg, about 75 mg to about 80 mg) of recombinant protein may result in capture on the chromatographic resin.

In some embodiments, the purity of capture, as determined by analytical size exclusion chromatography (SEC), is at least 45% (eg, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, At least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63 %, At least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, At least 76%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89 %, At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.2%, at least 99.4%, At least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%).

Duration In some embodiments of any of the methods provided herein, the step of contacting any of the pods provided herein with liquid cell culture is for a period of time (eg, about 30 seconds to about 2 weeks). ). In some embodiments of any of the methods provided herein, the step of elution of the recombinant protein from the chromatographic resin is after a period of time (eg, about 30 seconds to about 2 weeks).

In some embodiments, the duration of the contact and / or elution of the recombinant protein from the chromatographic resin is about 30 seconds to about 1 minute, about 30 seconds to 45 seconds, about 30 seconds to about 90 seconds, about. 30 seconds to about 2 minutes, about 30 seconds to about 150 seconds, about 30 seconds to about 3 minutes, about 30 seconds to about 210 seconds, about 30 seconds to about 4 minutes, about 30 seconds to about 270 seconds, about 30 seconds ~ About 5 minutes, about 1 minute ~ about 2 minutes, about 1 minute ~ about 3 minutes, about 1 minute ~ about 4 minutes, about 1 minute ~ about 5 minutes, about 2 minutes ~ about 3 minutes, about 2 minutes ~ about 4 minutes, about 2 to about 5 minutes, about 3 minutes to about 4 minutes, about 3 minutes to about 5 minutes, about 4 minutes to about 5 minutes, about 5 minutes to about 2 weeks, about 5 minutes to about 168 hours, About 5 minutes to about 162 hours, about 5 minutes to about 156 hours, about 5 minutes to about 150 hours, about 5 minutes to about 144 hours, about 5 minutes to about 138 hours, about 5 minutes to about 132 hours, about 5 Minutes to about 126 hours, about 5 minutes to about 120 hours, about 5 minutes to about 114 hours, about 5 minutes to about 108 hours, about 5 minutes to about 102 hours, about 5 minutes to about 96 hours, about 5 minutes About 90 hours, about 5 minutes to about 84 hours, about 5 minutes to about 78 hours, about 5 minutes to about 72 hours, about 5 minutes to about 66 hours, about 5 minutes to about 60 hours, about 5 minutes to about 54 Time, about 5 minutes to about 48 hours, about 5 minutes to about 42 hours, about 5 minutes to about 36 hours, about 5 minutes to about 30 hours, about 5 minutes to about 24 hours, about 5 minutes to about 18 hours, About 5 minutes to about 12 hours, about 5 minutes to about 6 hours, about 5 minutes to about 5 hours, about 5 minutes to about 240 minutes, about 5 minutes to about 210 minutes, about 5 minutes to about 180 minutes, about 5 Minutes to about 150 minutes, about 5 minutes to about 120 minutes, about 5 minutes to about 90 minutes, about 5 minutes to about 60 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 20 minutes, about 5 minutes About 15 minutes, about 5 minutes to about 10 minutes; about 10 minutes to about 2 weeks, about 10 minutes to about 168 hours, about 10 minutes to about 162 hours, about 10 minutes to about 156 hours, about 10 minutes to about 150 Time, about 10 minutes to about 144 hours, about 10 minutes to about 138 hours, about 10 minutes to about 132 hours, about 10 minutes to about 126 hours, about 10 minutes to about 120 hours, about 10 minutes to about 114 hours, About 10 minutes to about 108 hours, about 10 minutes to about 102 hours, about 10 minutes to about 96 hours, about 10 minutes to about 90 hours, about 10 minutes to about 84 hours, about 10 minutes to about 78 hours, about 10 Minutes to about 72 hours, about 10 minutes to about 66 hours, about 10 minutes to about 60 hours, about 10 minutes to about 54 hours, About 10 minutes to about 48 hours, about 10 minutes to about 42 hours, about 10 minutes to about 36 hours, about 10 minutes to about 30 hours, about 10 minutes to about 24 hours, about 10 minutes to about 18 hours, about 10 Minutes to about 12 hours, about 10 minutes to about 6 hours, about 10 minutes to about 5 hours, about 10 minutes to about 240 minutes, about 10 minutes to about 210 minutes, about 10 minutes to about 180 minutes, about 10 minutes About 150 minutes, about 10 minutes to about 120 minutes, about 10 minutes to about 90 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 15 minutes, about 10 minutes to about 20 Minutes, about 10 minutes to about 25 minutes; about 20 minutes to about 2 weeks, about 20 minutes to about 168 hours, about 20 minutes to about 162 hours, about 20 minutes to about 156 hours, about 20 minutes to about 150 hours, About 20 minutes to about 144 hours, about 20 minutes to about 138 hours, about 20 minutes to about 132 hours, about 20 minutes to about 126 hours, about 20 minutes to about 120 hours, about 20 minutes to about 114 hours, about 20 Minutes to about 108 hours, about 20 minutes to about 102 hours, about 20 minutes to about 96 hours, about 20 minutes to about 90 hours, about 20 minutes to about 84 hours, about 20 minutes to about 78 hours, about 20 minutes About 72 hours, about 20 minutes to about 66 hours, about 20 minutes to about 60 hours, about 20 minutes to about 54 hours, about 20 minutes to about 48 hours, about 20 minutes to about 42 hours, about 20 minutes to about 36 Time, about 20 minutes to about 30 hours, about 20 minutes to about 24 hours, about 20 minutes to about 18 hours, about 20 minutes to about 12 hours, about 20 minutes to about 6 hours, about 20 minutes to about 5 hours, About 20 minutes to about 240 minutes, about 20 minutes to about 210 minutes, about 20 minutes to about 180 minutes, about 20 minutes to about 150 minutes, about 20 minutes to about 120 minutes, about 20 minutes to about 90 minutes, about 20 Minutes to about 60 minutes, about 20 minutes to about 30 minutes, about 20 minutes to about 25 minutes; about 30 minutes to about 2 weeks, about 30 minutes to about 168 hours, about 30 minutes to about 162 hours, about 30 minutes About 156 hours, about 30 minutes to about 150 hours, about 30 minutes to about 144 hours, about 30 minutes to about 138 hours, about 30 minutes to about 132 hours, about 30 minutes to about 126 hours, about 30 minutes to about 120 Time, about 30 minutes to about 114 hours, about 30 minutes to about 108 hours, about 30 minutes to about 102 hours, about 30 minutes to about 96 hours, about 30 minutes to about 90 hours, about 30 minutes to about 84 hours, About 30 minutes to about 78 hours, about 30 minutes to about 72 hours, about 30 minutes to about 66 hours, about 30 minutes to about 60 hours, about 30 minutes to about 54 hours, about 30 minutes to about 48 hours, About 30 minutes to about 42 hours, about 30 minutes to about 36 hours, about 30 minutes to about 30 hours, about 30 minutes to about 24 hours, about 30 minutes to about 18 hours, about 30 minutes to about 12 hours, about 30 Minutes to about 6 hours, about 30 minutes to about 5 hours, about 30 minutes to about 240 minutes, about 30 minutes to about 210 minutes, about 30 minutes to about 180 minutes, about 30 minutes to about 150 minutes, about 30 minutes About 120 minutes, about 30 minutes to about 90 minutes, about 30 minutes to about 60 minutes; about 40 minutes to about 2 weeks, about 40 minutes to about 168 hours, about 40 minutes to about 162 hours, about 40 minutes to about 156 Time, about 40 minutes to about 150 hours, about 40 minutes to about 144 hours, about 40 minutes to about 138 hours, about 40 minutes to about 132 hours, about 40 minutes to about 126 hours, about 40 minutes to about 120 hours, About 40 minutes to about 114 hours, about 40 minutes to about 108 hours, about 40 minutes to about 102 hours, about 40 minutes to about 96 hours, about 40 minutes to about 90 hours, about 40 minutes to about 84 hours, about 40 Minutes to about 78 hours, about 40 minutes to about 72 hours, about 40 minutes to about 66 hours, about 40 minutes to about 60 hours, about 40 minutes to about 54 hours, about 40 minutes to about 48 hours, about 40 minutes About 42 hours, about 40 minutes to about 36 hours, about 40 minutes to about 30 hours, about 40 minutes to about 24 hours, about 40 minutes to about 18 hours, about 40 minutes to about 12 hours, about 40 minutes to about 6 Time, about 40 minutes to about 5 hours, about 40 minutes to about 240 minutes, about 40 minutes to about 210 minutes, about 40 minutes to about 180 minutes, about 40 minutes to about 150 minutes, about 40 minutes to about 120 minutes, About 40 minutes to about 90 minutes, about 40 minutes to about 60 minutes, about 40 minutes to about 50 minutes, about 40 minutes to about 45 minutes; about 45 minutes to about 2 weeks, about 45 minutes to about 168 hours, about 45 Minutes to about 162 hours, about 45 minutes to about 156 hours, about 45 minutes to about 150 hours, about 45 minutes to about 144 hours, about 45 minutes to about 138 hours, about 45 minutes to about 132 hours, about 45 minutes About 126 hours, about 45 minutes to about 120 hours, about 45 minutes to about 114 hours, about 45 minutes to about 108 hours, about 45 minutes to about 102 hours, about 45 minutes to about 96 hours, about 45 minutes to about 90 Time, about 45 minutes to about 84 hours, about 45 minutes to about 78 hours, about 45 minutes to about 72 hours, about 45 minutes to about 66 hours, about 45 minutes to about 60 hours, about 45 minutes to about 54 hours, About 45 minutes to about 48 hours, about 45 minutes to about 42 hours, about 45 minutes to about 36 hours, about 45 minutes to about 30 hours, about 45 minutes to about 24 hours, about 45 minutes to about 18 hours, About 45 minutes to about 12 hours, about 45 minutes to about 6 hours, about 45 minutes to about 5 hours, about 45 minutes to about 240 minutes, about 45 minutes to about 210 minutes, about 45 minutes to about 180 minutes, about 45 Minutes-about 150 minutes, about 45 minutes-about 120 minutes, about 45 minutes-about 90 minutes, about 45 minutes-about 60 minutes, about 45 minutes-about 50 minutes, about 45 minutes-about 55 minutes; about 60 minutes- About 2 weeks, about 60 minutes to about 168 hours, about 60 minutes to about 162 hours, about 60 minutes to about 156 hours, about 60 minutes to about 150 hours, about 60 minutes to about 144 hours, about 60 minutes to about 138 Time, about 60 minutes to about 132 hours, about 60 minutes to about 126 hours, about 60 minutes to about 120 hours, about 60 minutes to about 114 hours, about 60 minutes to about 108 hours, about 60 minutes to about 102 hours, About 60 minutes to about 96 hours, about 60 minutes to about 90 hours, about 60 minutes to about 84 hours, about 60 minutes to about 78 hours, about 60 minutes to about 72 hours, about 60 minutes to about 66 hours, about 60 Minutes to about 60 hours, about 60 minutes to about 54 hours, about 60 minutes to about 48 hours, about 60 minutes to about 42 hours, about 60 minutes to about 36 hours, about 60 minutes to about 30 hours, about 60 minutes About 24 hours, about 60 minutes to about 18 hours, about 60 minutes to about 12 hours, about 60 minutes to about 6 hours, about 60 minutes to about 5 hours, about 60 minutes to about 240 minutes, about 60 minutes to about 210 Minutes, about 60 minutes to about 180 minutes, about 60 minutes to about 150 minutes, about 60 minutes to about 120 minutes, about 60 minutes to about 90 minutes, about 60 minutes to about 85 minutes, about 60 minutes to about 80 minutes, About 60 minutes to about 75 minutes, about 60 minutes to about 70 minutes; about 70 minutes to about 2 weeks, about 70 minutes to about 168 hours, about 70 minutes to about 162 hours, about 70 minutes to about 156 hours, about 70 Minutes to about 150 hours, about 70 minutes to about 144 hours, about 70 minutes to about 138 hours, about 70 minutes to about 132 hours, about 70 minutes to about 126 hours, about 70 minutes to about 120 hours, about 70 minutes About 114 hours, about 70 minutes to about 108 hours, about 70 minutes to about 102 hours, about 70 minutes to about 96 hours, about 70 minutes to about 90 hours, about 70 minutes to about 84 hours, about 70 minutes to about 78 Time, about 70 minutes to about 72 hours, about 70 minutes to about 66 hours, about 70 minutes to about 60 hours, about 70 minutes to about 54 hours, about 70 minutes to about 48 hours, about 70 minutes to about 42 hours, About 70 minutes to about 36 hours, about 70 minutes to about 30 hours, about 70 minutes to about 24 hours, about 70 minutes to about 18 hours, about 70 minutes to about 12 hours, about 70 minutes to about 6 hours, about 70 minutes to about 5 hours, about 70 minutes to about 240 minutes, about 70 minutes to about 210 minutes, about 70 minutes to about 180 minutes, about 70 minutes to about 150 minutes, about 70 minutes to about 120 minutes, about 70 minutes ~ About 90 minutes, about 70 minutes ~ about 85 minutes, about 70 minutes ~ about 80 minutes; about 75 minutes ~ about 80 minutes, about 75 minutes ~ about 85 minutes, about 75 minutes ~ about 90 minutes; about 90 minutes ~ about 2 weeks, about 90 minutes to about 168 hours, about 90 minutes to about 162 hours, about 90 minutes to about 156 hours, about 90 minutes to about 150 hours, about 90 minutes to about 144 hours, about 90 minutes to about 138 hours , About 90 minutes to about 132 hours, about 90 minutes to about 126 hours, about 90 minutes to about 120 hours, about 90 minutes to about 114 hours, about 90 minutes to about 108 hours, about 90 minutes to about 102 hours, about 90 minutes to about 96 hours, about 90 minutes to about 90 hours, about 90 minutes to about 84 hours, about 90 minutes to about 78 hours, about 90 minutes to about 72 hours, about 90 minutes to about 66 hours, about 90 minutes ~ About 60 hours, about 90 minutes ~ about 54 hours, about 90 minutes ~ about 48 hours, about 90 minutes ~ about 42 hours, about 90 minutes ~ about 36 hours, about 90 minutes ~ about 30 hours, about 90 minutes ~ about 24 hours, about 90 minutes to about 18 hours, about 90 minutes to about 12 hours, about 90 minutes to about 6 hours, about 90 minutes to about 5 hours, about 90 minutes to about 240 minutes, about 90 minutes to about 210 minutes , About 90 minutes to about 180 minutes, about 90 minutes to about 150 minutes, about 90 minutes to about 120 minutes, about 90 minutes to about 100 minutes, about 90 minutes to about 110 minutes, about 100 minutes to about 110 minutes, about 100 minutes to about 120 minutes; about 120 minutes to about 2 weeks, about 120 minutes to about 168 hours, about 120 minutes to about 162 hours, about 120 minutes to about 156 hours, about 120 minutes to about 150 hours, about 120 minutes ~ About 144 hours, about 120 minutes ~ about 138 hours, about 120 minutes ~ about 132 hours, about 120 minutes ~ about 126 hours, about 120 minutes ~ about 120 hours, about 120 minutes ~ about 114 hours, about 120 minutes ~ about 108 hours, about 120 minutes to about 102 hours, about 120 minutes to about 96 hours, about 120 minutes to about 90 hours, about 120 minutes to about 84 hours, about 120 minutes to about 78 hours, about 120 minutes to about 72 hours , About 120 minutes to about 66 hours, about 120 minutes to about 60 hours, about 120 minutes to about 54 hours, about 120 minutes to about 48 hours, about 120 minutes to about 42 hours, about 120 minutes to about 36 hours, about 120 minutes to about 30 hours, about 120 minutes to about 24 hours, about 120 minutes to about 18 hours, about 12 0 minutes to about 12 hours, about 120 minutes to about 6 hours, about 120 minutes to about 5 hours, about 120 minutes to about 240 minutes, about 120 minutes to about 210 minutes, about 120 minutes to about 180 minutes, about 120 minutes ~ About 150 minutes, about 120 minutes ~ about 130 minutes, about 120 minutes ~ about 140 minutes, about 130 minutes ~ about 140 minutes, about 130 minutes ~ about 150 minutes, about 140 minutes ~ about 150 minutes; about 150 minutes ~ about 2 weeks, about 150 minutes to about 168 hours, about 150 minutes to about 162 hours, about 150 minutes to about 156 hours, about 150 minutes to about 150 hours, about 150 minutes to about 144 hours, about 150 minutes to about 138 hours , About 150 minutes to about 132 hours, about 150 minutes to about 126 hours, about 150 minutes to about 120 hours, about 150 minutes to about 114 hours, about 150 minutes to about 108 hours, about 150 minutes to about 102 hours, about 150 minutes to about 96 hours, about 150 minutes to about 90 hours, about 150 minutes to about 84 hours, about 150 minutes to about 78 hours, about 150 minutes to about 72 hours, about 150 minutes to about 66 hours, about 150 minutes ~ About 60 hours, about 150 minutes ~ about 54 hours, about 150 minutes ~ about 48 hours, about 150 minutes ~ about 42 hours, about 150 minutes ~ about 36 hours, about 150 minutes ~ about 30 hours, about 150 minutes ~ about 24 hours, about 150 minutes to about 18 hours, about 150 minutes to about 12 hours, about 150 minutes to about 6 hours, about 150 minutes to about 5 hours, about 150 minutes to about 240 minutes, about 150 minutes to about 210 minutes , About 150 minutes to about 180 minutes; about 180 minutes to about 2 weeks, about 180 minutes to about 168 hours, about 180 minutes to about 162 hours, about 180 minutes to about 156 hours, about 180 minutes to about 150 hours, about 180 minutes to about 144 hours, about 180 minutes to about 138 hours, about 180 minutes to about 132 hours, about 180 minutes to about 126 hours, about 180 minutes to about 120 hours, about 180 minutes to about 114 hours, about 180 minutes ~ About 108 hours, about 180 minutes ~ about 102 hours, about 180 minutes ~ about 96 hours, about 180 minutes ~ about 90 hours, about 180 minutes ~ about 84 hours, about 180 minutes ~ about 78 hours, about 180 minutes ~ about 72 hours, about 180 minutes to about 66 hours, about 180 minutes to about 60 hours, about 180 minutes to about 54 hours, about 180 minutes to about 48 hours, about 180 minutes to about 42 hours, about 180 minutes to about 36 hours , About 180 minutes to about 30 hours, about 180 minutes to about 24 hours, about 180 minutes to about 18 hours, about 180 minutes to about 12 hours, about 180 minutes to about 6 hours, about 180 minutes to about 5 hours, About 180 minutes to about 240 minutes, about 180 minutes to about 210 minutes, about 180 minutes to about 200 minutes, about 180 minutes to about 190 minutes, about 190 minutes to about 200 minutes, about 190 minutes to about 210 minutes; about 210 Minutes to about 2 weeks, about 210 minutes to about 168 hours, about 210 minutes to about 162 hours, about 210 minutes to about 156 hours, about 210 minutes to about 150 hours, about 210 minutes to about 144 hours, about 210 minutes About 138 hours, about 210 minutes to about 132 hours, about 210 minutes to about 126 hours, about 210 minutes to about 120 hours, about 210 minutes
About 114 hours, about 210 minutes to about 108 hours, about 210 minutes to about 102 hours, about 210 minutes to about 96 hours, about 210 minutes to about 90 hours, about 210 minutes to about 84 hours, about 210 minutes to about 78 Time, about 210 minutes to about 72 hours, about 210 minutes to about 66 hours, about 210 minutes to about 60 hours, about 210 minutes to about 54 hours, about 210 minutes to about 48 hours, about 210 minutes to about 42 hours, About 210 minutes to about 36 hours, about 210 minutes to about 30 hours, about 210 minutes to about 24 hours, about 210 minutes to about 18 hours, about 210 minutes to about 12 hours, about 210 minutes to about 6 hours, about 210 Minutes to about 5 hours, about 210 minutes to about 240 minutes, about 210 minutes to about 230 minutes, about 210 minutes to about 220 minutes; about 240 minutes to about 2 weeks, about 240 minutes to about 168 hours, about 240 minutes About 162 hours, about 240 minutes to about 156 hours, about 240 minutes to about 150 hours, about 240 minutes to about 144 hours, about 240 minutes to about 138 hours, about 240 minutes to about 132 hours, about 240 minutes to about 126 Time, about 240 minutes to about 120 hours, about 240 minutes to about 114 hours, about 240 minutes to about 108 hours, about 240 minutes to about 102 hours, about 240 minutes to about 96 hours, about 240 minutes to about 90 hours, About 240 minutes to about 84 hours, about 240 minutes to about 78 hours, about 240 minutes to about 72 hours, about 240 minutes to about 66 hours, about 240 minutes to about 60 hours, about 240 minutes to about 54 hours, about 240 Minutes to about 48 hours, about 240 minutes to about 42 hours, about 240 minutes to about 36 hours, about 240 minutes to about 30 hours, about 240 minutes to about 24 hours, about 240 minutes to about 18 hours, about 240 minutes About 12 hours, about 240 minutes to about 6 hours, about 240 minutes to about 5 hours, about 240 minutes to about 280 minutes, about 240 minutes to about 270 minutes, about 240 minutes to about 260 minutes, about 240 minutes to about 250 Minutes; about 270 minutes to about 280 minutes, about 270 minutes to about 290 minutes, about 270 minutes to about 5 hours; about 5 hours to about 2 weeks, about 5 hours to about 168 hours, about 5 hours to about 162 hours, About 5 hours to about 156 hours, about 5 hours to about 150 hours, about 5 hours to about 144 hours, about 5 hours to about 138 hours, about 5 hours to about 132 hours, about 5 hours to about 126 hours, about 5 Hours ~ about 120 hours, about 5 hours ~ about 114 hours, about 5 hours ~ about 108 hours, about 5 hours ~ about 102 hours, about 5 hours ~ about 96 hours, about 5 hours ~ about 90 hours, about 5 hours ~ About 84 hours, about 5 hours to about 78 hours, about 5 hours to about 72 hours, About 5 hours to about 66 hours, about 5 hours to about 60 hours, about 5 hours to about 54 hours, about 5 hours to about 48 hours, about 5 hours to about 42 hours, about 5 hours to about 36 hours, about 5 Time ~ about 30 hours, about 5 hours ~ about 24 hours, about 5 hours ~ about 18 hours, about 5 hours ~ about 12 hours, about 5 hours ~ about 11 hours, about 5 hours ~ about 10 hours, about 5 hours ~ About 9 hours, about 5 hours to about 8 hours, about 5 hours to about 7 hours, about 5 hours to about 6 hours; about 6 hours to about 12 hours, about 6 hours to about 8 hours, about 6 hours to about 10 Time, about 8 hours to about 10 hours, about 8 hours to about 12 hours; about 8 hours to about 2 weeks, about 8 hours to about 168 hours, about 8 hours to about 162 hours, about 6 hours to about 156 hours, About 6 hours to about 150 hours, about 6 hours to about 144 hours, about 6 hours to about 138 hours, about 6 hours to about 132 hours, about 6 hours to about 126 hours, about 6 hours to about 120 hours, about 6 Hours ~ about 114 hours, about 6 hours ~ about 108 hours, about 6 hours ~ about 102 hours, about 6 hours ~ about 96 hours, about 6 hours ~ about 90 hours, about 6 hours ~ about 84 hours, about 6 hours ~ About 78 hours, about 6 hours to about 72 hours, about 6 hours to about 66 hours, about 6 hours to about 60 hours, about 6 hours to about 54 hours, about 6 hours to about 48 hours, about 6 hours to about 42 Time, about 6 hours to about 36 hours, about 6 hours to about 30 hours, about 6 hours to about 24 hours, about 6 hours to about 18 hours, about 6 hours to about 12 hours, about 6 hours to about 11 hours, About 6 hours to about 10 hours, about 6 hours to about 9 hours, about 6 hours to about 8 hours, about 6 hours to about 7 hours, about 7 hours to about 8 hours; about 12 hours to about 2 weeks, about 12 Hours ~ about 168 hours, about 12 hours ~ about 162 hours, about 12 hours ~ about 156 hours, about 12 hours ~ about 150 hours, about 12 hours ~ about 144 hours, about 12 hours ~ about 138 hours, about 12 hours ~ About 132 hours, about 12 hours to about 126 hours, about 12 hours to about 120 hours, about 12 hours to about 114 hours, about 12 hours to about 108 hours, about 12 hours to about 102 hours, about 12 hours to about 96 Time, about 12 hours to about 90 hours, about 12 hours to about 84 hours, about 12 hours to about 78 hours, about 12 hours to about 72 hours, about 12 hours to about 66 hours, about 12 hours to about 60 hours, About 12 hours to about 54 hours, about 12 hours to about 48 hours, about 12 hours to about 42 hours, about 12 hours to about 36 hours, about 12 hours to about 30 hours, about 12 hours to about 24 hours, about 12 Hours ~ about 18 hours, about 12 hours ~ about 14 hours, about 12 hours ~ about 16 hours, about 14 hours ~ about 16 hours, about 14 hours ~ about 18 hours, about 14 hours ~ about 24:00 Between, about 14 hours to about 36 hours, about 14 hours to about 48 hours, about 14 hours to about 36 hours, about 16 hours to about 18 hours, about 16 hours to about 20 hours, about 16 hours to about 24 hours, About 16 hours to about 36 hours, about 16 hours to about 48 hours, about 18 hours to about 24 hours, about 18 hours to about 36 hours, about 18 hours to about 48 hours; about 24 hours to about 2 weeks, about 24 Hours ~ about 168 hours, about 24 hours ~ about 162 hours, about 24 hours ~ about 156 hours, about 24 hours ~ about 150 hours, about 24 hours ~ about 144 hours, about 24 hours ~ about 138 hours, about 24 hours ~ About 132 hours, about 24 hours to about 126 hours, about 24 hours to about 120 hours, about 24 hours to about 114 hours, about 24 hours to about 108 hours, about 24 hours to about 102 hours, about 24 hours to about 96 Time, about 24 hours to about 90 hours, about 24 hours to about 84 hours, about 24 hours to about 78 hours, about 24 hours to about 72 hours, about 24 hours to about 66 hours, about 24 hours to about 60 hours, About 24 hours to about 54 hours, about 24 hours to about 48 hours, about 24 hours to about 42 hours, about 24 hours to about 36 hours, about 24 hours to about 30 hours, about 30 hours to about 36 hours; about 26 Hours ~ about 28 hours, about 26 hours ~ about 30 hours, about 26 hours ~ about 36 hours, about 26 hours ~ about 48 hours, about 28 hours ~ about 30 hours, about 28 hours ~ about 36 hours, about 28 hours ~ About 48 hours, about 30 hours to about 36 hours, about 30 hours to about 48 hours; about 36 hours to about 2 weeks, about 36 hours to about 168 hours, about 36 hours to about 162 hours, about 36 hours to about 156 Time, about 36 hours to about 150 hours, about 36 hours to about 144 hours, about 36 hours to about 138 hours, about 36 hours to about 132 hours, about 36 hours to about 126 hours, about 36 hours to about 120 hours, About 36 hours to about 114 hours, about 36 hours to about 108 hours, about 36 hours to about 102 hours, about 36 hours to about 96 hours, about 36 hours to about 90 hours, about 36 hours to about 84 hours, about 36 Hours ~ about 78 hours, about 36 hours ~ about 72 hours, about 36 hours ~ about 66 hours, about 36 hours ~ about 60 hours, about 36 hours ~ about 54 hours, about 36 hours ~ about 48 hours, about 36 hours ~ About 42 hours, about 36 hours to about 40 hours, about 40 hours to about 42 hours; about 48 hours to about 2 weeks, about 48 hours to about 168 hours, about 48 hours to about 162 hours, about 48 hours to about 156 Time, about 48 hours to about 150 hours, about 48 hours to about 144 hours, about 48 hours to about 138 hours, about 48 hours to about 132 hours, about 48 hours to about 126 hours, about 48 hours to about 12 0 hours, about 48 hours to about 114 hours, about 48 hours to about 108 hours, about 48 hours to about 102 hours, about 48 hours to about 96 hours, about 48 hours to about 90 hours, about 48 hours to about 84 hours , About 48 hours to about 78 hours, about 48 hours to about 72 hours, about 48 hours to about 66 hours, about 48 hours to about 60 hours, about 48 hours to about 54 hours; about 60 hours to about 2 weeks, about 60 hours to about 168 hours, about 60 hours to about 162 hours, about 60 hours to about 156 hours, about 60 hours to about 150 hours, about 60 hours to about 144 hours, about 60 hours to about 138 hours, about 60 hours ~ About 132 hours, about 60 hours ~ about 126 hours, about 60 hours ~ about 120 hours, about 60 hours ~ about 114 hours, about 60 hours ~ about 108 hours, about 60 hours ~ about 102 hours, about 60 hours ~ about 96 hours, about 60 hours to about 90 hours, about 60 hours to about 84 hours, about 60 hours to about 78 hours, about 60 hours to about 72 hours, about 60 hours to about 66 hours; about 72 hours to about 2 weeks , About 72 hours to about 168 hours, about 72 hours to about 162 hours, about 72 hours to about 156 hours, about 72 hours to about 150 hours, about 72 hours to about 144 hours, about 72 hours to about 138 hours, about 72 hours to about 132 hours, about 72 hours to about 126 hours, about 72 hours to about 120 hours, about 72 hours to about 114 hours, about 72 hours to about 108 hours, about 72 hours to about 102 hours, about 72 hours ~ About 96 hours, about 72 hours ~ about 90 hours, about 72 hours ~ about 84 hours, about 72 hours ~ about 78 hours; about 84 hours ~ about 2 weeks, about 84 hours ~ about 168 hours, about 84 hours ~ about 162 hours, about 84 hours to about 156 hours, about 84 hours to about 150 hours, about 84 hours to about 144 hours, about 84 hours to about 138 hours, about 84 hours to about 132 hours, about 84 hours to about 126 hours , About 84 hours to about 120 hours, about 84 hours to about 114 hours, about 84 hours to about 108 hours, about 84 hours to about 102 hours, about 84 hours to about 96 hours, about 84 hours to about 90 hours; about 96 hours to about 2 weeks, about 96 hours to about 168 hours, about 96 hours to about 162 hours, about 96 hours to about 156 hours, about 96 hours to about 150 hours, about 96 hours to about 144 hours, about 96 hours ~ 138 hours, about 96 hours ~ about 132 hours, about 96 hours ~ about 126 hours, about 96 hours ~ about 120 hours, about 96 hours ~ about 114 hours, about 96 hours ~ about 108 hours, about 96 hours ~ about 102 hours; about 108 hours to about 2 weeks, about 108 hours to about 168 hours, about 108 hours to about 162 hours, about 108 hours to about 1 56 hours, about 108 hours to about 150 hours, about 108 hours to about 144 hours, about 108 hours to about 138 hours, about 108 hours to about 132 hours, about 108 hours to about 126 hours, about 108 hours to about 120 hours , About 108 hours to about 114 hours; about 120 hours to about 2 weeks, about 120 hours to about 168 hours, about 120 hours to about 162 hours, about 120 hours to about 156 hours, about 120 hours to about 150 hours, about 120 hours to about 144 hours, about 120 hours to about 138 hours, about 120 hours to about 132 hours, about 120 hours to about 126 hours; about 132 hours to about 2 weeks, about 132 hours to about 168 hours, about 132 hours ~ About 162 hours, about 132 hours ~ about 156 hours, about 132 hours ~ about 150 hours, about 132 hours ~ about 144 hours, about 132 hours ~ about 138 hours; about 144 hours ~ about 2 weeks, about 144 hours ~ about 168 hours, about 144 hours to about 162 hours, about 144 hours to about 156 hours, about 144 hours to about 150 hours; about 156 hours to about 2 weeks, about 156 hours to about 168 hours, about 156 hours to about 162 hours Or about 168 hours to about 2 weeks.

In some aspects of the contact phase of any of the methods provided herein, the period occurs when the liquid cell culture is in an exponential growth phase. In some aspects of the contact stage of any of the methods provided herein, the period occurs when the liquid cell culture is in a steady growth phase. In some embodiments, the contact step may occur when the liquid cell culture is in the dying phase. In some aspects of the contact stage of any of the methods provided herein, the period occurs on day 4 of cell culture and continues until day 5.

In some embodiments of any of the methods described herein, the stage of contact is one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10) pods. May include steps of contacting the liquid cell cultures sequentially or simultaneously over a period of time (eg, any period described herein).

Recombinant Proteins The recombinant proteins described herein are associated with receptors, antibodies, antigen-binding antibody fragments, antibody fragments, fusion proteins, ligands, secretory proteins, enzymes, recombinant protein preparations, and tags. Or, but is not limited to, any recombinant protein without it. Non-limiting examples of tags include fluorescent proteins (eg, green fluorescent protein, red fluorescent protein, cyanide fluorescent protein, yellow fluorescent protein), radioactive isotopes, His-tag, FLAG-tag, c-myc tag, maltose binding. Protein (MBP) tag, albumin-binding protein (ABP) tag, bacteriophage T7 epitope (T7-tag), biotin carboxyl carrier protein (BCCP) tag, blue tongue virus tag (B-tag), carmodulin-binding peptide tag, cellulose-binding Domain tag, chitin binding domain tag, choline binding domain tag, galactose binding protein tag, Glu-Glu epitope tag, human influenza hemagglutinin tag, Halo Tag®, histidine affinity tag (HAT), polyarginine tag (Asp) -Tag), Polyphenylalanine tag (Phe-tag), S-TAG, Streptavidin-binding peptide, Streptavidin tag, Strep-tag, Enzymes (eg, alkali phosphatase, chloramphenicol acetyltransferase (CAT), glutathione S-transferase) ) Is included. In some embodiments, the recombinant protein is a multimeric protein (eg, a heterodimer protein, a homodimer protein, or a trimer protein).

Exemplary recombinant antibodies and recombinant antibody fragments that may be expressed in any of the cells described herein include recombinant IgG1 (eg, recombinant human IgG1), recombinant IgG2 (eg, recombinant human IgG2). ), Recombinant IgG3 (eg, recombinant human IgG3), recombinant IgG4 (eg, recombinant human IgG4), recombinant IgM (eg, recombinant human IgM), recombinant IgE (eg, recombinant human IgE), recombinant IgD ( For example, recombinant human IgD), recombinant IgA (eg recombinant human IgA), recombinant Fab, recombinant scFv, recombinant VNAR, recombinant VHH, recombinant diabody (eg recombinant bispecific dia). Body), recombinant scFv-Fc, recombinant IgNAR, recombinant bispecific diabody, recombinant light chain variable domain, recombinant heavy chain variable domain, recombinant triabody, recombinant tetrabody ), Recombinant tandem scFv, Recombinant tandem scFv-Fc, Recombine, ant scFv-Fc Knobbs-into-holes, Recombinant scFv-Fc-scFv, Recombinant F (ab') ₂ , recombinant Fab-scFv, recombinant (Fab'scFv) ₂ , recombinant scDiabody-Fc, recombinant scDiabody, recombinant scDiabody-CH3, recombinant IgG-scFv, recombinant V-IgG, recombinant IgG-V , Recombinant two-in-one IgG, recombinant dual V-region IgG, and recombinant knob-in-to-hole with a common light chain may be included.

In some embodiments, the recombinant protein is an antibody. As used herein, the term "antibody" refers to a whole antibody or intact antibody (eg, IgM, IgG, IgA, IgD, or IgE) molecule. Antibodies are essentially tetramers containing two identical heavy chain protein moieties and two identical light chain protein moieties, which are held together by disulfide bridges in the hinge region and together. Relatedly take a "Y" -shaped conformation. The term "antibody" includes monoclonal antibodies, chimeric or chimeric antibodies, humanized antibodies, deimmunized human antibodies, and fully human antibodies. Antibodies include mammals such as humans, non-human primates (eg monkeys, baboons, or chimpanzees), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. It may be made or derived from any of a variety of species, such as. In some embodiments, the antibody, eg, Fuh et al., J. Mol. Biol. 340: 1073-1093, 2004 and Knappik et al, the entire disclosure of which is incorporated herein by reference. They may be synthetically produced, for example via synthetically produced oligonucleotides, as described in., J. Mol. Biol. 296 (11): 57-86, 2000. In some embodiments, the antibody may be produced by antibody phage display in yeast and / or human cells.

As used herein, the terms "antibody fragment", "antigen-binding fragment", or similar terms are, for example, single chain antibody (scFv), Fd fragment, Fab fragment, Fab. A fragment of an antibody that retains the ability to bind an antigen, such as a'fragment or an F (ab') ₂ fragment. scFv is a single polypeptide chain that contains both the heavy and light chain variable regions of the antibody from which the scFv is derived. In addition, Diabodies, all of which are incorporated herein by reference, Poljak, Structure 2 (12): 1121-1123, 1994; Hudson et al., J. Immunol. Methods 23 (1). -2): 177-189, 1999), minibody, triabody (Schoonooghe et al., BMC Biotechnol. 9:70, 2009), domain antibody (also as "heavy chain immunoglobulin" or camelid antibody Known; Holt et al., Trends Biotechnol. 21 (11): 484-490, 2003); and intrabody, Huston, the entire disclosure of which is incorporated herein by reference. et al., Human Antibodies 10 (3-4): 127-142, 2001; Wheeler et al., Mol. Ther. 8 (3): 355-366, 2003; Stocks, Drug Discov. Today 9 (22): 960-966, 2004) are also included in the definition of antibody fragments and may be incorporated into the compositions described herein and used in the methods described herein.

In some embodiments, the recombinant protein is a bispecific antibody. Bispecific antibodies are monoclonal, preferably human or humanized antibodies that have binding specificity for at least two different antigens.

Traditionally, recombinant production of bispecific antibodies has been based on co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy / light chain pairs have different specificities (Milstein and Cuello, Nature 305: 537-539, 1983). An antibody variable domain with the desired binding specificity (antibody-antigen binding site) can be fused to an immunoglobulin constant domain sequence. The fusion of the heavy chain variable region is preferably a fusion with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge region, CH2 region, and CH3 region. For more information on currently known exemplary methods for producing bispecific antibodies, see, eg, Suresh et al., Methods Enzymol. 121: 210, 1986; WO 96/27011; Brennan et al., Science 229: 81, 1985; Shalaby et al., J. Exp. Med. 175: 217-225, 1992; Kostelny et al., J. Immunol. 148 (5): 1547-1553, 1992; Hollinger et al. , Proc. Natl. Acad. Sci. USA. 90: 6444-6448, 1993; Gruber et al., J. Immunol. 152: 5368, 1994; and Tutt et al., J. Immunol. 147: 60, 1991. Please refer.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell cultures have also been described. For example, leucine zippers have been used to produce bispecific antibodies. See, for example, Kostelny et al., J. Immunol. 148 (5): 1547-1553, 1992. Leucine zipper peptides derived from the Fos and Jun proteins may be linked to the Fab'portion of two different antibodies by gene fusion. The antibody homodimer may be reduced in the hinge region to form a monomer and then reoxidized to form an antibody heterodimer. This method may also be utilized for the production of antibody homodimers. The "diabody" technique described in Hollinger et al., Proc. Natl. Acad. Sci. USA. 90: 6444-6448, 1993 provides an alternative mechanism for making bispecific antibody fragments. ing. The fragment contains a heavy chain variable domain (VH) linked to the light chain variable domain (VL) by a linker; the linker is short to allow pairing between those two domains on the same chain. Too much. Therefore, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, which forms two antigen binding sites. Another strategy for making bispecific antibody fragments by using a single chain Fv (scFv) dimer has also been reported. See, for example, Gruber et al., J. Immunol. 152: 5368, 1994. Alternatively, the antibody may be a "linear antibody", as described, for example, in Zapata et al., Protein Eng. 8 (10): 1057-1062, 1995. Briefly, these antibodies include a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen binding regions. Linear antibodies may be bispecific or unispecific.

Antibodies with a valency greater than 2 (eg, trispecific antibodies) are assumed and described, for example, in Tutt et al., J. Immunol. 147: 60, 1991.

The disclosure also includes variants of multispecific antibodies, such as the dual variable domain immunoglobulin (DVD-Ig) molecule described in Wu et al., Nat Biotechnol. 25 (11): 1290-1297, 2007. To do. In the DVD-Ig molecule, two different light chain variable domains (VL) derived from two different parent antibodies are linked in tandem form directly or via a short linker by recombinant DNA techniques, and the light chain is defined therein. Designed to be followed by a constant domain. Similarly, the heavy chain also contains two different heavy chain variable domains (VHs) linked in tandem, followed by the CH1 and Fc regions of the constant domain. Methods for making DVD-Ig molecules from the two parent antibodies are further described, for example, in WO 08/024188 and WO 07/024715. In some embodiments, the bispecific antibody is a tandem Fab immunoglobulin in which a light chain variable region with a second specificity is fused to the heavy chain variable region of all antibodies. Such antibodies are described, for example, in WO 2015/103072.

In some embodiments, the recombinant protein is a Nanobody such as a camelid or dromedary antibody (eg, an antibody derived from Bactrian camel, Calelus dromaderius, or Lama paccos). is there. Such antibodies generally lack a light chain, unlike typical 2-chain or 4-chain antibodies (total antibodies) from the majority of mammals. US Pat. No. 5,759,808; Stijlemans et al., J. Biol. Chem. 279: 1256-1261, 2004; Dumoulin et al., Nature 424: 783-788, 2003; and Pleschberger et al., Bioconjugate Chem. 14 See: 440-448, 2003. Similar to other antibodies of non-human origin, the amino acid sequence of the Camelid antibody may be recombinantly modified to obtain a sequence that is closer and more similar to the human sequence; May further reduce the potential immunogenicity of the antibody.

In some embodiments, the recombinant protein is a non-antibody scaffold protein. These proteins are generally obtained by adapting existing antigen-binding proteins based on combinatorial chemistry. For example, to create a diverse library of transferrin variants in which parts of the library have acquired affinity for different antigens, the systems described herein are used to diversify the binding sites of human transferrin to the human transferrin receptor. You may. Ali et al., J. Biol. Chem. 274: 24066-24073, 1999. The portion of human transferrin that is not involved in receptor binding remains unchanged and acts as a scaffold to present the mutant binding site, such as the framework region of an antibody.

The scaffold portion of the non-antibody scaffold protein is, for example, the Z domain of S. aureus protein A, human transferase, human 10th fibronectin type III domain, human trypsin inhibitor Knitz domain, human CTLA-4, ankyrin repeat. It will be appreciated by those skilled in the art that the protein, human lipocalin (eg, anticarin), human crystallin, human ubiquitin, or all or part of the trypsin inhibitor from E. elaterium may be included.

In some embodiments, the recombinant protein is a fusion protein. Non-limiting examples of fusion proteins that may be produced by any of the cells described herein include chimeric antibodies (eg, humanized antibodies); labeled antibodies (eg, labeled with an enzyme or fluorescent protein tag). Antibodies); protein ligands labeled with protein cytotoxicity that specifically bind to surface receptors in cancer cells; antibodies with fusion partners that prolong the half-life of the antibody in vivo (eg, to human albumin). Fused antibody); one light chain variable domain and one heavy chain variable domain bind to an antigen on immune cells (eg, T cells, macrophages, neutrophils, or monospheres) and the other light A bispecific antibody in which the chain variable domain and the other heavy chain variable domain bind to antigens on target cells (eg, cancer cells or virus-infected cells) or pathogens (eg, viruses, mycobacteria, or parasites). , Is included.

Further examples of recombinant proteins include ligands, enzymes, peptides, scFvs, non-antibody scaffold proteins, and chimeric antigen receptor molecules in which diversity elements are fused in frame with one or more constant elements. However, it is not limited to that. In some embodiments, the recombinant protein is a Fab, total antibody, MHC class I molecule, MHC class I molecule, T cell receptor molecule, bispecific antibody, trispecific antibody, integrin, tumor growth factor receptor. Dimers, and multimeric proteins such as the G protein-conjugated receptor, but are not limited thereto.

The cell liquid cell culture medium may contain any cells, such as eukaryotic cells. As used herein, the term "eukaryotic cell" refers to a cell with a well-defined nucleus surrounded by a membrane. Such cells may include, for example, cells of mammals (eg rodents, non-human primates, or humans), insects, fungi, or plants. In some embodiments, the eukaryotic cell is a yeast cell such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryotic cell, such as a mammalian, avian, plant, or insect cell.

Non-limiting examples of mammalian cells capable of producing recombinant proteins include, for example, rodent (eg, mouse, rat, or hamster) or human cells. In some embodiments, the mammalian cells are Chinese hamster ovary (CHO) cells, human retinal PER.C6 cells, human aminoocyte (aminocyte) CAP cells, mouse melanoma NS0 cells or Sp2 / 0 cells, duck EB66 cells, hamsters. BHK cells, human fetal kidney (HEK 293) cells, COS cells, or chicken-derived B cell line DT40. In some embodiments, the HEK 293 cells are Freestyle HEK 293-F cells, HEK 293 6F, HEK 293FT, HEK 293T cells, or Expi 293F cells. CHO cells are epithelial cells that grow as an adhesive monolayer or in suspension. CHO cell lines derived from the original cells isolated by Tjio and Puck (Tjio et al., J. Exp. Med. 108: 259-271, 1958) have nucleic acids compared to the original CHO cell lines. May contain point mutations, deletions, or integrations of the sequence. The CHO cell subgroup includes CHO DP-12 cells, CHO-K1 cells, CHO / dhfr ^- cells, CHO-S cells, CHO-GS cells, CHO-K1 DUX B11 cells, CHO pro3 ^- cells, CHO-DG44. It can include, but is not limited to, cells, CHO GAT ^- cells, or Freestyle CHO-S cells. In some embodiments, the eukaryotic cell is a primary cell or cell line. In some embodiments, the mammalian cell is a cancer cell. In some embodiments, the mammalian cell is a "Cancer Cell Line Encyclopedia" (Barretina et al., Nature 483 (7391): 603-607, 2012, or Forbes et al., Curr. Protoc. Hum. Genet. 2008). Human cancer cell lines, including those described in. In some embodiments, eukaryotic cells are T lymphocytes (eg, primary T cells or T cell lines), T cell-derived cell lines lacking TCR expression (eg, Torres et al., J. Immunol. 2003). ), Or pro-B cells (see, eg, Urashima et al., Am. J. Hematol. 1994), pre-B cells (see, eg, Adachi et al., DNA Cell Biol. 2006), and B cells, etc. B lymphoid cells, as well as cell lines derived from them. In some embodiments, the B-line cell line is a mouse pre-B cell line, such as 1624-5 (Holms et al., J. Exp. Med. 1986; Principato et al., Mol. Cell Biol. 1990). Alternatively, it is a pro-B cell line such as Ba / F3 (commercially available from Thermo Scientific). Mammalian cells may express a secretory version of the recombinant protein. In some embodiments, the secretory version of the recombinant protein is transiently expressed. In some embodiments, the secretory version of the recombinant protein is stably expressed.

In some embodiments, the liquid cell culture medium is an insect cell (eg, baculovirus-insect cell, Drosophila cell (eg, Drosophila S2 cell), Trichoplusia ni cell, scaly cell), yeast cell. (Eg yeast cells, Pichia pastoris cells), plant cells, or bacterial cells (eg E. coli cells, Baccillus brevis cells, Bacillus megaterium cells, Bacillus subtilis cells) , Caulobacter crescentis cells) may be included.

Those skilled in the art will appreciate that the proteins and vectors described herein may be introduced into cells (eg, mammalian cells, insect cells, yeast cells, bacterial cells, or plant cells). Non-limiting examples of vectors and methods for introducing vectors into cells are described herein.

Methods for culturing cells Methods for culturing cells are well known in the art. Cells can be maintained in vitro under conditions favorable for growth, proliferation, sustained viability, and differentiation. In general, cells can be cultured by contacting the cells with a liquid medium containing growth factors and dietary supplements necessary to support the viability and growth of the cells. The term "liquid culture medium" means a fluid containing sufficient nutrients for cells (eg, any of the cells described herein) to grow or proliferate in vitro. For example, liquid media include amino acids (eg, 20 amino acids), purines (eg, hypoxanthin), pyrimidine (eg, thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, niacinamide, pyridoxine, riboflavin, thymidine, cyanocobalamine, pyruvin. It may contain one or more of acid salts, lipoic acid, magnesium, glucose, sodium, potassium, iron, copper, zinc, and sodium bicarbonate.

In some embodiments, the liquid medium may contain mammalian-derived serum. In some embodiments, the liquid medium does not contain mammalian-derived serum or another extract (limited liquid medium). In some embodiments, the liquid medium may contain trace metals, mammalian growth hormone, and / or mammalian growth factors. Another example of a liquid medium is a minimal medium (eg, a medium containing only inorganic salts, carbon sources, and water). Non-limiting examples of liquid media are described herein. Further examples of liquid media are known in the art and are commercially available. The liquid cell medium may contain cells of any density as described herein (eg, mammalian cells of any density).

As used herein, the term "animal-derived component free liquid culture medium" means a liquid medium that does not contain any components of mammalian origin (eg, protein or serum). .. As used herein, the term "serum-free liquid culture medium" means a liquid medium that does not contain mammalian serum. As used herein, the term "serum-containing liquid culture medium" means a liquid medium containing mammalian serum. The term "chemically-defined liquid culture medium" as used herein is a technical term and means a liquid medium in which all chemical components are known. For example, liquid synthetic media do not contain fetal bovine serum, bovine serum albumin, or human serum albumin; because these formulations typically contain a complex mixture of albumin and lipids. As used herein, the term "protein-free liquid culture medium" means a liquid medium that does not contain any protein (eg, any detectable protein).

In some embodiments, the cells are described, for example, from about 1 day to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, About 45 days, about 40 days, about 35 days, about 30 days, about 20 days, about 25 days, about 20 days, about 15 days, about 10 days, about 8 days, about 6 days, about 4 days, or about 2 days (inclusive); about 2 to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days , About 40 days, about 35 days, about 30 days, about 20 days, about 25 days, about 20 days, about 15 days, about 10 days, about 8 days, about 6 days, or about 4 days (inclusive); About 4 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 Days, about 30 days, about 20 days, about 25 days, about 20 days, about 15 days, about 10 days, about 8 days, or about 6 days (inclusive); about 6 days to about 90 days, about 85 days , About 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 days, about 30 days, about 20 days, about 25 days, about 20 days, about 15 days, about 10 days, or about 8 days (inclusive); about 8 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 Days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 days, about 30 days, about 20 days, about 25 days, about 20 days, about 15 days, or about 10 days (Inclusive); about 10 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 days, about 30 days, about 20 days, about 25 days, about 20 days, or about 15 days (inclusive); about 15 to about 90 days, about 85 days, about 80 days, about 75 Days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 days, about 30 days, about 20 days, about 25 days, or about 20 days (Inclusive); about 20 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 days, about 30 days, about 20 days, or Is about 25 days (inclusive); about 25 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 days, or about 30 days (inclusive); about 30 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 Days, about 55 days, about 50 days, about 45 days, about 40 days, or about 35 days (inclusive); about 35 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days , About 65 days, about 60 days, about 55 days, about 50 days, about 45 days, or about 40 days (inclusive); about 40 days to about 90 days, about 85 days, about 80 days, about 75 days, About 70 days, about 65 days, about 60 days, about 55 days, or about 50 days (inclusive); about 50 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, or about 55 days (inclusive); about 55 to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, or about 60 days (included) ); About 60 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, or about 65 days (inclusive); about 65 days to about 90 days, about 85 days, about 80 Days, about 75 days, or about 70 days (inclusive); about 70 to about 90 days, about 85 days, about 80 days, or about 75 days (inclusive); about 75 to about 90 days, about 85 Approximately 20 ° C. for long periods of time, such as days, or approximately 80 days (inclusive); approximately 80 to approximately 90 days, or approximately 85 days (inclusive); or approximately 85 to approximately 90 days (inclusive)). ~ About 40 ° C (eg, about 39 ° C, about 38 ° C, about 37 ° C, about 36 ° C, or about 35 ° C (inclusive); about 34 ° C to about 40 ° C, about 39 ° C, about 38 ° C, about 37 ° C , About 36 ° C, about 34 ° C, about 32 ° C, about 30 ° C, about 28 ° C, about 26 ° C, about 24 ° C, about 22 ° C, about 20 ° C (inclusive); about 20 ° C to about 40 ° C, about 20 ° C to about 38 ° C, about 20 ° C to about 36 ° C, about 20 ° C to about 34 ° C, about 20 ° C to about 32 ° C, about 20 ° C to about 30 ° C, about 20 ° C to about 28 ° C, about 20 ° C ~ About 26 ℃, about 20 ℃ ~ about 24 ℃, about 20 ℃ ~ about 22 ℃ (inclusive); about 22 ℃ ~ about 40 ℃, about 22 ℃ ~ about 38 ℃, about 22 ℃ ~ about 36 ℃, about 22 ° C to about 34 ° C, about 22 ° C to about 32 ° C, about 22 ° C to about 30 ° C, about 22 ° C to about 28 ° C, about 22 ° C to about 26 ° C, about 22 ° C to about 24 ° C (inclusive); about 24 ° C to about 40 ° C, about 24 ° C to about 38 ° C, about 24 ° C to about 36 ° C, about 24 ° C to about 34 ° C, about 24 ° C to about 32 ° C, About 24 ° C to about 30 ° C, about 24 ° C to about 28 ° C, about 24 ° C to about 26 ° C (inclusive); about 26 ° C to about 40 ° C, about 26 ° C to about 38 ° C, about 26 ° C to about 36 ℃, about 26 ℃ to about 34 ℃, about 26 ℃ to about 32 ℃, about 26 ℃ to about 30 ℃, about 26 ℃ to about 28 ℃ (inclusive); about 28 ℃ to about 40 ℃, about 28 ℃ ~ About 38 ° C, about 28 ° C to about 36 ° C, about 28 ° C to about 34 ° C, about 28 ° C to about 32 ° C, about 28 ° C to about 30 ° C, about 30 ° C to about 40 ° C, about 30 ° C to about 38 ℃, about 30 ℃ to about 36 ℃, about 30 ℃ to about 34 ℃, about 30 ℃ to about 32 ℃ (inclusive); about 32 ℃ to about 40 ℃, about 32 ℃ to about 38 ℃, about 32 ℃ ~ About 36 ° C, about 32 ° C to about 34 ° C, about 34 ° C to about 40 ° C, about 34 ° C to about 38 ° C, about 34 ° C to about 36 ° C, about 36 ° C to about 40 ° C, about 36 ° C to about 38 ℃, about 38 ° C to about 40 ° C (inclusive); about 35 ° C to about 40 ° C, about 39 ° C, about 38 ° C, or about 37 ° C (inclusive); about 36 ° C to about 40 ° C, about 39 ° C , Or about 38 ° C (inclusive); about 37 ° C to about 40 ° C, or about 39 ° C (inclusive); or about 38 ° C to about 40 ° C (inclusive), about 5% to about 12% (eg,). About 5% to about 10%, about 5% to about 8%, about 5% to about 7%, about 6% to about 8%, about 6% to about 10%, or about 8% to about 10%) It may be cultured in CO ₂ . In some embodiments, the liquid cell culture medium is agitated during the culture for a period of time (eg, persistently, periodically). The term "agitation" means agitating or otherwise moving a portion of the liquid medium in a bioreactor. This is done, for example, to increase the dissolved oxygen concentration in the liquid medium in the bioreactor. Stirring may be performed using any method known in the art, such as an instrument (eg, a shaking, rocking, or tilting platform) or a propeller. Illustrative devices and methods that can be used to agitate a portion of the liquid medium in a bioreactor are known in the art.

In some embodiments of any of the methods described herein, the liquid cell culture is a perfusion culture. The term "perfusion culture" is a technical term; and the first liquid medium is removed periodically or continuously, and at the same time or shortly thereafter, a second of substantially the same volume. By adding a liquid medium to a culture vessel, it refers to culturing cells existing in the culture vessel (for example, any of the culture vessels described herein). In some examples, during the culture period (eg, the resupply rate of the medium is daily), an incremental period (eg, a period of about 24 hours, a period of about 1 minute to about 24 hours, or There is an incremental change (eg, increase or decrease) in the volume of the first liquid medium that is removed and added over a period of more than 24 hours). The proportion of medium removed and replaced each day may vary depending on the particular cells being cultured, the initial seeding density, and the cell density over a particular time period.

In some embodiments of any of the methods described herein, the liquid cell culture is a fed batch culture. The term "fed batch culture" is a technical term; and without substantial or significant removal of the first or second liquid medium from the liquid cell culture medium. It refers to culturing cells existing in a culture vessel by adding a second liquid medium to the first liquid medium periodically or continuously. The second liquid medium may be the same as the first liquid medium. In some examples of fed batch culture, the second liquid medium is a concentrated form of the first liquid medium. In some examples of fed batch culture, the second liquid medium is added as dry powder.

The liquid cell culture medium may be placed in a culture vessel. Non-limiting examples of culture vessels include shaking flasks, shaking tubes, wave bags, T-flakes, triple flasks, spinner flasks, roller bottles, bioreactors (eg perfusion bioreactors, fed batch bioreactors), or mulch. Well plates (eg, 4-well cell culture plate, 6-well cell culture plate, 8-well cell culture plate, 12-well cell culture plate, 24-well cell culture plate, 24-well cell culture plate) are included.

In some embodiments, the culture vessel is about 0.2 mL to 10,000 L (eg, about 0.2 mL to about 9,000 L, about 0.2 mL to about 8,000 L, about 0.2 mL to about 7,000 L, about 0.2 mL to about 6,000 L, About 0.2 mL to about 5,000 L, about 0.2 mL to about 4,000 L, about 0.2 mL to about 3,000 L, about 0.2 mL to about 2,000 L, about 0.2 mL to about 1,000 L, about 0.2 mL to about 950 L, about 0.2 mL ~ about 900 L, about 0.2 mL ~ about 850 L, about 0.2 mL ~ about 800 L, about 0.2 mL ~ about 750 mL, about 0.2 mL ~ about 700 L, about 0.2 mL ~ about 650 mL, about 0.2 mL ~ About 600 L, about 0.2 mL to about 550 L, about 0.2 mL to about 500 L, about 0.2 mL to about 450 L, about 0.2 mL to about 400 L, about 0.2 mL to about 300 L, about 0.2 mL to about 200 L, about 0.2 mL to about 190 L, about 0.2 mL to about 180 L, about 0.2 mL to about 170 L, about 0.2 mL to about 160 L, about 0.2 mL to about 150 L, about 0.2 mL to about 140 L, About 0.2 mL to about 130 L, about 0.2 mL to about 120 L, about 0.2 mL to about 110 L, about 0.2 mL to about 100 L, about 0.2 mL to about 90 L, about 0.2 mL to about 85 L, about 0.2 mL ~ about 80 L, about 0.2 mL ~ about 75 L, about 0.2 mL ~ about 70 L, about 0.2 mL ~ about 65 L, about 0.2 mL ~ about 60 L, about 0.2 mL ~ about 55 L, about 0.2 mL ~ About 50 L, about 0.2 mL to about 45 L, about 0.2 mL to about 40 L, about 0.2 mL to about 35 L, about 0.2 mL to about 30 L, about 0.2 mL to about 25 L, about 0.2 mL to about 20 L, about 0.2 mL to about 15 L, about 0.2 mL to about 10 L, about 0.2 mL to about 5 L, about 0.2 mL to about 4 L, about 0.2 mL to about 4 L, about 0.2 mL to about 3 L, About 0.2 mL to about 2 L, about 0.2 mL to about 1 L; about 0.2 mL to about 900 mL, about 0.2 mL to about 800 mL, about 0.2 mL to about 700 mL, about 0.2 mL to about 600 mL, about 0.2 mL ~ about 500 mL, about 0.2 mL ~ about 400 mL, about 0.2 mL ~ about 300 mL, about 0.2 mL ~ about 250 mL, about 0. 2 mL to about 100 mL, about 0.2 mL to about 90 mL, about 0.2 mL to about 80 mL, about 0.2 mL to about 70 mL, about 0.2 mL to about 60 mL, about 0.2 mL to about 50 mL, about 0.2 mL ~ About 40 mL, about 0.2 mL ~ about 30 mL, about 0.2 mL ~ about 25 mL, about 0.2 mL ~ about 20 mL, about 0.2 mL ~ about 15 mL, about 0.2 mL ~ about 10 mL, about 0.2 mL ~ about 5 mL, about 0.2 mL to about 4 mL, about 0.2 mL to about 3 mL, about 0.2 mL to about 2 mL, about 0.2 mL to about 1 mL; about 0.4 mL to 10,000 L, about 0.4 mL to about 9,000 L, About 0.4 mL to about 8,000 L, about 0.4 mL to about 7,000 L, about 0.4 mL to about 6,000 L, about 0.4 mL to about 5,000 L, about 0.4 mL to about 4,000 L, about 0.4 mL to about 3,000 L, about 0.4 mL ~ about 2,000 L, about 0.4 mL ~ about 1,000 L, about 0.4 mL ~ about 950 L, about 0.4 mL ~ about 900 L, about 0.4 mL ~ about 850 L, about 0.4 mL ~ about 800 L, about 0.4 mL ~ About 750 L, about 0.4 mL to about 700 L, about 0.4 mL to about 650 L, about 0.4 mL to about 600 L, about 0.4 mL to about 550 L, about 0.4 mL to about 500 L, about 0.4 mL to about 450 L, about 0.4 mL to about 400 L, about 0.4 mL to about 350 L, about 0.4 mL to about 300 L, about 0.4 mL to about 250 L, about 0.4 mL to about 200 L, about 0.4 mL to about 190 L, About 0.4 mL to about 180 L, about 0.4 mL to about 170 L, about 0.4 mL to about 160 L, about 0.4 mL to about 150 L, about 0.4 mL to about 140 L, about 0.4 mL to about 130 L, about 0.4 mL ~ about 120 L, about 0.4 mL ~ about 110 L, about 0.4 mL ~ about 100 L, about 0.4 mL ~ about 90 L, about 0.4 mL ~ about 85 L, about 0.4 mL ~ about 80 L, about 0.4 mL ~ About 75 L, about 0.4 mL to about 70 L, about 0.4 mL to about 65 L, about 0.4 mL to about 60 L, about 0.4 mL to about 55 L, about 0.4 mL to about 50 L, about 0.4 mL to about 45 L, about 0.4 mL to about 40 L, about 0.4 mL to about 35 L, about 0.4 mL to about 30 L, about 0.4 mL to about 2 5 L, about 0.4 mL to about 20 L, about 0.4 mL to about 15 L, about 0.4 mL to about 10 L, about 0.4 mL to about 5 L, about 0.4 mL to about 4 L, about 0.4 mL to about 4 L , About 0.4 mL to about 3 L, about 0.4 mL to about 2 L, about 0.4 mL to about 1 L; about 0.4 mL to about 900 mL, about 0.4 mL to about 800 mL, about 0.4 mL to about 700 mL, about 0.4 mL to about 600 mL, about 0.4 mL to about 500 mL, about 0.4 mL to about 400 mL, about 0.4 mL to about 300 mL, about 0.4 mL to about 250 mL, about 0.4 mL to about 100 mL, about 0.4 mL ~ About 90 mL, about 0.4 mL ~ about 80 mL, about 0.4 mL ~ about 70 mL, about 0.4 mL ~ about 60 mL, about 0.4 mL ~ about 50 mL, about 0.4 mL ~ about 40 mL, about 0.4 mL ~ about 30 mL, about 0.4 mL to about 25 mL, about 0.4 mL to about 20 mL, about 0.4 mL to about 15 mL, about 0.4 mL to about 10 mL, about 0.4 mL to about 5 mL, about 0.4 mL to about 4 mL , About 0.4 mL to about 3 mL, about 0.4 mL to about 2 mL, about 0.4 mL to about 1 mL; about 0.6 mL to 10,000 L, about 0.6 mL to about 9,000 L, about 0.6 mL to about 8,000 L, about 0.6 mL ~ about 7,000 L, about 0.6 mL ~ about 6,000 L, about 0.6 mL ~ about 5,000 L, about 0.6 mL ~ about 4,000 L, about 0.6 mL ~ about 3,000 L, about 0.6 mL ~ about 2,000 L, about 0.6 mL ~ About 1,000 L, about 0.6 mL to about 950 L, about 0.6 mL to about 900 L, about 0.6 mL to about 850 L, about 0.6 mL to about 800 L, about 0.6 mL to about 750 L, about 0.6 mL to about 700 L, about 0.6 mL to about 650 L, about 0.6 mL to about 600 L, about 0.6 mL to about 550 L, about 0.6 mL to about 500 L, about 0.6 mL to about 450 L, about 0.6 mL to about 400 L, About 0.6 mL to about 350 L, about 0.6 mL to about 300 L, about 0.6 mL to about 250 L, about 0.6 mL to about 200 L, about 0.6 mL to about 190 L, about 0.6 mL to about 180 L, about 0.6 mL ~ about 170 L, about 0.6 mL ~ about 160 L, about 0.6 mL ~ about 150 L, about 0.6 mL ~ about 140 L, about 0.6 mL to about 130 L, about 0.6 mL to about 120 L, about 0.6 mL to about 110 L, about 0.6 mL to about 100 L, about 0.6 mL to about 90 L, about 0.6 mL to about 85 L, About 0.6 mL to about 80 L, about 0.6 mL to about 75 L, about 0.6 mL to about 70 L, about 0.6 mL to about 65 L, about 0.6 mL to about 60 L, about 0.6 mL to about 55 L, about 0.6 mL ~ about 50 L, about 0.6 mL ~ about 45 L, about 0.6 mL ~ about 40 L, about 0.6 mL ~ about 35 L, about 0.6 mL ~ about 30 L, about 0.6 mL ~ about 25 L, about 0.6 mL ~ About 20 L, about 0.6 mL to about 15 L, about 0.6 mL to about 10 L, about 0.6 mL to about 5 L, about 0.6 mL to about 4 L, about 0.6 mL to about 4 L, about 0.6 mL to about 3 L, about 0.6 mL to about 2 L, about 0.6 mL to about 1 L; about 0.6 mL to about 900 mL, about 0.6 mL to about 800 mL, about 0.6 mL to about 700 mL, about 0.6 mL to about 600 mL, About 0.6 mL to about 500 mL, about 0.6 mL to about 400 mL, about 0.6 mL to about 300 mL, about 0.6 mL to about 250 mL, about 0.6 mL to about 100 mL, about 0.6 mL to about 90 mL, about 0.6 mL ~ about 80 mL, about 0.6 mL ~ about 70 mL, about 0.6 mL ~ about 60 mL, about 0.6 mL ~ about 50 mL, about 0.6 mL ~ about 40 mL, about 0.6 mL ~ about 30 mL, about 0.6 mL ~ About 25 mL, about 0.6 mL to about 20 mL, about 0.6 mL to about 15 mL, about 0.6 mL to about 10 mL, about 0.6 mL to about 5 mL, about 0.6 mL to about 4 mL, about 0.6 mL to about 3 mL, about 0.6 mL to about 2 mL, about 0.6 mL to about 1 mL; about 0.8 mL to 10,000 L, about 0.8 mL to about 9,000 L, about 0.8 mL to about 8,000 L, about 0.8 mL to about 7,000 L, about 0.8 mL to about 6,000 L, about 0.8 mL to about 5,000 L, about 0.8 mL to about 4,000 L, about 0.8 mL to about 3,000 L, about 0.8 mL to about 2,000 L, about 0.8 mL to about 1,000 L, about 0.5 mL ~ 950 L, about 0.8 mL ~ about 900 L, about 0.8 mL ~ about 850 L, about 0.8 mL ~ about 800 L, about 0.8 mL ~ about 750 L, about 0.8 mL to about 700 L, about 0.8 mL to about 650 L, about 0.8 mL to about 600 L, about 0.8 mL to about 550 L, about 0.8 mL to about 500 L, about 0.8 mL to about 450 L, About 0.8 mL to about 400 L, about 0.8 mL to about 350 L, about 0.8 mL to about 300 L, about 0.8 mL to about 250 L, about 0.8 mL to about 200 L, about 0.8 mL to about 190 L, about 0.8 mL ~ about 180 L, about 0.8 mL ~ about 170 L, about 0.8 mL ~ about 160 L, about 0.8 mL ~ about 150 L, about 0.8 mL ~ about 140 L, about 0.8 mL ~ about 130 L, about 0.8 mL ~ About 120 L, about 0.8 mL to about 110 L, about 0.8 mL to about 100 L, about 0.8 mL to about 90 L, about 0.8 mL to about 85 L, about 0.8 mL to about 80 L, about 0.8 mL to about 75 L, about 0.8 mL to about 70 L, about 0.8 mL to about 65 L, about 0.8 mL to about 60 L, about 0.8 mL to about 55 L, about 0.8 mL to about 50 L, about 0.8 mL to about 45 L, About 0.8 mL to about 40 L, about 0.8 mL to about 35 L, about 0.8 mL to about 30 L, about 0.8 mL to about 25 L, about 0.8 mL to about 20 L, about 0.8 mL to about 15 L, about 0.8 mL ~ about 10 L, about 0.8 mL ~ about 5 L, about 0.8 mL ~ about 4 L, about 0.8 mL ~ about 4 L, about 0.8 mL ~ about 3 L, about 0.8 mL ~ about 2 L, about 0.8 mL ~ About 1 L; about 0.8 mL to about 900 mL, about 0.8 mL to about 800 mL, about 0.8 mL to about 700 mL, about 0.8 mL to about 600 mL, about 0.8 mL to about 500 mL, about 0.8 mL to about 400 mL, about 0.8 mL to about 300 mL, about 0.8 mL to about 250 mL, about 0.8 mL to about 100 mL, about 0.8 mL to about 90 mL, about 0.8 mL to about 80 mL, about 0.8 mL to about 70 mL, About 0.8 mL to about 60 mL, about 0.8 mL to about 50 mL, about 0.8 mL to about 40 mL, about 0.8 mL to about 30 mL, about 0.8 mL to about 25 mL, about 0.8 mL to about 20 mL, about 0.8 mL ~ about 15 mL, about 0.8 mL ~ about 10 mL, about 0.8 mL ~ about 5 mL, about 0.8 mL ~ about 4 mL, about 0.8 mL ~ about 3 mL, about 0.8 mL ~ about 2 mL, about 0.8 mL ~ about 1 mL; about 1 mL ~ 10,000 L, about 1 mL ~ about 9,000 L, about 1 mL ~ about 8,000 L, about 1 mL ~ about 7,000 L, about 1 mL ~ about 6,000 L, about 1 mL ~ About 5,000 L, about 1 mL to about 4,000 L, about 1 mL to about 3,000 L, about 1 mL to about 2,000 L, about 1 mL to about 1,000 L, about 1 mL to about 950 L, about 1 mL to about 900 L, about 1 mL to about 850 L, about 1 mL to about 800 L, about 1 mL to about 750 L, about 1 mL to about 700 L, about 1 mL to about 650 L, about 1 mL to about 600 L, About 1 mL to about 550 L, about 1 mL to about 500 L, about 1 mL to about 450 L, about 1 mL to about 400 L, about 1 mL to about 350 L, about 1 mL to about 300 L, about 1 mL ~ about 250 L, about 1 mL ~ about 200 L, about 1 mL ~ about 190 L, about 1 mL ~ about 180 L, about 1 mL ~ about 170 L, about 1 mL ~ about 160 L, about 1 mL ~ About 150 L, about 1 mL to about 140 L, about 1 mL to about 130 L, about 1 mL to about 120 L, about 1 mL to about 110 L, about 1 mL to about 100 L, about 1 mL to about 90 L, about 1 mL to about 80 L, about 1 mL to about 70 L, about 1 mL to about 60 L, about 1 mL to about 50 L, about 1 mL to about 40 L, about 1 mL to about 30 L, About 1 mL to about 20 L, about 1 mL to about 10 L, about 1 mL to about 5 L, about 1 mL to about 4 L, about 1 mL to about 3 L, about 1 mL to about 2 L, about 1 mL ~ about 1 L, about 1 mL ~ about 900 mL, about 1 mL ~ about 800 mL, about 1 mL ~ about 700 mL, about 1 mL ~ about 600 mL, about 1 mL ~ about 500 mL, about 1 mL ~ About 400 mL, about 1 mL to about 300 mL, about 1 mL to about 250 mL, about 1 mL to about 100 mL, about 1 mL to about 90 mL, about 1 mL to about 80 mL, about 1 mL to about 70 mL, about 1 mL to about 60 mL, about 1 mL to about 50 mL, about 1 mL to about 40 mL, about 1 mL to about 30 mL, about 1 mL to about 25 mL, about 1 mL to about 20 mL, About 1 mL to about 15 mL, about 1 mL to about 10 mL, about 1 mL to about 5 mL; about 1 mL to about 4 mL, about 1 mL to about 3 mL, about 1 mL to about 2 mL; about 2 mL ~ 10,000 L, about 2 m L ~ about 9,000 L, about 2 mL ~ about 8,000 L, about 2 mL ~ about 7,000 L, about 2 mL ~ about 6,000 L, about 2 mL ~ about 5,000 L, about 2 mL ~ about 4,000 L, about 2 mL ~ About 3,000 L, about 2 mL to about 2,000 L, about 2 mL to about 1,000 L, about 2 mL to about 950 L, about 2 mL to about 900 L, about 2 mL to about 850 L, about 2 mL to about 800 L, about 2 mL to about 750 L, about 2 mL to about 700 L, about 2 mL to about 650 L, about 2 mL to about 600 L, about 2 mL to about 550 L, about 2 mL to about 500 L, About 2 mL to about 450 L, about 2 mL to about 400 L, about 2 mL to about 350 L, about 2 mL to about 300 L, about 2 mL to about 250 L, about 2 mL to about 200 L; about 2 mL ~ about 190 L, about 2 mL ~ about 180 L, about 2 mL ~ about 170 L, about 2 mL ~ about 160 L, about 2 mL ~ about 150 L, about 2 mL ~ about 140 L, about 2 mL ~ About 130 L, about 2 mL to about 120 L, about 2 mL to about 110 L, about 2 mL to about 100 L, about 2 mL to about 90 L, about 2 mL to about 80 L, about 2 mL to about 70 L, about 2 mL to about 60 L, about 2 mL to about 50 L, about 2 mL to about 40 L, about 2 mL to about 30 L, about 2 mL to about 20 L, about 2 mL to about 10 L, About 2 mL to about 5 L, about 2 mL to about 4 L, about 2 mL to about 3 L, about 2 mL to about 2 L, about 2 mL to about 1 L, about 2 mL to about 900 mL, about 2 mL ~ about 800 mL, about 2 mL ~ about 700 mL, about 2 mL ~ about 600 mL, about 2 mL ~ about 500 mL, about 2 mL ~ about 400 mL, about 2 mL ~ about 300 mL, about 2 mL ~ About 250 mL, about 2 mL to about 100 mL, about 2 mL to about 90 mL, about 2 mL to about 80 mL, about 2 mL to about 70 mL, about 2 mL to about 60 mL, about 2 mL to about 50 mL, about 2 mL to about 40 mL, about 2 mL to about 30 mL, about 2 mL to about 25 mL, about 2 mL to about 20 mL, about 2 mL to about 15 mL, about 2 mL to about 10 mL, About 2 mL to about 5 mL; about 5 mL to 10,000 L, about 5 mL to about 9,000 L, about 5 mL to about 8,000 L, about 5 mL to about 7,000 L, about 5 mL to about 6,000 L, about 5 mL ~ About 5,000 L, about 5 mL to about 4,000 L, about 5 mL to about 3,000 L, about 5 mL to about 2,000 L, about 5 mL to about 1,000 L, about 5 mL to about 950 L, about 5 mL to about 900 L, About 5 mL to about 850 L, about 5 mL to about 800 L, about 5 mL to about 750 L, about 5 mL to about 700 L, about 5 mL to about 650 L, about 5 mL to about 600 L, about 5 mL ~ about 550 L, about 5 mL ~ about 500 L, about 5 mL ~ about 450 L, about 5 mL ~ about 400 L, about 5 mL ~ about 350 L, about 5 mL ~ about 300 L, about 5 mL ~ About 250 L, about 5 mL to about 200 L; about 5 mL to about 190 L, about 5 mL to about 180 L, about 5 mL to about 170 L, about 5 mL to about 160 L, about 5 mL to about 150 L, about 5 mL to about 140 L, about 5 mL to about 130 L, about 5 mL to about 120 L, about 5 mL to about 110 L, about 5 mL to about 100 L, about 5 mL to about 90 L, About 5 mL to about 80 L, about 5 mL to about 70 L, about 5 mL to about 60 L, about 5 mL to about 50 L, about 5 mL to about 40 L, about 5 mL to about 30 L, about 5 mL ~ about 20 L, about 5 mL ~ about 10 L, about 5 mL ~ about 5 L, about 5 mL ~ about 4 L, about 5 mL ~ about 3 L, about 5 mL ~ about 5 L, about 5 mL ~ About 1 L, about 5 mL to about 900 mL, about 5 mL to about 800 mL, about 5 mL to about 700 mL, about 5 mL to about 600 mL, about 5 mL to about 500 mL, about 5 mL to about 400 mL, about 5 mL to about 300 mL, about 5 mL to about 250 mL, about 5 mL to about 100 mL, about 5 mL to about 90 mL, about 5 mL to about 80 mL, about 5 mL to about 70 mL, About 5 mL to about 60 mL, about 5 mL to about 50 mL, about 5 mL to about 40 mL, about 5 mL to about 30 mL, about 5 mL to about 25 mL, about 5 mL to about 20 mL, about 5 mL ~ about 15 mL, about 5 mL ~ about 10 mL; about 10 mL ~ 10,000 L, about 10 mL ~ about 9,000 L, about 10 mL ~ about 8,000 L, about 10 mL ~ about 7,000 L, about 10 mL ~ about 6,000 L, about 10 mL to about 5,000 L, about 10 mL to about 4,000 L, about 10 mL to about 3,000 L, about 10 mL to about 2,000 L, about 10 mL to about 1,000
L, about 10 mL to about 950 L, about 10 mL to about 900 L, about 10 mL to about 850 L, about 10 mL to about 800 L, about 10 mL to about 750 L, about 10 mL to about 700 L, About 10 mL to about 650 L, about 10 mL to about 600 L, about 10 mL to about 550 L, about 10 mL to about 500 L, about 10 mL to about 450 L, about 10 mL to about 400 L, about 10 mL ~ about 350 L, about 10 mL ~ about 300 L, about 10 mL ~ about 250 L, about 10 mL ~ about 200 L, about 10 mL ~ about 190 L, about 10 mL ~ about 180 L, about 10 mL ~ About 170 L, about 10 mL to about 160 L, about 10 mL to about 150 L, about 10 mL to about 140 L, about 10 mL to about 130 L, about 10 mL to about 120 L, about 10 mL to about 110 L, about 10 mL to about 100 L, about 10 mL to about 90 L, about 10 mL to about 80 L, about 10 mL to about 70 L, about 10 mL to about 60 L, about 10 mL to about 50 L, About 10 mL to about 40 L, about 10 mL to about 30 L, about 10 mL to about 20 mL, about 10 mL to about 10 L, about 10 mL to about 5 L, about 10 mL to about 4 L, about 10 mL ~ about 3 L, about 10 mL ~ about 5 L, about 10 mL ~ about 1 L, about 10 mL ~ about 900 mL, about 10 mL ~ about 800 mL, about 10 mL ~ about 700 mL, about 10 mL ~ About 600 mL, about 10 mL to about 500 mL, about 10 mL to about 400 mL, about 10 mL to about 300 mL, about 10 mL to about 250 mL, about 10 mL to about 100 mL, about 10 mL to about 90 mL, about 10 mL to about 80 mL, about 10 mL to about 70 mL, about 10 mL to about 60 mL, about 10 mL to about 50 mL, about 10 mL to about 40 mL, about 10 mL to about 30 mL, About 10 mL to about 25 mL, about 10 mL to about 20 mL, about 10 mL to about 15 mL; about 25 mL to 10,000 L, about 25 mL to about 9,000 L, about 25 mL to about 8,000 L, about 25 mL ~ 7,000 L, about 25 mL ~ about 6,000 L, about 25 mL ~ about 5,000 L, about 25 mL ~ about 4,000 L, about 25 mL ~ about 3,000 L, about 25 mL ~ about 2,000 L, about 25 mL ~ about 1,000 L, about 25 mL ~ about 950 L, about 25 mL ~ About 900 L, about 25 mL to about 850 L, about 25 mL to about 800 L, about 25 mL to about 750 L, about 25 mL to about 700 L, about 25 mL to about 650 L, about 25 mL to about 600 L, about 25 mL to about 550 L, about 25 mL to about 500 L, about 25 mL to about 450 L, about 25 mL to about 400 L, about 25 mL to about 350 L, about 25 mL to about 300 L, About 25 mL to about 250 L, about 25 mL to about 200 L, about 25 mL to about 190 L, about 25 mL to about 180 L, about 25 mL to about 170 L, about 25 mL to about 160 L, about 25 mL ~ about 150 L, about 25 mL ~ about 140 L, about 25 mL ~ about 130 L, about 25 mL ~ about 120 L, about 25 mL ~ about 110 L, about 25 mL ~ about 100 L, about 25 mL ~ About 90 L, about 25 mL to about 80 L, about 25 mL to about 70 L, about 25 mL to about 60 L, about 25 mL to about 50 L, about 25 mL to about 40 L, about 25 mL to about 30 L, about 25 mL to about 20 L, about 25 mL to about 10 L, about 25 mL to about 5 L, about 25 mL to about 4 L, about 25 mL to about 3 L, about 25 mL to about 5 L, About 25 mL to about 1 L, about 25 mL to about 900 mL, about 25 mL to about 800 mL, about 25 mL to about 700 mL, about 25 mL to about 600 mL, about 25 mL to about 500 mL, about 25 mL ~ about 400 mL, about 25 mL ~ about 300 mL, about 25 mL ~ about 250 mL, about 25 mL ~ about 100 mL, about 25 mL ~ about 90 mL, about 25 mL ~ about 80 mL, about 25 mL ~ About 70 mL, about 25 mL to about 60 mL, about 25 mL to about 50 mL, about 25 mL to about 40 mL, about 25 mL to about 30 mL; about 50 mL to 10,000 L, about 50 mL to about 9,000 L , About 50 mL to about 8,000 L, about 50 mL to about 7,000 L, about 50 mL to about 6,000 L, about 50 mL to about 5,000 L, about 50 mL to about 4,000 L, about 50 mL to about 3,000 L, about 50 mL to about 2,000 L, about 50 mL to about 1,000 L, about 50 mL to about 950 L, about 50 mL to about 900 L, about 50 mL to about 850 L, about 50 mL to about 800 L, about 50 mL ~ About 750 L, about 50 mL ~ About 700 L, about 50 mL ~ about 650 L, about 50 mL ~ about 600 L, about 50 mL ~ about 550 L, about 50 mL ~ about 500 L, about 50 mL ~ about 450 L, about 50 mL ~ about 400 L, about 50 mL ~ About 350 L, about 50 mL to about 300 L, about 50 mL to about 250 L, about 50 mL to about 200 L, about 50 mL to about 190 L, about 50 mL to about 180 L, about 50 mL to about 170 L, about 50 mL to about 160 L, about 50 mL to about 150 L, about 50 mL to about 140 L, about 50 mL to about 130 L, about 50 mL to about 120 L, about 50 mL to about 110 L, About 50 mL to about 100 L, about 50 mL to about 90 L, about 50 mL to about 80 L, about 50 mL to about 70 L, about 50 mL to about 60 L, about 50 mL to about 50 L, about 50 mL ~ about 40 L, about 50 mL ~ about 30 L, about 50 mL ~ about 20 L, about 50 mL ~ about 10 L, about 50 mL ~ about 5 L, about 50 mL ~ about 4 L, about 50 mL ~ About 3 L, about 50 mL to about 5 L, about 50 mL to about 1 L, about 50 mL to about 900 mL, about 50 mL to about 800 mL, about 50 mL to about 700 mL, about 50 mL to about 600 mL, about 50 mL to about 500 mL, about 50 mL to about 400 mL, about 50 mL to about 300 mL, about 50 mL to about 250 mL, about 50 mL to about 100 mL, about 50 mL to about 90 mL, About 50 mL to about 80 mL, about 50 mL to about 70 mL, about 50 mL to about 60 mL; about 75 mL to 10,000 L, about 75 mL to about 9,000 L, about 75 mL to about 8,000 L, about 75 mL ~ 7,000 L, about 75 mL ~ about 6,000 L, about 75 mL ~ about 5,000 L, about 75 mL ~ about 4,000 L, about 75 mL ~ about 3,000 L, about 75 mL ~ about 2,000 L, about 75 mL ~ about 1,000 L, about 75 mL to about 950 L, about 75 mL to about 900 L, about 75 mL to about 850 L, about 75 mL to about 800 L, about 75 mL to about 750 L, about 75 mL to about 700 L , About 75 mL-about 650 L, about 75 mL-about 600 L, about 75 mL-about 550 L, about 75 mL-about 500 L, about 75 mL-about 450 L, about 75 mL-about 400 L, about 75 mL to about 350 L, about 75 mL to about 300 L , About 75 mL to about 250 L, about 75 mL to about 200 L, about 75 mL to about 190 L, about 75 mL to about 180 L, about 75 mL to about 170 L, about 75 mL to about 160 L, about 75 mL to about 150 L, about 75 mL to about 140 L, about 75 mL to about 130 L, about 75 mL to about 120 L, about 75 mL to about 110 L, about 75 mL to about 100 L, about 75 mL ~ About 90 L, about 75 mL ~ about 80 L, about 75 mL ~ about 70 L, about 75 mL ~ about 60 L, about 75 mL ~ about 50 L, about 75 mL ~ about 40 L, about 75 mL ~ about 30 L, about 75 mL to about 20 L, about 75 mL to about 10 L, about 75 mL to about 5 L, about 75 mL to about 4 L, about 75 mL to about 3 L, about 75 mL to about 5 L , About 75 mL ~ about 1 L, about 75 mL ~ about 900 mL, about 75 mL ~ about 800 mL, about 75 mL ~ about 700 mL, about 75 mL ~ about 600 mL, about 75 mL ~ about 500 mL, about 75 mL-about 400 mL, about 75 mL-about 300 mL, about 75 mL-about 250 mL, about 75 mL-about 100 mL, about 75 mL-about 90 mL; about 100 mL-10,000 L, about 100 mL- About 9,000 L, about 100 mL to about 8,000 L, about 100 mL to about 7,000 L, about 100 mL to about 6,000 L, about 100 mL to about 5,000 L, about 100 mL to about 4,000 L, about 100 mL to about 3,000 L, about 100 mL to about 2,000 L, about 100 mL to about 1,000 L, about 100 mL to about 950 L, about 100 mL to about 900 L, about 100 mL to about 850 L, about 100 mL to about 800 L, About 100 mL to about 750 L, about 100 mL to about 700 L, about 100 mL to about 650 L, about 100 mL to about 600 L, about 100 mL to about 550 L, about 100 mL to about 500 L, about 100 mL ~ about 450 L, about 100 mL ~ about 400 L, about 100 mL ~ about 350 L, about 100 mL ~ about 300 L, about 100 mL ~ about 250 L, about 100 mL ~ about 200 L, about 100 mL ~ About 190 L, about 100 mL to about 180 L, about 100 mL to about 170 L, about 100 mL to about 160 L, about 100 mL to about 150 L, about 100 mL to about 140 L, about 100 mL ~ about 130 L, about 100 mL ~ about 120 L, about 100 mL ~ about 110 L, about 100 mL ~ about 100 L, about 100 mL ~ about 90 L, about 100 mL ~ about 80 L, about 100 mL ~ About 70 L, about 100 mL to about 60 L, about 100 mL to about 50 L, about 100 mL to about 40 L, about 100 mL to about 30 L, about 100 mL to about 20 L, about 100 mL to about 10 L, about 100 mL to about 5 L, about 100 mL to about 4 L, about 100 mL to about 3 L, about 100 mL to about 5 L, about 100 mL to about 1 L, about 100 mL to about 900 mL, About 100 mL to about 800 mL, about 100 mL to about 700 mL, about 100 mL to about 600 mL, about 100 mL to about 500 mL, about 100 mL to about 400 mL, about 100 mL to about 300 mL, about 100 mL ~ about 250 mL; about 250 mL ~ 10,000 L, about 250 mL ~ about 9,000 L, about 250 mL ~ about 8,000 L, about 250 mL ~ about 7,000 L, about 250 mL ~ about 6,000 L, about 250 mL ~ about 5,000 L, about 250 mL to about 4,000 L, about 250 mL to about 3,000 L, about 250 mL to about 2,000 L, about 250 mL to about 1,000 L, about 250 mL to about 950 L, about 250 mL to about 900 L , About 250 mL to about 850 L, about 250 mL to about 800 L, about 250 mL to about 750 L, about 250 mL to about 700 L, about 250 mL to about 650 L, about 250 mL to about 600 L, about 250 mL to about 550 L, about 250 mL to about 500 L, about 250 mL to about 450 L, about 250 mL to about 400 L, about 250 mL to about 350 L, about 250 mL to about 300 L, about 250 mL ~ About 250 L, about 250 mL ~ about 200 L, about 250 mL ~ about 190 L, about 250 mL ~ about 180 L, about 250 mL ~ about 170 L, about 250 mL ~ about 160 L, about 250 mL ~ about 150 L, about 250 mL to about 140 L, about 250 mL to about 130 L, about 250 mL to about 120 L, about 250 mL to about 110 L, about 250 mL to about 100 L, about 250 mL to about 90 L , About 250 mL ~ about 80 L, about 250 mL ~ about 70 L, about 250 mL ~ about 60 L, about 250 mL ~ about 5 0 L, about 250 mL to about 40 L, about 250 mL to about 30 L, about 250 mL to about 20 L, about 250 mL to about 10 L, about 250 mL to about 5 L, about 250 mL to about 4 L , About 250 mL ~ about 3 L, about 250 mL ~ about 5 L, about 250 mL ~ about 1 L, about 250 mL ~ about 900 mL, about 250 mL ~ about 800 mL, about 250 mL ~ about 700 mL, about 250 mL-about 600 mL, about 250 mL-about 500 mL, about 250 mL-about 400 mL, about 250 mL-about 300 mL; about 300 mL-10,000 L, about 300 mL-about 9,000 L, about 300 mL- About 8,000 L, about 300 mL to about 7,000 L, about 300 mL to about 6,000 L, about 300 mL to about 5,000 L, about 300 mL to about 4,000 L, about 300 mL to about 3,000 L, about 300 mL to about 2,000 L, about 300 mL to about 1,000 L, about 300 mL to about 950 L, about 300 mL to about 900 L, about 300 mL to about 850 L, about 300 mL to about 800 L, about 300 mL to about 750 L, About 300 mL to about 700 L, about 300 mL to about 650 L, about 300 mL to about 600 L, about 300 mL to about 550 L, about 300 mL to about 500 L, about 300 mL to about 450 L, about 300 mL ~ about 400 L, about 300 mL ~ about 350 L, about 300 mL ~ about 300 L, about 300 mL ~ about 250 L, about 300 mL ~ about 200 L, about 300 mL ~ about 190 L, about 300 mL ~ About 180 L, about 300 mL to about 170 L, about 300 mL to about 160 L, about 300 mL to about 150 L, about 300 mL to about 140 L, about 300 mL to about 130 L, about 300 mL to about 120 L, about 300 mL to about 110 L, about 300 mL to about 100 L, about 300 mL to about 90 L, about 300 mL to about 80 L, about 300 mL to about 70 L, about 300 mL to about 60 L, About 300 mL to about 50 L, about 300 mL to about 40 L, about 300 mL to about 30 L, about 300 mL to about 20 L, about 300 mL to about 10 L, about 300 mL to about 5 L, about 300 mL ~ about 4 L, about 300 mL ~ about 3 L, about 300 mL ~ about 5 L, about 300 mL ~ about 1 L, about 300 mL ~ about 900 mL, about 300 mL to about 800 mL, about 300 mL to about 700 mL, about 300 mL to about 600 mL, about 300 mL to about 500 mL, about 300 mL to about 400 mL; about 450 mL to 10,000 L, about 450 mL to about 9,000 L, about 450 mL to about 8,000 L, about 450 mL to about 7,000 L, about 450 mL to about 6,000 L, about 450 mL to about 5,000 L, about 450 mL to about 4,000 L, about 450 mL ~ 3,000 L, about 450 mL ~ about 2,000 L, about 450 mL ~ about 1,000 L, about 450 mL ~ about 950 L, about 450 mL ~ about 900 L, about 450 mL ~ about 850 L, about 450 mL ~ about 800 L, about 450 mL to about 750 L, about 450 mL to about 700 L, about 450 mL to about 650 L, about 450 mL to about 600 L, about 450 mL to about 550 L, about 450 mL to about 500 L , About 450 mL to about 450 L, about 450 mL to about 400 L, about 450 mL to about 350 L, about 450 mL to about 300 L, about 450 mL to about 250 L, about 450 mL to about 200 L, about 450 mL to about 190 L, about 450 mL to about 180 L, about 450 mL to about 170 L, about 450 mL to about 160 L, about 450 mL to about 150 L, about 450 mL to about 140 L, about 450 mL ~ About 130 L, about 450 mL ~ about 120 L, about 450 mL ~ about 110 L, about 450 mL ~ about 100 L, about 450 mL ~ about 90 L, about 450 mL ~ about 80 L, about 450 mL ~ about 70 L, about 450 mL to about 60 L, about 450 mL to about 50 L, about 450 mL to about 40 L, about 450 mL to about 30 L, about 450 mL to about 20 L, about 450 mL to about 10 L , About 450 mL ~ about 5 L, about 450 mL ~ about 4 L, about 450 mL ~ about 3 L, about 450 mL ~ about 5 L, about 450 mL ~ about 1 L, about 450 mL ~ about 900 mL, about 450 mL-about 800 mL, about 450 mL-about 700 mL, about 450 mL-about 600 mL; about 500 mL-10,000 L, about 500 mL-about 9,000 L, about 500 mL-about 8,000 L, about 500 mL- About 7,000 L, about 500 mL to about 6,000 L, about 500 mL to about 5,000 L , About 500 mL to about 4,000 L, about 500 mL to about 3,000 L, about 500 mL to about 2,000 L, about 500 mL to about 1,000 L, about 500 mL to about 950 L, about 500 mL to about 900 L, about 500 mL to about 850 L, about 500 mL to about 800 L, about 500 mL to about 750 L, about 500 mL to about 700 L, about 500 mL to about 650 L, about 500 mL to about 600 L, about 500 mL ~ About 550 L, about 500 mL ~ about 500 L, about 500 mL ~ about 450 L, about 500 mL ~ about 400 L, about 500 mL ~ about 350 L, about 500 mL ~ about 300 L, about 500 mL ~ about 250 L, about 500 mL to about 200 L, about 500 mL to about 190 L, about 500 mL to about 180 L, about 500 mL to about 170 L, about 500 mL to about 160 L, about 500 mL to about 150 L , About 500 mL to about 140 L, about 500 mL to about 130 L, about 500 mL to about 120 L, about 500 mL to about 110 L, about 500 mL to about 100 L, about 500 mL to about 90 L, about 500 mL to about 80 L, about 500 mL to about 70 L, about 500 mL to about 60 L, about 500 mL to about 50 L, about 500 mL to about 40 L, about 500 mL to about 30 L, about 500 mL ~ About 20 L, about 500 mL ~ about 10 L, about 500 mL ~ about 5 L, about 500 mL ~ about 4 L, about 500 mL ~ about 3 L, about 500 mL ~ about 5 L, about 500 mL ~ about 1 L, about 500 mL to about 900 mL, about 500 mL to about 800 mL, about 500 mL to about 700 mL, about 500 mL to about 600 mL; about 750 mL to 10,000 L, about 750 mL to about 9,000 L, About 750 mL to about 8,000 L, about 750 mL to about 7,000 L, about 750 mL to about 6,000 L, about 750 mL to about 5,000 L, about 750 mL to about 4,000 L, about 750 mL to about 3,000 L, about 750 mL ~ about 2,000 L, about 750 mL ~ about 1,000 L, about 750 mL ~ about 950 L, about 750 mL ~ about 900 L, about 750 mL ~ about 850 L, about 750 mL ~ about 800 L, about 750 mL ~ About 750 L, about 750 mL to about 700 L, about 750 mL to about 650 L, about 750 mL to about 60 0 L, about 750 mL to about 550 L, about 750 mL to about 500 L, about 750 mL to about 450 L, about 750 mL
~ About 400 L, about 750 mL ~ about 350 L, about 750 mL ~ about 300 L, about 750 mL ~ about 250 L, about 750 mL ~ about 200 L, about 750 mL ~ about 190 L, about 750 mL ~ about 180 L, about 750 mL to about 170 L, about 750 mL to about 160 L, about 750 mL to about 150 L, about 750 mL to about 140 L, about 750 mL to about 130 L, about 750 mL to about 120 L , About 750 mL to about 110 L, about 750 mL to about 100 L, about 750 mL to about 90 L, about 750 mL to about 80 L, about 750 mL to about 70 L, about 750 mL to about 60 L, about 750 mL to about 50 L, about 750 mL to about 40 L, about 750 mL to about 30 L, about 750 mL to about 20 L, about 750 mL to about 10 L, about 750 mL to about 5 L, about 750 mL ~ About 4 L, about 750 mL ~ about 3 L, about 750 mL ~ about 5 L, about 750 mL ~ about 1 L, about 750 mL ~ about 900 mL, about 750 mL ~ about 800 mL; about 1 L ~ 10,000 L, about 1 L to about 9,000 L, about 1 L to about 8,000 L, about 1 L to about 7,000 L, about 1 L to about 6,000 L, about 1 L to about 5,000 L, about 1 L to about 4,000 L, About 1 L to about 3,000 L, about 1 L to about 2,000 L, about 1 L to about 1,000 L, about 1 L to about 950 L, about 1 L to about 900 L, about 1 L to about 850 L, about 1 L ~ about 800 L, about 1 L ~ about 750 L, about 1 L ~ about 700 L, about 1 L ~ about 650 L, about 1 L ~ about 600 L, about 1 L ~ about 550 L, about 1 L ~ About 500 L, about 1 L to about 450 L, about 1 L to about 400 L, about 1 L to about 350 L, about 1 L to about 300 L, about 1 L to about 250 L, about 1 L to about 200 L, about 1 L to about 190 L, about 1 L to about 180 L, about 1 L to about 170 L, about 1 L to about 160 L, about 1 L to about 150 L, about 1 L to about 140 L, About 1 L to about 130 L, about 1 L to about 120 L, about 1 L to about 110 L, about 1 L to about 100 L, about 1 L to about 90 L, about 1 L to about 80 L, about 1 L ~ about 70 L, about 1 L ~ about 60 L, about 1 L ~ about 50 L, about 1 L ~ about 40 L, about 1 L ~ about 30 L, about 1 L ~ about 20 L, about 1 L ~ About 10 L, about 1 L to about 5 L, about 1 L to about 4 L, about 1 L to about 3 L, about 1 L to about 2 L; about 2 L to 10,000 L, about 2 L to about 9,000 L, about 2 L to about 8,000 L, about 2 L to about 7,000 L, about 2 L to about 6,000 L, about 2 L to about 5,000 L, about 2 L to about 4,000 L, about 2 L to about 3,000 L, about 2 L to about 2,000 L, about 2 L ~ About 1,000 L, About 2 L ~ About 950 L, About 2 L ~ About 900 L, About 2 L ~ About 850 L, About 2 L ~ About 800 L, About 2 L ~ About 750 L, About 2 L ~ About 700 L, about 2 L to about 650 L, about 2 L to about 600 L, about 2 L to about 550 L, about 2 L to about 500 L, about 2 L to about 450 L, about 2 L to about 400 L , About 2 L to about 350 L, about 2 L to about 300 L, about 2 L to about 250 L, about 2 L to about 200 L, about 2 L to about 190 L, about 2 L to about 180 L, about 2 L to about 170 L, about 2 L to about 160 L, about 2 L to about 150 L, about 2 L to about 140 L, about 2 L to about 130 L, about 2 L to about 120 L, about 2 L ~ 110 L, about 2 L ~ about 100 L, about 2 L ~ about 90 L, about 2 L ~ about 80 L, about 2 L ~ about 70 L, about 2 L ~ about 60 L, about 2 L ~ about 50 L, about 2 L to about 40 L, about 2 L to about 30 L, about 2 L to about 20 L, about 2 L to about 10 L, about 2 L to about 5 L, about 2 L to about 4 L , About 2 L to about 3 L; about 4 L to 10,000 L, about 4 L to about 9,000 L, about 4 L to about 8,000 L, about 4 L to about 7,000 L, about 4 L to about 6,000 L, about 4 L ~ about 5,000 L, about 4 L ~ about 4,000 L, about 4 L ~ about 3,000 L, about 4 L ~ about 2,000 L, about 4 L ~ about 1,000 L, about 4 L ~ about 950 L, about 4 L ~ About 900 L, about 4 L to about 850 L, about 4 L to about 800 L, about 4 L to about 750 L, about 4 L to about 700 L, about 4 L to about 650 L, about 4 L to about 600 L, about 4 L to about 550 L, about 4 L to about 500 L, about 4 L to about 450 L, about 4 L to about 400 L, about 4 L to about 350 L, about 4 L to about 300 L, About 4 L to about 250 L, about 4 L to about 200 L, about 4 L to about 190 L, about 4 L to about 180 L, about 4 L to about 170 L, about 4 L to about 160 L, about 4 L ~ about 1 50 L, about 4 L to about 140 L, about 4 L to about 130 L, about 4 L to about 120 L, about 4 L to about 110 L, about 4 L to about 100 L, about 4 L to about 90 L , About 4 L to about 80 L, about 4 L to about 70 L, about 4 L to about 60 L, about 4 L to about 50 L, about 4 L to about 40 L, about 4 L to about 30 L, about 4 L ~ about 20 L, about 4 L ~ about 10 L, about 4 L ~ about 5 L; about 5 L ~ 10,000 L, about 5 L ~ about 9,000 L, about 5 L ~ about 8,000 L, about 5 L ~ About 7,000 L, about 5 L to about 6,000 L, about 5 L to about 5,000 L, about 5 L to about 4,000 L, about 5 L to about 3,000 L, about 5 L to about 2,000 L, about 5 L to about 1,000 L, about 5 L to about 950 L, about 5 L to about 900 L, about 5 L to about 850 L, about 5 L to about 800 L, about 5 L to about 750 L, about 5 L to about 700 L, About 5 L to about 650 L, about 5 L to about 600 L, about 5 L to about 550 L, about 5 L to about 500 L, about 5 L to about 450 L, about 5 L to about 400 L, about 5 L ~ about 350 L, about 5 L ~ about 300 L, about 5 L ~ about 250 L, about 5 L ~ about 200 L, about 5 L ~ about 190 L, about 5 L ~ about 180 L, about 5 L ~ About 170 L, about 5 L to about 160 L, about 5 L to about 150 L, about 5 L to about 140 L, about 5 L to about 130 L, about 5 L to about 120 L, about 5 L to about 110 L, about 5 L to about 100 L, about 5 L to about 90 L, about 5 L to about 80 L, about 5 L to about 70 L, about 5 L to about 60 L, about 5 L to about 50 L, About 5 L to about 40 L, about 5 L to about 30 L, about 5 L to about 20 L, about 5 L to about 10 L, about 6 L to about 10 L, about 6 L to about 8 L, about 8 L ~ about 10 L; about 7.5 L ~ 10,000 L, about 7.5 L ~ about 9,000 L, about 7.5 L ~ about 8,000 L, about 7.5 L ~ about 7,000 L, about 7.5 L ~ about 6,000 L, about 7.5 L ~ about 5,000 L, about 7.5 L to about 4,000 L, about 7.5 L to about 3,000 L, about 7.5 L to about 2,000 L, about 7.5 L to about 1,000 L, about 7.5 L to about 950 L, about 7.5 L to about 900 L , About 7.5 L to about 850 L, about 7.5 L to about 800 L, about 7.5 L to about 750 L, about 7.5 L to about 700 L , About 7.5 L to about 650 L, about 7.5 L to about 600 L, about 7.5 L to about 550 L, about 7.5 L to about 500 L, about 7.5 L to about 450 L, about 7.5 L to about 400 L, about 7.5 L to about 350 L, about 7.5 L to about 300 L, about 7.5 L to about 250 L, about 7.5 L to about 200 L, about 7.5 L to about 190 L, about 7.5 L to about 180 L, about 7.5 L ~ 170 L, about 7.5 L ~ about 160 L, about 7.5 L ~ about 150 L, about 7.5 L ~ about 140 L, about 7.5 L ~ about 130 L, about 7.5 L ~ about 120 L, about 7.5 L ~ about 110 L, about 7.5 L to about 100 L, about 7.5 L to about 90 L, about 7.5 L to about 80 L, about 7.5 L to about 70 L, about 7.5 L to about 60 L, about 7.5 L to about 50 L , About 7.5 L to about 40 L, about 7.5 L to about 30 L, about 7.5 L to about 20 L, about 7.5 L to about 10 L; about 10 L to 10,000 L, about 10 L to about 9,000 L, about 10 L ~ about 8,000 L, about 10 L ~ about 7,000 L, about 10 L ~ about 6,000 L, about 10 L ~ about 5,000 L, about 10 L ~ about 4,000 L, about 10 L ~ about 3,000 L, about 10 L ~ About 2,000 L, about 10 L to about 1,000 L, about 10 L to about 950 L, about 10 L to about 900 L, about 10 L to about 850 L, about 10 L to about 800 L, about 10 L to about 750 L, about 10 L to about 700 L, about 10 L to about 650 L, about 10 L to about 600 L, about 10 L to about 550 L, about 10 L to about 500 L, about 10 L to about 450 L, About 10 L to about 400 L, about 10 L to about 350 L, about 10 L to about 300 L, about 10 L to about 250 L, about 10 L to about 200 L, about 10 L to about 190 L, about 10 L ~ about 180 L, about 10 L ~ about 170 L, about 10 L ~ about 160 L, about 10 L ~ about 150 L, about 10 L ~ about 140 L, about 10 L ~ about 130 L, about 10 L ~ About 120 L, about 10 L to about 110 L, about 10 L to about 100 L, about 10 L to about 90 L, about 10 L to about 80 L, about 10 L to about 70 L, about 10 L to about 60 L, about 10 L to about 50 L, about 10 L to about 40 L, about 10 L to about 30 L, about 10 L to about 20 L, about 10 L to about 15 L, about 15 L ~ About 20 L, about 15 L to about 25 L, about 15 L to about 30 L; about 15 L to about 40 L, about 15 L to about 50 L, about 15 L to about 60 L, about 15 L to about 70 L, about 15 L to about 80 L, about 15 L to about 90 L, about 15 L to about 100 L, about 15 L to about 110 L, about 15 L to about 120 L, about 15 L to about 130 L, About 15 L to about 140 L, about 15 L to about 150 L, about 15 L to about 160 L, about 15 L to about 170 L, about 15 L to about 180 L, about 15 L to about 190 L; about 15 L ~ about 200 L, about 15 L ~ about 250 L, about 15 L ~ about 300 L, about 15 L ~ about 350 L, about 15 L ~ about 400 L, about 15 L ~ about 450 L, about 15 L ~ About 500 L, about 15 L to about 550 L, about 15 L to about 600 L, about 15 L to about 650 L, about 15 L to about 700 L, about 15 L to about 750 L, about 15 L to about 800 L, about 15 L to about 850 L, about 15 L to about 900 L, about 15 L to about 950 L, about 15 L to about 1,000 L, about 15 L to about 2,000 L, about 15 L to about 3,000 L, About 15 L to about 4,000 L, about 15 L to about 5,000 L, about 15 L to about 6,000 L, about 15 L to about 7,000 L, about 15 L to about 8,000 L, about 15 L to about 9,000 L, about 15 L ~ 10,000 L; about 20 L ~ about 25 L, about 20 L ~ about 30 L, about 20 L ~ about 40 L, about 20 L ~ about 50 L, about 20 L ~ about 60 L, about 20 L ~ about 70 L, about 20 L to about 80 L, about 20 L to about 90 L, about 20 L to about 100 L, about 20 L to about 110 L, about 20 L to about 120 L, about 20 L to about 130 L , About 20 L to about 140 L, about 20 L to about 150 L, about 20 L to about 160 L, about 20 L to about 170 L, about 20 L to about 180 L, about 20 L to about 190 L, about 20 L to about 200 L, about 20 L to about 250 L, about 20 L to about 300 L, about 20 L to about 350 L, about 20 L to about 400 L, about 20 L to about 450 L, about 20 L ~ About 500 L, About 20 L ~ About 550 L, About 20 L ~ About 600 L, About 20 L ~ About 650 L, About 20 L ~ About 700 L, About 20 L ~ About 750 L, About 20 L ~ About 800 L, about 20 L to about 850 L, about 2 0 L to about 900 L, about 20 L to about 950 L, about 20 L to about 1,000 L, about 20 L to about 2,000 L, about 20 L to about 3,000 L, about 20 L to about 4,000 L, about 20 L ~ 5,000 L, about 20 L ~ about 6,000 L, about 20 L ~ about 7,000 L, about 20 L ~ about 8,000 L, about 20 L ~ about 9,000 L, about 20 L ~ 10,000 L; about 25 L ~ about 30 L; about 25 L to about 40 L, about 25 L to about 50 L, about 25 L to about 60 L, about 25 L to about 70 L, about 25 L to about 80 L, about 25 L to about 90 L, About 25 L to about 100 L, about 25 L to about 110 L, about 25 L to about 120 L, about 25 L to about 130 L, about 25 L to about 140 L, about 25 L to about 150 L, about 25 L ~ about 160 L, about 25 L ~ about 170 L, about 25 L ~ about 180 L, about 25 L ~ about 190 L, about 25 L ~ about 200 L, about 25 L ~ about 250 L, about 25 L ~ About 300 L, about 25 L to about 350 L, about 25 L to about 400 L, about 25 L to about 450 L, about 25 L to about 500 L, about 25 L to about 550 L, about 25 L to about 600 L, about 25 L to about 650 L, about 25 L to about 700 L, about 25 L to about 750 L, about 25 L to about 800 L, about 25 L to about 850 L, about 25 L to about 900 L, About 25 L to about 950 L, about 25 L to about 1,000 L, about 25 L to about 2,000 L, about 25 L to about 3,000 L, about 25 L to about 4,000 L, about 25 L to about 5,000 L, about 25 L ~ about 6,000 L, about 25 L ~ about 7,000 L, about 25 L ~ about 8,000 L, about 25 L ~ about 9,000 L, about 25 L ~ 10,000 L; about 30 L ~ about 50 L, about 30 L ~ about 75 L, about 30 L to about 100 L, about 30 L to about 150 L, about 30 L to about 200 L, about 30 L to about 250 L, about 30 L to about 300 L, about 30 L to about 350 L , About 30 L to about 400 L, about 30 L to about 450 L, about 30 L to about 500 L, about 30 L to about 550 L, about 30 L to about 600 L, about 30 L to about 650 L, about 30 L to about 700 L, about 30 L to about 750 L, about 30 L to about 800 L, about 30 L to about 850 L, about 30 L to about 900 L, about 30 L to about 95 0 L, about 30 L to about 1,000 L, about 30 L to about 2,000 L, about 30 L to about 3,000 L, about 30 L to about 4,000 L, about 30 L to about 5,000 L, about 30 L to about 6,000 L , About 30 L to about 7,000 L, about 30 L to about 8,000 L, about 30 L to about 9,000 L, about 30 L to 10,000 L; about 40 L to about 75 L, about 40 L to about 100 L, about 40 L ~ about 150 L, about 40 L ~ about 200 L; about 40 L ~ about 250 L, about 40 L ~ about 300 L, about 40 L ~ about 350 L, about 40 L ~ about 400 L, about 40 L ~ About 450 L, about 40 L to about 500 L, about 40 L to about 550 L, about 40 L to about 600 L, about 40 L to about 650 L, about 40 L to about 700 L, about 40 L to about 750 L, about 40 L to about 800 L, about 40 L to about 850 L, about 40 L to about 900 L, about 40 L to about 950 L, about 40 L to about 1,000 L, about 40 L to about 2,000 L, About 40 L to about 3,000 L, about 40 L to about 4,000 L, about 40 L to about 5,000 L, about 40 L to about 6,000 L, about 40 L to about 7,000 L, about 40 L to about 8,000 L, about 40 L ~ about 9,000 L, about 40 L ~ 10,000 L; about 50 L ~ about 60 L, about 50 L ~ about 70 L, about 50 L ~ about 80 L, about 50 L ~ about 90 L, about 50 L ~ about 100 L, about 50 L to about 110 L, about 50 L to about 120 L, about 50 L to about 130 L, about 50 L to about 140 L, about 50 L to about 150 L, about 50 L to about 160 L , About 50 L to about 170 L, about 50 L to about 180 L, about 50 L to about 190 L, about 50 L to about 200 L, about 50 L to about 250 L, about 50 L to about 300 L, about 50 L to about 350 L, about 50 L to about 400 L, about 50 L to about 450 L, about 50 L to about 500 L, about 50 L to about 550 L, about 50 L to about 600 L, about 50 L ~ About 650 L, About 50 L ~ About 700 L, About 50 L ~ About 750 L, About 50 L ~ About 800 L, About 50 L ~ About 850 L, About 50 L ~ About 900 L, About 50 L ~ About 950 L, about 50 L to about 1,000 L, about 50 L to about 2,000 L, about 50 L to about 3,000 L, about 50 L to about 4,000 L, about 50 L ~ about 5,000 L, about 50 L ~ about 6,000 L, about 50 L ~ about 7,000 L, about 50 L ~ about 8,000 L, about 50 L ~ about 9,000 L, about 50 L ~ 10,000 L; about 75 L ~ about 100 L, about 75 L to about 110 L, about 75 L to about 120 L, about 75 L to about 130 L, about 75 L to about 140 L, about 75 L to about 150 L, about 75 L to about 160 L , About 75 L to about 170 L, about 75 L to about 180 L, about 75 L to about 190 L, about 75 L to about 200 L, about 75 L to about 250 L, about 75 L to about 300 L, about 75 L to about 350 L, about 75 L to about 400 L, about 75 L to about 450 L, about 75 L to about 500 L, about 75 L to about 550 L, about 75 L to about 600 L, about 75 L ~ About 650 L, About 75 L ~ About 700 L, About 75 L ~ About 750 L, About 75 L ~ About 800 L, About 75 L ~ About 850 L, About 75 L ~ About 900 L, About 75 L ~ About 950 L, about 75 L to about 1,000 L, about 75 L to about 2,000 L, about 75 L to about 3,000 L, about 75 L to about 4,000 L, about 75 L to about 5,000 L, about 75 L to about 6,000 L , About 75 L to about 7,000 L, about 75 L to about 8,000 L, about 75 L to about 9,000 L, about 75 L to 10,000 L; about 100 L to about 110 L, about 100 L to about 120 L, about 100 L ~ about 130 L, about 100 L ~ about 140 L, about 100 L ~ about 150 L, about 100 L ~ about 160 L, about 100 L ~ about 170 L, about 100 L ~ about 180 L, about 100 L ~ About 190 L, about 100 L to about 200 L, about 100 L to about 250 L, about 100 L to about 300 L, about 100 L to about 350 L, about 100 L to about 400 L, about 100 L to about 450 L, about 100 L to about 500 L, about 100 L to about 550 L, about 100 L to about 600 L, about 100 L to about 650 L, about 100 L to about 700 L, about 100
L ~ about 750 L, about 100 L ~ about 800 L, about 100 L ~ about 850 L, about 100 L ~ about 900 L, about 100 L ~ about 950 L, about 100 L ~ about 1,000 L, about 100 L ~ About 2,000 L, about 100 L to about 3,000 L, about 100 L to about 4,000 L, about 100 L to about 5,000 L, about 100 L to about 6,000 L, about 100 L to about 7,000 L, about 100 L to about 8,000 L, about 100 L to about 9,000 L, about 100 L to 10,000 L; about 120 L to about 130 L, about 120 L to about 140 L, about 120 L to about 150 L, about 120 L to about 160 L, about 120 L to about 170 L, about 120 L to about 180 L, about 120 L to about 190 L, about 120 L to about 200 L, about 120 L to about 250 L, about 120 L to about 300 L, about 120 L ~ About 350 L, About 120 L ~ About 400 L, About 120 L ~ About 450 L, About 120 L ~ About 500 L, About 120 L ~ About 550 L, About 120 L ~ About 600 L, About 120 L ~ About 650 L, about 120 L to about 700 L, about 120 L to about 750 L, about 120 L to about 800 L, about 120 L to about 850 L, about 120 L to about 900 L, about 120 L to about 950 L , About 120 L to about 1,000 L, about 120 L to about 2,000 L, about 120 L to about 3,000 L, about 120 L to about 4,000 L, about 120 L to about 5,000 L, about 120 L to about 6,000 L, about 120 L ~ about 7,000 L, about 120 L ~ about 8,000 L, about 120 L ~ about 9,000 L, about 120 L ~ 10,000 L; about 150 L ~ about 160 L, about 150 L ~ about 170 L, about 150 L ~ About 180 L, about 150 L to about 190 L, about 150 L to about 200 L, about 150 L to about 250 L, about 150 L to about 300 L, about 150 L to about 350 L, about 150 L to about 400 L, about 150 L to about 450 L, about 150 L to about 500 L, about 150 L to about 550 L, about 150 L to about 600 L, about 150 L to about 650 L, about 150 L to about 700 L, About 150 L to about 750 L, about 150 L to about 800 L, about 150 L to about 850 L, about 150 L to about 900 L, about 150 L to about 950 L, about 150 L to about 1,000 L, about 150 L to about 2,000 L, about 150 L to about 3,000 L, about 150 L to about 4,000 L, about 150 L to about 5,000 L, about 150 L to about 6,000 L, about 150 L to about 7,000 L, About 150 L to about 8,000 L, about 150 L to about 9,000 L, about 150 L to 10,000 L; about 200 L to about 250 L, about 200 L to about 300 L, about 200 L to about 350 L, about 200 L ~ About 400 L, About 200 L ~ About 450 L, About 200 L ~ About 500 L, About 200 L ~ About 550 L, About 200 L ~ About 600 L, About 200 L ~ About 650 L, About 200 L ~ About 700 L, about 200 L to about 750 L, about 200 L to about 800 L, about 200 L to about 850 L, about 200 L to about 900 L, about 200 L to about 950 L, about 200 L to about 1,000 L , About 200 L to about 2,000 L, about 200 L to about 3,000 L, about 200 L to about 4,000 L, about 200 L to about 5,000 L, about 200 L to about 6,000 L, about 200 L to about 7,000 L, about 200 L ~ about 8,000 L, about 200 L ~ about 9,000 L, about 200 L ~ 10,000 L; about 250 L ~ about 300 L, about 250 L ~ about 350 L, about 250 L ~ about 400 L, about 250 L ~ About 450 L, about 250 L to about 500 L, about 250 L to about 550 L, about 250 L to about 600 L, about 250 L to about 650 L, about 250 L to about 700 L, about 250 L to about 750 L, about 250 L to about 800 L, about 250 L to about 850 L, about 250 L to about 900 L, about 250 L to about 950 L, about 250 L to about 1,000 L, about 250 L to about 2,000 L, About 250 L to about 3,000 L, about 250 L to about 4,000 L, about 250 L to about 5,000 L, about 250 L to about 6,000 L, about 250 L to about 7,000 L, about 250 L to about 8,000 L, about 250 L ~ about 9,000 L, about 250 L ~ 10,000 L; about 300 L ~ about 350 L, about 300 L ~ about 400 L, about 300 L ~ about 450 L, about 300 L ~ about 500 L, about 300 L ~ about 550 L, about 300 L to about 600 L, about 300 L to about 650 L, about 300 L to about 700 L, about 300 L to about 750 L, about 300 L to about 800 L, about 300 L to about 850 L, about 300 L to about 900 L, about 300 L to about 950 L, about 300 L to about 1,000 L, about 300 L to about 2,000 L, about 300 L ~ About 3,000 L, About 300 L ~ About 4,000 L, About 300 L ~ About 5,000 L, About 300 L ~ About 6,000 L, About 300 L ~ About 7,000 L, About 300 L ~ About 8,000 L, About 300 L ~ About 9,000 L, about 300 L to 10,000 L; about 350 L to about 400 L, about 350 L to about 450 L, about 350 L to about 500 L, about 350 L to about 550 L, about 350 L to about 600 L, About 350 L to about 650 L, about 350 L to about 700 L, about 350 L to about 750 L, about 350 L to about 800 L, about 350 L to about 850 L, about 350 L to about 900 L, about 350 L ~ about 950 L, about 350 L ~ about 1,000 L, about 350 L ~ about 2,000 L, about 350 L ~ about 3,000 L, about 350 L ~ about 4,000 L, about 350 L ~ about 5,000 L, about 350 L ~ About 6,000 L, about 350 L to about 7,000 L, about 350 L to about 8,000 L, about 350 L to about 9,000 L, about 350 L to 10,000 L; about 400 L to about 450 L, about 400 L to about 500 L , About 400 L to about 550 L, About 400 L to about 600 L, About 400 L to about 650 L, About 400 L to about 700 L, About 400 L to about 750 L, About 400 L to about 800 L, About 400 L to about 850 L, about 400 L to about 900 L, about 400 L to about 950 L, about 400 L to about 1,000 L, about 400 L to about 2,000 L, about 400 L to about 3,000 L, about 400 L ~ 4,000 L, about 400 L ~ about 5,000 L, about 400 L ~ about 6,000 L, about 400 L ~ about 7,000 L, about 400 L ~ about 8,000 L, about 400 L ~ about 9,000 L, about 400 L ~ 10,000 L; about 450 L to about 500 L, about 450 L to about 550 L, about 450 L to about 600 L, about 450 L to about 650 L, about 450 L to about 700 L, about 450 L to about 750 L, About 450 L to about 800 L, about 450 L to about 850 L, about 450 L to about 900 L, about 450 L to about 950 L, about 45 0 L to about 1,000 L, about 450 L to about 2,000 L, about 450 L to about 3,000 L, about 450 L to about 4,000 L, about 450 L to about 5,000 L, about 450 L to about 6,000 L, about 450 L ~ 7,000 L, about 450 L ~ about 8,000 L, about 450 L ~ about 9,000 L, about 450 L ~ 10,000 L; about 500 L ~ about 550 L, about 500 L ~ about 600 L, about 500 L ~ about 650 L, about 500 L to about 700 L, about 500 L to about 750 L, about 500 L to about 800 L, about 500 L to about 850 L, about 500 L to about 900 L, about 500 L to about 950 L, About 500 L to about 1,000 L, about 500 L to about 2,000 L, about 500 L to about 3,000 L, about 500 L to about 4,000 L, about 500 L to about 5,000 L, about 500 L to about 6,000 L, about 500 L ~ about 7,000 L, about 500 L ~ about 8,000 L, about 500 L ~ about 9,000 L, about 500 L ~ 10,000 L; about 600 L ~ about 650 L, about 600 L ~ about 700 L, about 600 L ~ about 750 L, about 600 L to about 800 L, about 600 L to about 850 L, about 600 L to about 900 L, about 600 L to about 950 L, about 600 L to about 1,000 L, about 600 L to about 2,000 L , About 600 L to about 3,000 L, about 600 L to about 4,000 L, about 600 L to about 5,000 L, about 600 L to about 6,000 L, about 600 L to about 7,000 L, about 600 L to about 8,000 L, about 600 L ~ about 9,000 L, about 600 L ~ 10,000 L; about 700 L ~ about 750 L, about 700 L ~ about 800 L, about 700 L ~ about 850 L, about 700 L ~ about 900 L, about 700 L ~ About 950 L, about 700 L to about 1,000 L, about 700 L to about 2,000 L, about 700 L to about 3,000 L, about 700 L to about 4,000 L, about 700 L to about 5,000 L, about 700 L to about 6,000 L, about 700 L to about 7,000 L, about 700 L to about 8,000 L, about 700 L to about 9,000 L, about 700 L to 10,000 L; about 800 L to about 850 L, about 800 L to about 900 L, about 800 L to about 950 L, about 800 L to about 1,000 L, about 800 L to about 2,0 00 L, about 800 L to about 3,000 L, about 800 L to about 4,000 L, about 800 L to about 5,000 L, about 800 L to about 6,000 L, about 800 L to about 7,000 L, about 800 L to about 8,000 L , About 800 L ~ about 9,000 L, about 800 L ~ 10,000 L; about 900 L ~ about 950 L, about 900 L ~ about 1,000 L, about 900 L ~ about 2,000 L, about 900 L ~ about 3,000 L, about 900 L ~ about 4,000 L, about 900 L ~ about 5,000 L, about 900 L ~ about 6,000 L, about 900 L ~ about 7,000 L, about 900 L ~ about 8,000 L, about 900 L ~ about 9,000 L, about 900 L ~ It has a total internal volume of 10,000 L; about 950 L to about 10,000 L).

Vectors Provided herein are cells in a liquid cell culture medium that may contain nucleic acids or vectors encoding a recombinant protein (eg, any recombinant protein described herein). Nucleic acids encoding any of the recombinant proteins described herein may be included within the expression vector. One of ordinary skill in the art will be able to select a suitable expression vector for making the recombinant protein described herein and use that expression vector to express the recombinant protein in cells. For example, in the selection of an expression vector, the cell must be considered as it may be necessary for the vector to replicate in the cell and / or be integrated into the chromosome of the cell. An exemplary expression vector that can be used to produce recombinant proteins will also be described below.

A "vector" according to the present disclosure is a polynucleotide capable of inducing expression of a recombinant protein (eg, any recombinant protein described herein) in a cell. The vector acts like a "molecular carrier" that delivers a fragment of nucleic acid into a host cell (eg, any cell described herein). The vector may include an expression cassette containing a control sequence. The foreign polynucleotide may be inserted into the expression cassette of the vector nucleic acid for expression from it. The vector based on the present invention may exist in a circular or linear form. The term "vector" also includes artificial chromosomes or similar respective (respective) polynucleotides that allow the transfer of foreign nucleic acid fragments. Each vector may be used as an expression vector for intracellularly producing a recombinant protein as described herein.

A method of carrying DNA encoding a recombinant protein described herein and constructing a suitable vector suitable for transformation (eg, eukaryotic cells, mammalian cells, insect cells, yeast cells, bacterial cells). Is well known in the art. See, for example, Ausubel et al., Eds. "Current Protocols in Molecular Biology" Current Protocols, 1993; and Sambrook et al., Eds. "Molecular Cloning: A Laboratory Manual," 2nd ed., Cold Spring Harbor Press, 1989. I want to be.

Methods for Introducing Nucleic Acids into Cells To introduce any of the nucleic acids and expression vectors disclosed herein into cells (eg, eukaryotic cells, plant cells), various different methods known in the art are available. May be used. Non-limiting examples of methods that can be used to introduce nucleic acids into cells include lipoffection, transfection, electroporation, microinjection, calcium phosphate transfection, dendrimer-based transfection, cation polymer transfection, Includes cell squeezing, sonoporation, transformation, phototransfection, impalection, hydrodynamic delivery, magnetfection, viral transfection (eg adenovirus and lentivirus transduction), and nanoparticle transfection Is done. These and other methods of introducing nucleic acids into cells are well known in the art. In some embodiments, the nucleic acid introduced into the cell is transiently expressed. In some embodiments, the nucleic acid introduced into the cell is integrated into the cell genome.

The stage of contacting the pod with the liquid cell culture solution The stage of contact is at least a portion of the at least one area (eg, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, At least 70%) results in contact with liquid cell culture medium. In some embodiments, the contact step is performed under conditions that result in the capture of the recombinant protein by the chromatographic resin. In some embodiments, the contact step is performed under sterile conditions. In some embodiments, the contact step may be performed during agitation of the liquid cell culture (eg, by rocking, tilting, rotary agitation (eg, with a propeller), vortex, and / or aeration). .. In some embodiments, the contact step may occur when the liquid cell culture is in an exponential or steady growth phase. In some embodiments, the contact step may occur when the liquid cell culture is in the dying phase. In some embodiments of any of the methods described herein, the contact step may occur in the clear supernatant of the liquid cell culture medium.

In some embodiments, the contact stage is about 20 ° C to about 40 ° C (eg, about 20 ° C to about 40 ° C, about 20 ° C to about 25 ° C, about 20 ° C to about 30 ° C, about 20 ° C to about 35). ℃, about 20 ℃ to about 37 ℃, about 20 ℃ to about 38 ℃, about 20 ℃ to about 39 ℃, about 27 ℃ to about 30 ℃, about 25 ℃ to about 30 ℃, about 25 ℃ to about 37 ℃, About 25 ° C to about 38 ° C, about 25 ° C to about 39 ° C, about 25 ° C to about 40 ° C, about 27 ° C to about 30 ° C, about 27 ° C to about 35 ° C, about 27 ° C to about 37 ° C, about 27 ℃ ~ 38 ℃, about 27 ℃ ~ 39 ℃, about 30 ℃ ~ 35 ℃, about 30 ℃ ~ 37 ℃, about 30 ℃ ~ 38 ℃, about 30 ℃ ~ 40 ℃, about 35 ℃ ~ It is carried out at a temperature of about 38 ° C, or about 32 ° C to about 38 ° C).

In some embodiments, the contact stage is about 4 ° C to about 18 ° C (eg, about 4 ° C to about 16 ° C, about 4 ° C to about 14 ° C, about 4 ° C to about 12 ° C, about 4 ° C to about 10). ℃, about 4 ℃ ~ about 8 ℃, about 4 ℃ ~ about 6 ℃, about 6 ℃ ~ about 18 ℃, about 6 ℃ ~ about 16 ℃, about 6 ℃ ~ about 14 ℃, about 6 ℃ ~ about 12 ℃, About 6 ° C to about 10 ° C, about 6 ° C to about 8 ° C, about 8 ° C to about 18 ° C, about 8 ° C to about 16 ° C, about 8 ° C to about 14 ° C, about 8 ° C to about 12 ° C, about 8 ℃ ~ about 10 ℃, about 10 ℃ ~ about 18 ℃, about 10 ℃ ~ about 16 ℃, about 10 ℃ ~ about 14 ℃, about 10 ℃ ~ about 12 ℃, about 12 ℃ ~ about 18 ℃, about 12 ℃ ~ It is carried out at a temperature of about 16 ° C, about 12 ° C to about 14 ° C, about 14 ° C to about 18 ° C, about 14 ° C to about 16 ° C, or about 16 ° C to about 18 ° C).

In some embodiments, the contact step is such that the pod is at an angle of 90 ° ± 50 ° (eg, angle) from the horizontal (eg, from the bottom of the culture vessel (eg, any of the culture vessels described herein)). 50 ° to 140 °, angle 50 ° to 135 °, angle 50 ° to 130 °, angle 50 ° to 125 °, angle 50 ° to 120 °, angle 50 ° to 115 °, angle 50 ° to 110 °, angle 50 ° ~ 105 °, angle 50 ° ~ 100 °, angle 50 ° ~ 95 °, angle 50 ° ~ 90 °, angle 50 ° ~ 85 °, angle 50 ° ~ 80 °, angle 50 ° ~ 75 °, angle 50 ° ~ 70 °, angle 50 ° ~ 65 °, angle 50 ° ~ 60 °, angle 55 ° ~ 140 °, angle 55 ° ~ 135 °, angle 55 ° ~ 130 °, angle 55 ° ~ 125 °, angle 55 ° ~ 120 °, angle 55 ° to 115 °, angle 55 ° to 110 °, angle 55 ° to 105 °, angle 55 ° to 100 °, angle 55 ° to 95 °, angle 55 ° to 90 °, angle 55 ° to 85 °, angle 55 ° to 80 °, angle 55 ° to 75 °, angle 55 ° to 70 °, angle 55 ° to 65 °, angle 55 ° to 60 °, angle 60 ° to 140 °, angle 60 ° to 135 ° , Angle 60 ° ~ 130 °, Angle 60 ° ~ 125 °, Angle 60 ° ~ 120 °, Angle 60 ° ~ 115 °, Angle 60 ° ~ 110 °, Angle 60 ° ~ 105 °, Angle 60 ° ~ 100 °, Angle 60 ° ~ 95 °, Angle 60 ° ~ 90 °, Angle 60 ° ~ 85 °, Angle 60 ° ~ 80 °, Angle 60 ° ~ 75 °, Angle 60 ° ~ 70 °, Angle 60 ° ~ 65 °, Angle 65 ° to 140 °, angle 65 ° to 135 °, angle 65 ° to 130 °, angle 65 ° to 125 °, angle 65 ° to 120 °, angle 65 ° to 115 °, angle 65 ° to 110 °, angle 65 ° ~ 105 °, angle 65 ° ~ 100 °, angle 65 ° ~ 95 °, angle 65 ° ~ 90 °, angle 65 ° ~ 85 °, angle 65 ° ~ 80 °, angle 65 ° ~ 75 °, angle 65 ° ~ 70 °, angle 70 ° ~ 140 °, angle 70 ° ~ 135 °, angle 70 ° ~ 130 °, angle 70 ° ~ 125 °, angle 70 ° ~ 120 °, angle 70 ° ~ 115 °, angle 70 ° ~ 110 °, angle 70 ° ~ 105 °, angle 70 ° ~ 100 °, angle 70 ° ~ 95 °, angle 70 ° ~ 90 °, angle 70 ° ~ 85 °, angle 70 ° ~ 80 °, angle 70 ° ~ 75 °, angle 75 ° to 140 °, angle 75 ° to 135 °, angle 75 ° to 130 °, angle 75 ° to 125 °, angle 75 ° to 120 °, angle 75 ° to 115 °, angle 75 ° to 110 ° , Angle 75 ° ~ 10 5 °, angle 75 ° ~ 100 °, angle 75 ° ~ 95 °, angle 75 ° ~ 90 °, angle 75 ° ~ 85 °, angle 75 ° ~ 80 °, angle 80 ° ~ 140 °, angle 80 ° ~ 135 °, angle 80 ° to 130 °, angle 80 ° to 125 °, angle 80 ° to 120 °, angle 80 ° to 115 °, angle 80 ° to 110 °, angle 80 ° to 105 °, angle 80 ° to 100 ° , Angle 80 ° -95 °, Angle 80 ° -90 °, Angle 80 ° -85 °, Angle 85 ° -140 °, Angle 85 ° -135 °, Angle 85 ° -130 °, Angle 85 ° -125 °, Angle 85 ° ~ 120 °, Angle 85 ° ~ 115 °, Angle 85 ° ~ 110 °, Angle 85 ° ~ 105 °, Angle 85 ° ~ 100 °, Angle 85 ° ~ 95 °, Angle 85 ° ~ 90 °, Angle 90 ° to 140 °, angle 90 ° to 135 °, angle 90 ° to 130 °, angle 90 ° to 125 °, angle 90 ° to 120 °, angle 90 ° to 115 °, angle 90 ° to 110 °, angle 90 ° ~ 105 °, angle 90 ° ~ 100 °, angle 90 ° ~ 95 °, angle 95 ° ~ 140 °, angle 95 ° ~ 135 °, angle 95 ° ~ 130 °, angle 95 ° ~ 125 °, angle 95 ° ~ 120 °, angle 95 ° ~ 115 °, angle 95 ° ~ 110 °, angle 95 ° ~ 105 °, angle 95 ° ~ 100 °, angle 100 ° ~ 140 °, angle 100 ° ~ 135 °, angle 100 ° ~ 130 °, angle 100 ° ~ 125 °, angle 100 ° ~ 120 °, angle 100 ° ~ 115 °, angle 100 ° ~ 110 °, angle 100 ° ~ 105 °, angle 105 ° ~ 140 °, angle 105 ° ~ 135 °, angle 105 ° to 130 °, angle 105 ° to 125 °, angle 105 ° to 120 °, angle 105 ° to 115 °, angle 105 ° to 110 °, angle 110 ° to 140 °, angle 110 ° to 135 ° , Angle 110 ° ~ 130 °, Angle 110 ° ~ 125 °, Angle 110 ° ~ 120 °, Angle 110 ° ~ 115 °, Angle 115 ° ~ 140 °, Angle 115 ° ~ 135 °, Angle 115 ° ~ 130 °, Angle 115 ° -125 °, Angle 115 ° -120 °, Angle 120 ° -140 °, Angle 120 ° -135 °, Angle 120 ° -130 °, Angle 120 ° -125 °, Angle 125 ° -140 °, Angle 125 ° to 135 °, angle 125 ° to 130 °, angle 130 ° to 140 °, angle 130 ° to 135 °, angle 135 ° to 140 °, angle 50 °, angle 55 °, angle 60 °, angle 65 °, Angle 70 °, Angle 75 °, Angle 80 °, Angle 85 °, Angle 90 °, Angle 95 °, angle 100 °, angle 105 °, angle 110 °, angle 115 °, angle 120 °, angle 125 °, angle 130 °, angle 135 °, angle 140 °) may occur.

In some embodiments, the contact step is such that the pod is at an angle of 180 ° ± 35 ° (eg, angle) from the horizontal (eg, from the bottom of the culture vessel (eg, any of the culture vessels described herein)). 145 ° to 180 °, angle 145 ° to 175 °, angle 145 ° to 170 °, angle 145 ° to 165 °, angle 145 ° to 160 °, angle 145 ° to 155 °, angle 145 ° to 150 °, angle 150 ° ~ 180 °, angle 150 ° ~ 175 °, angle 150 ° ~ 170 °, angle 150 ° ~ 165 °, angle 150 ° ~ 160 °, angle 150 ° ~ 155 °, angle 155 ° ~ 180 °, angle 155 ° ~ 175 °, angle 155 ° ~ 170 °, angle 155 ° ~ 165 °, angle 155 ° ~ 160 °, angle 160 ° ~ 180 °, angle 160 ° ~ 175 °, angle 160 ° ~ 170 °, angle 160 ° ~ 165 °, angle 165 ° to 180 °, angle 165 ° to 175 °, angle 165 ° to 170 °, angle 170 ° to 180 °, angle 170 ° to 175 °, angle 175 ° to 180 °, angle 145 °, angle It may occur when at 150 °, angle 155 °, angle 160 °, angle 165 °, angle 170 °, angle 175 °, angle 180 °).

Sterilization of Liquid Medium and / or Pod Containing Chromatographic Resin In some embodiments, the pod containing chromatographic resin may be sterilized. Non-limiting examples of sterilization methods include filtration, gamma ray irradiation, electron beam irradiation, steam sterilization, autoclaving, and chemical sterilization (eg, hydrogen chloride, sodium-N-lauroyl sarcosinate, sodium hydroxide). , Or ethanol) is included.

In some embodiments, the chromatographic resin is sterile. As used herein, the term "sterile assurance level" refers to the probability that a single unit (eg, a chromatographic resin) will become non-sterile after being subjected to a sterilization process. In some embodiments, the chromatographic resin is about 1 x 10 ^-5 to about 1 x 10 ^-15 (eg, about 1 x 10 ^-5 to about 1 x 10 ^-10 , about 1 x 10 ^-5 to about 1 x). 10 ^-15 , about 1 x 10 ^-6 to about 1 x 10 ^-10 , about 1 x 10 ^-6 to about 1 x 10 ^-12 , about 1 x 10 ^-6 to about 1 x 10 ^-15 , about 1 x 10 ^-5, about 1 x 10 ^-6, about 1 x 10 ^-7 to about 1 x 10 ^-7 to about 1 x 10 ^-8 to about 1 x 10 ^-9, about 1 x 10 ^-10, about 1 x 10 ^{- It} has a sterility assurance level of ¹¹ , about 1 x 10 ^-12 , about 1 x 10 ^-13 , about 1 x 10 ^-14 , about 1 x 10 ^-15 ).

In some embodiments, the liquid cell medium present in the liquid cell culture medium may be sterile and / or sterile.

Steps of Washing Chromatographic Resin In some embodiments of any of the methods described herein, the method further comprises washing. In some embodiments, the cleaning step is performed between the contact step and the elution step. In some embodiments, the wash step is a single or multiple wash with wash buffer (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). including. In some embodiments, the washing step removes or substantially removes contaminants or non-specific proteins. In some embodiments, the washing step equilibrates the chromatographic resin of the pod before contacting it with the liquid cell culture medium.

In some embodiments, the wash step is performed prior to removing the pod from the liquid cell culture and / or culture vessel. In some embodiments, the washing step is performed after removing the pod from the liquid cell culture and / or culture vessel. In some embodiments, the wash step is performed after the pod has been removed from the liquid cell culture and / or culture vessel and may involve pipetting the wash buffer directly into the pod. ..

In some embodiments, the cleaning step is about 2 ° C to about 25 ° C (eg, 2 ° C to about 4 ° C, about 2 ° C to about 6 ° C, about 2 ° C to about 8 ° C, about 2 ° C to about 10 ° C. , About 2 ° C to about 12 ° C, about 4 ° C to about 6 ° C, about 4 ° C to about 8 ° C, about 4 ° C to about 10 ° C, about 4 ° C to about 12 ° C, about 6 ° C to about 8 ° C, about 6 ° C to about 10 ° C, about 6 ° C to about 12 ° C, about 10 ° C to about 12 ° C, about 10 ° C to about 25 ° C, about 10 ° C to about 22 ° C, about 10 ° C to about 20 ° C, about 10 ° C ~ About 18 ℃, about 10 ℃ ~ about 14 ℃, about 12 ℃ ~ about 14 ℃, about 12 ℃ ~ about 16 ℃, about 16 ℃ ~ about 18 ℃, about 16 ℃ ~ about 20 ℃, about 16 ℃ ~ about 25 ° C, about 20 ° C to about 22 ° C, about 20 ° C to about 25 ° C, or about 22 ° C to about 25 ° C).

In some embodiments, the washing step is about 2 mM to about 6 mM (eg, about 2 mM to about 5 mM, about 2 mM to about 4 mM, about 2 mM to about 3 mM, about 3 mM to about 6). mM, about 3 mM to about 5 mM, about 3 mM to about 4 mM, about 4 mM to about 6 mM, about 4 mM to about 5 mM, about 5 mM to about 6 mM, about 2 mM, about 3 mM, This is done with a wash buffer containing about 4 mM, about 5 mM, about 6 mM) ammonium acetate. In some embodiments, the wash buffer may further comprise salts and / or detergent. As is known in the art, wash buffers are not intended to separate recombinant proteins from chromatographic resins.

In some embodiments, the washing steps are about 0.05 mL to about 1,000 mL, about 0.05 mL to about 950 mL, about 0.05 mL to about 900 mL, about 0.05 mL to about 850 mL, about 0.05 mL to about 800 mL. , About 0.05 mL to about 750 mL, about 0.05 mL to about 700 mL, about 0.05 mL to about 650 mL, about 0.05 mL to about 600 mL, about 0.05 mL to about 550 mL, about 0.05 mL to about 500 mL, about 0.05 mL to about 450 mL, about 0.05 mL to about 400 mL, about 0.05 mL to about 350 mL, about 0.05 mL to about 300 mL, about 0.05 mL to about 250 mL, about 0.05 mL to about 200 mL, about 0.05 mL ~ About 150 mL, about 0.05 mL ~ about 100 mL, about 0.05 mL ~ about 90 mL, about 0.05 mL ~ about 80 mL, about 0.05 mL ~ about 70 mL, about 0.05 mL ~ about 60 mL, about 0.05 mL ~ about 50 mL, about 0.05 mL to about 45 mL, about 0.05 mL to about 40 mL, about 0.05 mL to about 35 mL, about 0.05 mL to about 30 mL, about 0.05 mL to about 25 mL, about 0.05 mL to about 20 mL , About 0.05 mL ~ about 15 mL, about 0.05 mL ~ about 10 mL, about 0.05 mL ~ about 8 mL, about 0.05 mL ~ about 6 mL, about 0.05 mL ~ about 5 mL, about 0.05 mL ~ about 4 mL, about 0.05 mL to about 3 mL, about 0.05 mL to about 2 mL, about 0.05 mL to about 1 mL, about 0.05 mL to about 0.5 mL, about 0.05 mL to about 0.1 mL, about 0.1 mL to about 1,000 mL, about 0.1 mL ~ About 950 mL, about 0.1 mL ~ about 900 mL, about 0.1 mL ~ about 850 mL, about 0.1 mL ~ about 800 mL, about 0.1 mL ~ about 750 mL, about 0.1 mL ~ about 700 mL, about 0.1 mL ~ about 650 mL, about 0.1 mL to about 600 mL, about 0.1 mL to about 550 mL, about 0.1 mL to about 500 mL, about 0.1 mL to about 450 mL, about 0.1 mL to about 400 mL, about 0.1 mL to about 350 mL , About 0.1 mL to about 300 mL, about 0.1 mL to about 250 mL, about 0.1 mL to about 200 mL, about 0.1 mL to about 150 mL, about 0.1 mL to about 100 mL, about 0. 1 mL to about 90 mL, about 0.1 mL to about 80 mL, about 0.1 mL to about 70 mL, about 0.1 mL to about 60 mL, about 0.1 mL to about 50 mL, about 0.1 mL to about 45 mL, about 0.1 mL ~ About 40 mL, about 0.1 mL ~ about 35 mL, about 0.1 mL ~ about 30 mL, about 0.1 mL ~ about 25 mL, about 0.1 mL ~ about 20 mL, about 0.1 mL ~ about 15 mL, about 0.1 mL ~ about 10 mL, about 0.1 mL to about 8 mL, about 0.1 mL to about 6 mL, about 0.1 mL to about 5 mL, about 0.1 mL to about 4 mL, about 0.1 mL to about 3 mL, about 0.1 mL to about 2 mL , About 0.1 mL to about 1 mL, about 0.1 mL to about 0.5 mL, about 0.5 mL to about 1,000 mL, about 0.5 mL to about 950 mL, about 0.5 mL to about 900 mL, about 0.5 mL to about 850 mL, about 0.5 mL to about 800 mL, about 0.5 mL to about 750 mL, about 0.5 mL to about 700 mL, about 0.5 mL to about 650 mL, about 0.5 mL to about 600 mL, about 0.5 mL to about 550 mL, about 0.5 mL ~ About 500 mL, about 0.5 mL ~ about 450 mL, about 0.5 mL ~ about 400 mL, about 0.5 mL ~ about 350 mL, about 0.5 mL ~ about 300 mL, about 0.5 mL ~ about 250 mL, about 0.5 mL ~ about 200 mL, about 0.5 mL to about 150 mL, about 0.5 mL to about 100 mL, about 0.5 mL to about 90 mL, about 0.5 mL to about 80 mL, about 0.5 mL to about 70 mL, about 0.5 mL to about 60 mL , About 0.5 mL to about 50 mL, about 0.5 mL to about 45 mL, about 0.5 mL to about 40 mL, about 0.5 mL to about 35 mL, about 0.5 mL to about 30 mL, about 0.5 mL to about 25 mL, about 0.5 mL to about 20 mL, about 0.5 mL to about 15 mL, about 0.5 mL to about 10 mL, about 0.5 mL to about 8 mL, about 0.5 mL to about 6 mL, about 0.5 mL to about 5 mL, about 0.5 mL ~ About 4 mL, about 0.5 mL ~ about 3 mL, about 0.5 mL ~ about 2 mL, about 0.5 mL ~ about 1 mL, 1 mL ~ about 1 L, about 1 mL ~ about 900 mL, about 1 mL ~ about 800 mL, about 1 mL to about 700 mL, about 1 mL to about 600 mL, about 1 mL to about 500 mL, about 1 mL to about 250 mL, about 1 mL to about 400 mL, about 1 mL to about 350 mL, about 1 mL to about 300 mL, about 1 mL to about 250 mL, about 1 mL to about 200 mL, about 1 mL to about 150 mL, about 1 mL ~ About 100 mL, about 1 mL ~ about 90 mL, about 1 mL ~ about 80 mL, about 1 mL ~ about 70 mL, about 1 mL ~ about 60 mL, about 1 mL ~ about 50 mL, about 1 mL ~ about 40 mL, about 1 mL to about 30 mL, about 1 mL to about 20 mL, about 1 mL to about 10 mL, about 1 mL to about 5 mL; about 2 mL to about 1 L, about 2 mL to about 900 mL , About 2 mL to about 800 mL, about 2 mL to about 700 mL, about 2 mL to about 600 mL, about 2 mL to about 500 mL, about 2 mL to about 250 mL, about 2 mL to about 400 mL, about 2 mL to about 350 mL, about 2 mL to about 300 mL, about 2 mL to about 250 mL, about 2 mL to about 200 mL, about 2 mL to about 150 mL, about 2 mL to about 100 mL, about 2 mL ~ About 90 mL, about 2 mL ~ about 80 mL, about 2 mL ~ about 70 mL, about 2 mL ~ about 60 mL, about 2 mL ~ about 50 mL, about 2 mL ~ about 40 mL, about 2 mL ~ about 30 mL, about 2 mL to about 20 mL, about 2 mL to about 10 mL, about 2 mL to about 5 mL; about 5 mL to about 1 L, about 5 mL to about 900 mL, about 5 mL to about 800 mL , About 5 mL to about 700 mL, about 5 mL to about 600 mL, about 5 mL to about 500 mL, about 5 mL to about 250 mL, about 5 mL to about 400 mL, about 5 mL to about 350 mL, about 5 mL to about 300 mL, about 5 mL to about 250 mL, about 5 mL to about 200 mL, about 5 mL to about 150 mL, about 5 mL to about 100 mL, about 5 mL to about 90 mL, about 5 mL ~ About 80 mL, about 5 mL ~ about 70 mL, about 5 mL ~ about 60 mL, about 5 mL ~ about 50 mL, about 5 mL ~ about 40 mL, about 5 mL ~ about 30 mL, about 5 mL ~ about 20 mL, about 5 mL to about 10 mL; about 10 mL to about 1 L, about 10 mL to about 900 mL, about 10 mL to about 800 mL, about 10 mL to about 700 mL, about 10 mL to about 600 mL , About 10 mL to about 500 mL, about 10 mL to about 450 mL, about 10 mL to about 400 mL, about 10 mL to about 350 mL, about 10 mL to about 30 0 mL, about 10 mL to about 250 mL, about 10 mL to about 200 mL, about 10 mL to about 150 mL, about 10 mL to about 100 mL, about 10 mL to about 90 mL, about 10 mL to about 80 mL , About 10 mL to about 70 mL, about 10 mL to about 60 mL, about 10 mL to about 50 mL, about 10 mL to about 40 mL, about 10 mL to about 30 mL, about 10 mL to about 20 mL; 20 mL to about 1 L, about 20 mL to about 900 mL, about 20 mL to about 800 mL, about 20 mL to about 700 mL, about 20 mL to about 600 mL, about 20 mL to about 500 mL, about 20 mL ~ About 450 mL, about 20 mL ~ about 400 mL, about 20 mL ~ about 350 mL, about 20 mL ~ about 300 mL, about 20 mL ~ about 250 mL, about 20 mL ~ about 200 mL, about 20 mL ~ about 150 mL, about 20 mL to about 100 mL, about 20 mL to about 90 mL, about 20 mL to about 80 mL, about 20 mL to about 70 mL, about 20 mL to about 60 mL, about 20 mL to about 50 mL , About 20 mL to about 40 mL, about 20 mL to about 30 mL; about 50 mL to about 1 L, about 50 mL to about 900 mL, about 50 mL to about 800 mL, about 50 mL to about 700 mL, about 50 mL to about 600 mL, about 50 mL to about 500 mL, about 50 mL to about 450 mL, about 50 mL to about 400 mL, about 50 mL to about 350 mL, about 50 mL to about 300 mL, about 50 mL ~ About 250 mL, about 50 mL ~ about 200 mL, about 50 mL ~ about 150 mL, about 50 mL ~ about 100 mL, about 50 mL ~ about 90 mL, about 50 mL ~ about 80 mL, about 50 mL ~ about 70 mL, about 50 mL to about 60 mL; about 100 mL to about 1 L, about 100 mL to about 900 mL, about 100 mL to about 800 mL, about 100 mL to about 700 mL, about 100 mL to about 600 mL , About 100 mL to about 500 mL, about 100 mL to about 450 mL, about 100 mL to about 400 mL, about 100 mL to about 350 mL, about 100 mL to about 300 mL, about 100 mL to about 250 mL, about 100 mL to about 200 mL, about 100 mL to about 150 mL; about 150 mL to about 1 L, about 150 mL to about 900 mL, about 150 mL to about 800 mL, about 150 mL ~ about 700 mL, about 150 mL ~ about 600 mL, about 150 mL ~ about 500 mL, about 150 mL ~ about 250 mL, about 150 mL ~ about 400 mL, about 150 mL ~ about 350 mL, about 150 mL ~ About 300 mL, about 150 mL to about 250 mL, about 150 mL to about 200 mL; about 200 mL to about 1 L, about 200 mL to about 900 mL, about 200 mL to about 800 mL, about 200 mL to about 700 mL, about 200 mL to about 600 mL, about 200 mL to about 500 mL, about 200 mL to about 450 mL, about 200 mL to about 400 mL, about 200 mL to about 350 mL, about 200 mL to about 300 mL, About 200 mL to about 250 mL; about 250 mL to about 1 L, about 250 mL to about 900 mL, about 250 mL to about 800 mL, about 250 mL to about 700 mL, about 250 mL to about 600 mL, about 250 mL ~ about 500 mL, about 250 mL ~ about 450 mL, about 250 mL ~ about 400 mL, about 250 mL ~ about 350 mL, about 250 mL ~ about 300 mL; about 300 mL ~ about 1 L, about 300 mL ~ About 900 mL, about 300 mL to about 800 mL, about 300 mL to about 700 mL, about 300 mL to about 600 mL, about 300 mL to about 500 mL, about 300 mL to about 450 mL, about 300 mL to about 400 mL, about 300 mL to about 350 mL; about 350 mL to about 1 L, about 350 mL to about 900 mL, about 350 mL to about 800 mL, about 350 mL to about 700 mL, about 350 mL to about 600 mL, About 350 mL to about 500 mL, about 350 mL to about 450 mL; about 350 mL to about 400 mL; about 400 mL to about 1 L, about 400 mL to about 900 mL, about 400 mL to about 800 mL, about 400 mL ~ about 700 mL, about 400 mL ~ about 600 mL, about 400 mL ~ about 500 mL, about 400 mL ~ about 450 mL; about 500 mL ~ about 1 L, about 500 mL ~ about 900 mL, about 500 mL ~ About 800 mL, about 500 mL to about 700 mL, about 500 mL to about 600 mL; about 550 mL to about 1 L, about 550 mL to about 900 mL, about 550 mL to about 800 mL, about 550 mL to about 700 mL, about 550 mL to about 600 mL; about 6 00 mL to about 1 L, about 600 mL to about 900 mL, about 600 mL to about 800 mL, about 600 mL to about 700 mL; about 600 mL to about 650 mL; about 650 mL to about 1 L, about 650 mL ~ About 900 mL, about 650 mL ~ about 800 mL, about 650 mL ~ about 700 mL; about 700 mL ~ about 1 L, about 700 mL ~ about 900 mL, about 700 mL ~ about 800 mL, about 700 mL ~ about 750 mL; about 750 mL to about 1 L, about 750 mL to about 900 mL, about 750 mL to about 800 mL; about 800 mL to about 1 L, about 800 mL to about 900 mL, about 800 mL to about 950 mL Approximately 850 mL to approximately 1 L, approximately 850 L to approximately 950 mL, approximately 850 mL to approximately 900 mL, approximately 900 mL to approximately 1 L, approximately 900 mL to approximately 950 mL) wash buffers (eg, herein). It is carried out using any of the cleaning buffers described in.

In some embodiments of any of the methods described herein, the cleaning steps are one or more (eg, two, three, four of any of the cleaning buffers described herein). One, five, six, seven, or eight) wash buffers may be included.

Steps of Elution of Recombinant Protein In some embodiments, any of the pods described herein may be removed from the liquid medium prior to the step of elution. In some embodiments, the pod affinity chromatography resin is a protein A resin. In some embodiments where the pod affinity chromatography resin is a protein A resin, the elution step is performed with an elution buffer with an acidic pH. In some embodiments, the elution buffer comprises arginine or citrate. In some embodiments, the elution buffer is about 0.01 M to about 1 M (eg, about 0.01 M to about 1 M, about 0.01 M to about 0.8 M, about 0.01 M to about 0.6 M, about 0.01 M to about 0.4). M, about 0.01 M to about 0.2 M, about 0.01 M to about 0.1 M, about 0.01 M to about 0.08 M, about 0.01 M to about 0.06 M, about 0.01 M to about 0.05 M, about 0.01 M to about 0.04 M, About 0.01 M to about 0.02 M; 0.02 M to about 1 M, about 0.02 M to about 0.8 M, about 0.02 M to about 0.6 M, about 0.02 M to about 0.4 M, about 0.02 M to about 0.2 M, about 0.02 M ~ About 0.1 M, about 0.02 M ~ about 0.08 M, about 0.02 M ~ about 0.06 M, about 0.02 M ~ about 0.05 M, about 0.02 M ~ about 0.04 M, 0.04 M ~ about 1 M, about 0.04 M ~ about 0.8 M, about 0.04 M to about 0.6 M, about 0.04 M to about 0.4 M, about 0.04 M to about 0.2 M, about 0.04 M to about 0.1 M, about 0.04 M to about 0.08 M, about 0.04 M to about 0.06 M, About 0.04 M to about 0.05 M, 0.05 M to about 1 M, about 0.05 M to about 0.8 M, about 0.05 M to about 0.6 M, about 0.05 M to about 0.4 M, about 0.05 M to about 0.2 M, about 0.05 M ~ About 0.1 M, about 0.05 M ~ about 0.08 M, about 0.05 M ~ about 0.06 M, 0.06 M ~ about 1 M, about 0.06 M ~ about 0.8 M, about 0.06 M ~ about 0.6 M, about 0.06 M ~ about 0.4 M, about 0.06 M to about 0.2 M, about 0.06 M to about 0.1 M, about 0.06 M to about 0.08 M, 0.08 M to about 1 M, about 0.08 M to about 0.8 M, about 0.08 M to about 0.6 M, about 0.08 M to about 0.4 M, about 0.08 M to about 0.2 M, about 0.08 M to about 0.1 M, 0.1 M to about 1 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.6 M, about 0.1 M ~ About 0.4 M, about 0.1 M to about 0.2 M, 0.2 M to about 1 M, about 0.2 M to about 0.8 M, about 0.2 M to about 0.6 M, about 0.2 M to about 0.4 M, 0.4 M to about 1 M, About 0.4 M to about 0.8 M, about 0.4 M to about 0.6 M, 0.6 M to about 1 M, about 0.6 M to about 0.8 M, 0.8 M to about 1 M, about 0.01 M , About 0.02 M, about 0.05 M, about 0.05 M, about 0.06 M, about 0.08 M, about 0.1 M, about 0.2 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.8 M, or about 1 M) Contains arginine or citrate. In some embodiments, the elution buffer is about 0.1 to about 1 M (eg, about 0.1 to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, About 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.1 M to about 0.2 M, about 0.2 M to about 1 M, about 0.2 M to about 0.9 M, about 0.2 M ~ about 0.8 M, about 0.2 M ~ about 0.7 M, about 0.2 M ~ about 0.6 M, about 0.2 M ~ about 0.5 M, about 0.2 M ~ about 0.4 M, about 0.2 M ~ about 0.3 M, about 0.3 M ~ About 1 M, about 0.3 M to about 0.9 M, about 0.3 M to about 0.8 M, about 0.3 M to about 0.7 M, about 0.3 M to about 0.6 M, about 0.3 M to about 0.5, about 0.3 M to about 0.4 M , About 0.4 M to about 1 M, about 0.4 M to about 0.9 M, about 0.4 to about 0.8 M, about 0.4 M to about 0.7 M, about 0.4 M to about 0.6 M, about 0.4 M to about 0.5 M, about 0.5 M ~ about 1 M, about 0.5 M ~ about 0.9 M, about 0.5 M ~ about 0.8 M, about 0.5 M ~ about 0.7 M, about 0.5 M ~ about 0.6 M, about 0.6 M ~ about 1 M, about 0.6 M ~ About 0.9 M, about 0.6 M to about 0.8 M, about 0.6 M to about 0.7 M, about 0.7 M to about 1 M, about 0.7 M to about 0.9 M, about 0.7 M to about 0.8 M, about 0.8 M to about 1 M, about 0.8 M to about 0.9 M, about 0.9 M to about 1 M) of acetic acid.

In some embodiments, the elution buffer comprises about 0.1 M glycine (pH 3.0) or about 0.1 M citric acid (pH 3.0). In some embodiments of any of the chromatographic resins used herein, the eluted recombinant protein is neutralized with a buffer containing approximately 1 M tris (pH 9.0). In some embodiments of any of the chromatographic resins used herein, the eluted recombinant protein is about 1 M HEPES (pH 7.0) (eg, about 0.01 M to about 1 M, about 0.01 M to about 0.8). M, about 0.01 M to about 0.6 M, about 0.01 M to about 0.4 M, about 0.01 M to about 0.2 M, about 0.01 to about 0.1 M, about 0.01 M to about 0.05 M; about 0.02 M to about 1 M, about 0.02 M to about 0.8 M, about 0.02 M to about 0.6 M, about 0.02 M to about 0.4 M, about 0.02 M to about 0.2 M, about 0.02 to about 0.1 M, about 0.05 M to about 1 M, about 0.05 M to About 0.8 M, about 0.05 M to about 0.6 M, about 0.05 M to about 0.4 M, about 0.05 M to about 0.2 M, about 0.05 to about 0.1 M, about 0.1 M to about 1 M, about 0.1 M to about 0.8 M , About 0.1 M to about 0.6 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.2 M, about 0.2 M to about 1 M, about 0.2 M to about 0.8 M, about 0.2 M to about 0.6 M, about 0.2 M to about 0.4 M, about 0.4 M to about 1 M, about 0.4 M to about 0.8 M, about 0.4 M to about 0.6 M, about 0.6 M to about 1 M, about 0.6 M to about 0.8 M, or about 0.8 Neutralized with a buffer containing M ~ about 1 M).

In some embodiments, elution is about 0.05 mL to about 1,000 mL, about 0.05 mL to about 950 mL, about 0.05 mL to about 900 mL, about 0.05 mL to about 850 mL, about 0.05 mL to about 800 mL, about. 0.05 mL to about 750 mL, about 0.05 mL to about 700 mL, about 0.05 mL to about 650 mL, about 0.05 mL to about 600 mL, about 0.05 mL to about 550 mL, about 0.05 mL to about 500 mL, about 0.05 mL ~ About 450 mL, about 0.05 mL ~ about 400 mL, about 0.05 mL ~ about 350 mL, about 0.05 mL ~ about 300 mL, about 0.05 mL ~ about 250 mL, about 0.05 mL ~ about 200 mL, about 0.05 mL ~ about 150 mL, about 0.05 mL to about 100 mL, about 0.05 mL to about 90 mL, about 0.05 mL to about 80 mL, about 0.05 mL to about 70 mL, about 0.05 mL to about 60 mL, about 0.05 mL to about 50 mL , About 0.05 mL to about 45 mL, about 0.05 mL to about 40 mL, about 0.05 mL to about 35 mL, about 0.05 mL to about 30 mL, about 0.05 mL to about 25 mL, about 0.05 mL to about 20 mL, about 0.05 mL to about 15 mL, about 0.05 mL to about 10 mL, about 0.05 mL to about 8 mL, about 0.05 mL to about 6 mL, about 0.05 mL to about 5 mL, about 0.05 mL to about 4 mL, about 0.05 mL ~ About 3 mL, about 0.05 mL ~ about 2 mL, about 0.05 mL ~ about 1 mL, about 0.05 mL ~ about 0.5 mL, about 0.05 mL ~ about 0.1 mL, about 0.1 mL ~ about 1,000 mL, about 0.1 mL ~ about 950 mL, about 0.1 mL to about 900 mL, about 0.1 mL to about 850 mL, about 0.1 mL to about 800 mL, about 0.1 mL to about 750 mL, about 0.1 mL to about 700 mL, about 0.1 mL to about 650 mL , About 0.1 mL to about 600 mL, about 0.1 mL to about 550 mL, about 0.1 mL to about 500 mL, about 0.1 mL to about 450 mL, about 0.1 mL to about 400 mL, about 0.1 mL to about 350 mL, about 0.1 mL to about 300 mL, about 0.1 mL to about 250 mL, about 0.1 mL to about 200 mL, about 0.1 mL to about 150 mL, about 0.1 mL to about 100 mL, about 0.1 m L ~ about 90 mL, about 0.1 mL ~ about 80 mL, about 0.1 mL ~ about 70 mL, about 0.1 mL ~ about 60 mL, about 0.1 mL ~ about 50 mL, about 0.1 mL ~ about 45 mL, about 0.1 mL ~ About 40 mL, about 0.1 mL to about 35 mL, about 0.1 mL to about 30 mL, about 0.1 mL to about 25 mL, about 0.1 mL to about 20 mL, about 0.1 mL to about 15 mL, about 0.1 mL to about 10 mL, about 0.1 mL to about 8 mL, about 0.1 mL to about 6 mL, about 0.1 mL to about 5 mL, about 0.1 mL to about 4 mL, about 0.1 mL to about 3 mL, about 0.1 mL to about 2 mL, About 0.1 mL to about 1 mL, about 0.1 mL to about 0.5 mL, about 0.5 mL to about 1,000 mL, about 0.5 mL to about 950 mL, about 0.5 mL to about 900 mL, about 0.5 mL to about 850 mL, about 0.5 mL ~ about 800 mL, about 0.5 mL ~ about 750 mL, about 0.5 mL ~ about 700 mL, about 0.5 mL ~ about 650 mL, about 0.5 mL ~ about 600 mL, about 0.5 mL ~ about 550 mL, about 0.5 mL ~ About 500 mL, about 0.5 mL to about 450 mL, about 0.5 mL to about 400 mL, about 0.5 mL to about 350 mL, about 0.5 mL to about 300 mL, about 0.5 mL to about 250 mL, about 0.5 mL to about 200 mL, about 0.5 mL to about 150 mL, about 0.5 mL to about 100 mL, about 0.5 mL to about 90 mL, about 0.5 mL to about 80 mL, about 0.5 mL to about 70 mL, about 0.5 mL to about 60 mL, About 0.5 mL to about 50 mL, about 0.5 mL to about 45 mL, about 0.5 mL to about 40 mL, about 0.5 mL to about 35 mL, about 0.5 mL to about 30 mL, about 0.5 mL to about 25 mL, about 0.5 mL ~ about 20 mL, about 0.5 mL ~ about 15 mL, about 0.5 mL ~ about 10 mL, about 0.5 mL ~ about 8 mL, about 0.5 mL ~ about 6 mL, about 0.5 mL ~ about 5 mL, about 0.5 mL ~ About 4 mL, about 0.5 mL to about 3 mL, about 0.5 mL to about 2 mL, about 0.5 mL to about 1 mL, 1 mL to about 1 L, about 1 mL to about 900 mL, about 1 mL to about 800 mL , About 1 mL to about 700 mL, about 1 mL to about 600 mL, about 1 mL to about 500 mL, about 1 mL to about 250 mL, about 1 m L ~ about 400 mL, about 1 mL ~ about 350 mL, about 1 mL ~ about 300 mL, about 1 mL ~ about 250 mL, about 1 mL ~ about 200 mL, about 1 mL ~ about 150 mL, about 1 mL ~ About 100 mL, about 1 mL to about 90 mL, about 1 mL to about 80 mL, about 1 mL to about 70 mL, about 1 mL to about 60 mL, about 1 mL to about 50 mL, about 1 mL to about 40 mL, about 1 mL to about 30 mL, about 1 mL to about 20 mL, about 1 mL to about 10 mL, about 1 mL to about 5 mL; about 2 mL to about 1 L, about 2 mL to about 900 mL, About 2 mL to about 800 mL, about 2 mL to about 700 mL, about 2 mL to about 600 mL, about 2 mL to about 500 mL, about 2 mL to about 250 mL, about 2 mL to about 400 mL, about 2 mL ~ about 350 mL, about 2 mL ~ about 300 mL, about 2 mL ~ about 250 mL, about 2 mL ~ about 200 mL, about 2 mL ~ about 150 mL, about 2 mL ~ about 100 mL, about 2 mL ~ About 90 mL, about 2 mL to about 80 mL, about 2 mL to about 70 mL, about 2 mL to about 60 mL, about 2 mL to about 50 mL, about 2 mL to about 40 mL, about 2 mL to about 30 mL, about 2 mL to about 20 mL, about 2 mL to about 10 mL, about 2 mL to about 5 mL; about 5 mL to about 1 L, about 5 mL to about 900 mL, about 5 mL to about 800 mL, About 5 mL to about 700 mL, about 5 mL to about 600 mL, about 5 mL to about 500 mL, about 5 mL to about 250 mL, about 5 mL to about 400 mL, about 5 mL to about 350 mL, about 5 mL ~ about 300 mL, about 5 mL ~ about 250 mL, about 5 mL ~ about 200 mL, about 5 mL ~ about 150 mL, about 5 mL ~ about 100 mL, about 5 mL ~ about 90 mL, about 5 mL ~ About 80 mL, about 5 mL to about 70 mL, about 5 mL to about 60 mL, about 5 mL to about 50 mL, about 5 mL to about 40 mL, about 5 mL to about 30 mL, about 5 mL to about 20 mL, about 5 mL to about 10 mL; about 10 mL to about 1 L, about 10 mL to about 900 mL, about 10 mL to about 800 mL, about 10 mL to about 700 mL, about 10 mL to about 600 mL, About 10 mL to about 500 mL, about 10 mL to about 450 mL, about 10 mL to about 400 mL, about 10 mL to about 350 mL, about 10 mL to about 300 m L, about 10 mL to about 250 mL, about 10 mL to about 200 mL, about 10 mL to about 150 mL, about 10 mL to about 100 mL, about 10 mL to about 90 mL, about 10 mL to about 80 mL, About 10 mL to about 70 mL, about 10 mL to about 60 mL, about 10 mL to about 50 mL, about 10 mL to about 40 mL, about 10 mL to about 30 mL, about 10 mL to about 20 mL; about 20 mL ~ about 1 L, about 20 mL ~ about 900 mL, about 20 mL ~ about 800 mL, about 20 mL ~ about 700 mL, about 20 mL ~ about 600 mL, about 20 mL ~ about 500 mL, about 20 mL ~ About 450 mL, about 20 mL to about 400 mL, about 20 mL to about 350 mL, about 20 mL to about 300 mL, about 20 mL to about 250 mL, about 20 mL to about 200 mL, about 20 mL to about 150 mL, about 20 mL to about 100 mL, about 20 mL to about 90 mL, about 20 mL to about 80 mL, about 20 mL to about 70 mL, about 20 mL to about 60 mL, about 20 mL to about 50 mL, About 20 mL to about 40 mL, about 20 mL to about 30 mL; about 50 mL to about 1 L, about 50 mL to about 900 mL, about 50 mL to about 800 mL, about 50 mL to about 700 mL, about 50 mL ~ about 600 mL, about 50 mL ~ about 500 mL, about 50 mL ~ about 450 mL, about 50 mL ~ about 400 mL, about 50 mL ~ about 350 mL, about 50 mL ~ about 300 mL, about 50 mL ~ About 250 mL, about 50 mL to about 200 mL, about 50 mL to about 150 mL, about 50 mL to about 100 mL, about 50 mL to about 90 mL, about 50 mL to about 80 mL, about 50 mL to about 70 mL, about 50 mL to about 60 mL; about 100 mL to about 1 L, about 100 mL to about 900 mL, about 100 mL to about 800 mL, about 100 mL to about 700 mL, about 100 mL to about 600 mL, About 100 mL to about 500 mL, about 100 mL to about 450 mL, about 100 mL to about 400 mL, about 100 mL to about 350 mL, about 100 mL to about 300 mL, about 100 mL to about 250 mL, about 100 mL ~ about 200 mL, about 100 mL ~ about 150 mL; about 150 mL ~ about 1 L, about 150 mL ~ about 900 mL, about 150 mL ~ about 800 mL, about 150 mL ~ About 700 mL, about 150 mL to about 600 mL, about 150 mL to about 500 mL, about 150 mL to about 250 mL, about 150 mL to about 400 mL, about 150 mL to about 350 mL, about 150 mL to about 300 mL, about 150 mL to about 250 mL, about 150 mL to about 200 mL; about 200 mL to about 1 L, about 200 mL to about 900 mL, about 200 mL to about 800 mL, about 200 mL to about 700 mL, About 200 mL to about 600 mL, about 200 mL to about 500 mL, about 200 mL to about 450 mL, about 200 mL to about 400 mL, about 200 mL to about 350 mL, about 200 mL to about 300 mL, about 200 mL ~ about 250 mL; about 250 mL ~ about 1 L, about 250 mL ~ about 900 mL, about 250 mL ~ about 800 mL, about 250 mL ~ about 700 mL, about 250 mL ~ about 600 mL, about 250 mL ~ About 500 mL, about 250 mL-about 450 mL, about 250 mL-about 400 mL, about 250 mL-about 350 mL, about 250 mL-about 300 mL; about 300 mL-about 1 L, about 300 mL-about 900 mL, about 300 mL to about 800 mL, about 300 mL to about 700 mL, about 300 mL to about 600 mL, about 300 mL to about 500 mL, about 300 mL to about 450 mL, about 300 mL to about 400 mL, About 300 mL to about 350 mL; about 350 mL to about 1 L, about 350 mL to about 900 mL, about 350 mL to about 800 mL, about 350 mL to about 700 mL, about 350 mL to about 600 mL, about 350 mL ~ about 500 mL, about 350 mL ~ about 450 mL; about 350 mL ~ about 400 mL; about 400 mL ~ about 1 L, about 400 mL ~ about 900 mL, about 400 mL ~ about 800 mL, about 400 mL ~ About 700 mL, about 400 mL to about 600 mL, about 400 mL to about 500 mL, about 400 mL to about 450 mL; about 500 mL to about 1 L, about 500 mL to about 900 mL, about 500 mL to about 800 mL, about 500 mL to about 700 mL, about 500 mL to about 600 mL; about 550 mL to about 1 L, about 550 mL to about 900 mL, about 550 mL to about 800 mL, about 550 mL to about 700 mL, About 550 mL-about 600 mL; about 600 mL ~ about 1 L, about 600 mL ~ about 900 mL, about 600 mL ~ about 800 mL, about 600 mL ~ about 700 mL; about 600 mL ~ about 650 mL; about 650 mL ~ about 1 L, about 650 mL ~ About 900 mL, about 650 mL to about 800 mL, about 650 mL to about 700 mL; about 700 mL to about 1 L, about 700 mL to about 900 mL, about 700 mL to about 800 mL, about 700 mL to about 750 mL; about 750 mL to about 1 L, about 750 mL to about 900 mL, about 750 mL to about 800 mL; about 800 mL to about 1 L, about 800 mL to about 900 mL, about 800 mL to about 950 mL; About 850 mL to about 1 L, about 850 L to about 950 mL, about 850 mL to about 900 mL, about 900 mL to about 1 L, or about 900 mL to about 950 mL) elution buffers (eg, herein). It is carried out using any of the elution buffers described in.

In some embodiments, the elution is about 2 ° C to about 25 ° C (eg, 2 ° C to about 4 ° C, about 2 ° C to about 6 ° C, about 2 ° C to about 8 ° C, about 2 ° C to about 10 ° C, about. 2 ° C to about 12 ° C, about 4 ° C to about 6 ° C, about 4 ° C to about 8 ° C, about 4 ° C to about 10 ° C, about 4 ° C to about 12 ° C, about 6 ° C to about 8 ° C, about 6 ° C ~ About 10 ℃, about 6 ℃ ~ about 12 ℃, about 10 ℃ ~ about 12 ℃, about 10 ℃ ~ about 25 ℃, about 10 ℃ ~ about 22 ℃, about 10 ℃ ~ about 20 ℃, about 10 ℃ ~ about 18 ℃, about 10 ℃ to about 14 ℃, about 12 ℃ to about 14 ℃, about 12 ℃ to about 16 ℃, about 16 ℃ to about 18 ℃, about 16 ℃ to about 20 ℃, about 16 ℃ to about 25 ℃ , About 20 ° C to about 22 ° C, about 20 ° C to about 25 ° C, about 22 ° C to about 25 ° C).

In some embodiments, the total amount of recombinant protein eluted is from about 0.05 mg to about 80 mg (eg, about 0.05 mg to about 0.1 mg, about 0.05 mg to about 0.2 mg, about 0.05 mg to about 0.5 mg, about. 0.05 mg to about 0.6 mg, about 0.05 mg to about 0.8 mg, about 0.05 mg to about 1 mg, about 0.05 mg to about 2 mg, about 0.05 mg to about 5 mg, about 0.05 mg to about 10 mg, about 0.05 mg ~ About 15 mg, about 0.05 mg ~ about 20 mg, about 0.05 mg ~ about 25 mg, about 0.05 mg ~ about 30 mg, about 0.05 mg ~ about 35 mg, about 0.05 mg ~ about 40 mg, about 0.05 mg ~ about 45 mg, about 0.05 mg to about 50 mg, about 0.05 mg to about 55 mg, about 0.05 mg to about 60 mg, about 0.05 mg to about 65 mg, about 0.05 mg to about 70 mg, about 0.05 mg to about 75 mg , About 0.05 mg to about 80 mg, about 0.1 mg to about 0.2 mg, about 0.1 mg to about 0.5 mg, about 0.1 mg to about 0.6 mg, about 0.1 mg to about 0.8 mg, about 0.1 mg to about 1 mg, about 0.1 mg to about 2 mg, about 0.1 mg to about 5 mg, about 0.1 mg to about 10 mg, about 0.1 mg to about 15 mg, about 0.1 mg to about 20 mg, about 0.1 mg to about 25 mg, about 0.1 mg ~ About 30 mg, about 0.1 mg ~ about 35 mg, about 0.1 mg ~ about 40 mg, about 0.1 mg ~ about 45 mg, about 0.1 mg ~ about 50 mg, about 0.1 mg ~ about 55 mg, about 0.1 mg ~ about 60 mg, about 0.1 mg to about 65 mg, about 0.1 mg to about 70 mg, about 0.1 mg to about 75 mg, about 0.1 mg to about 80 mg, about 0.2 mg to about 0.5 mg, about 0.2 mg to about 0.6 mg , About 0.2 mg to about 0.8 mg, about 0.2 mg to about 1 mg, about 0.2 mg to about 2 mg, about 0.2 mg to about 5 mg, about 0.2 mg to about 10 mg, about 0.2 mg to about 15 mg, about 0.2 mg to about 20 mg, about 0.2 mg to about 25 mg, about 0.2 mg to about 30 mg, about 0.2 mg to about 35 mg, about 0.2 mg to about 40 mg, about 0.2 mg to about 45 mg, about 0.2 mg ~ About 50 mg, about 0.2 mg ~ about 55 mg, about 0.2 mg ~ about 6 0 mg, about 0.2 mg to about 65 mg, about 0.2 mg to about 70 mg, about 0.2 mg to about 75 mg, about 0.2 mg to about 80 mg, about 0.5 mg to about 0.6 mg, about 0.5 mg to about 0.8 mg , About 0.5 mg to about 1 mg, about 0.5 mg to about 2 mg, about 0.5 mg to about 5 mg, about 0.5 mg to about 10 mg, about 0.5 mg to about 15 mg, about 0.5 mg to about 20 mg, about 0.5 mg to about 25 mg, about 0.5 mg to about 30 mg, about 0.5 mg to about 35 mg, about 0.5 mg to about 40 mg, about 0.5 mg to about 45 mg, about 0.5 mg to about 50 mg, about 0.5 mg ~ About 55 mg, about 0.5 mg ~ about 60 mg, about 0.5 mg ~ about 65 mg, about 0.5 mg ~ about 70 mg, about 0.5 mg ~ about 75 mg, about 0.5 mg ~ about 80 mg, about 0.6 mg ~ about 0.8 mg, about 0.6 mg to about 1 mg, about 0.6 mg to about 2 mg, about 0.6 mg to about 5 mg, about 0.6 mg to about 10 mg, about 0.6 mg to about 15 mg, about 0.6 mg to about 20 mg , About 0.6 mg to about 25 mg, about 0.6 mg to about 30 mg, about 0.6 mg to about 35 mg, about 0.6 mg to about 40 mg, about 0.6 mg to about 45 mg, about 0.6 mg to about 50 mg, about 0.6 mg to about 55 mg, about 0.6 mg to about 60 mg, about 0.6 mg to about 65 mg, about 0.6 mg to about 70 mg, about 0.6 mg to about 75 mg, about 0.6 mg to about 80 mg, about 0.8 mg ~ About 1 mg, about 0.8 mg ~ about 2 mg, about 0.8 mg ~ about 5 mg, about 0.8 mg ~ about 10 mg, about 0.8 mg ~ about 15 mg, about 0.8 mg ~ about 20 mg, about 0.8 mg ~ about 25 mg, about 0.8 mg to about 30 mg, about 0.8 mg to about 35 mg, about 0.8 mg to about 40 mg, about 0.8 mg to about 45 mg, about 0.8 mg to about 50 mg, about 0.8 mg to about 55 mg , About 0.8 mg to about 60 mg, about 0.8 mg to about 65 mg, about 0.8 mg to about 70 mg, about 0.8 mg to about 75 mg, about 0.8 mg to about 80 mg, about 1 mg to about 2 mg, about 1 mg to about 5 mg, about 1 mg to about 10 mg, about 1 mg to about 15 mg, about 1 mg to about 20 mg, about 1 mg to about 25 mg, about 1 mg to about 30 mg, about 1 mg to about 35 mg, about 1 mg to about 40 mg, about 1 mg to about 45 mg, about 1 mg to about 50 mg, about 1 mg to about 55 mg, about 1 mg to about 60 mg, About 1 mg to about 65 mg, about 1 mg to about 70 mg, about 1 mg to about 75 mg, about 1 mg to about 80 mg, about 2 mg to about 5 mg, about 2 mg to about 10 mg, about 2 mg ~ about 15 mg, about 2 mg ~ about 20 mg, about 2 mg ~ about 25 mg, about 2 mg ~ about 30 mg, about 2 mg ~ about 35 mg, about 2 mg ~ about 40 mg, about 2 mg ~ About 45 mg, about 2 mg to about 50 mg, about 2 mg to about 55 mg, about 2 mg to about 60 mg, about 2 mg to about 65 mg, about 2 mg to about 70 mg, about 2 mg to about 75 mg, about 2 mg to about 80 mg, about 5 mg to about 10 mg, about 5 mg to about 15 mg, about 5 mg to about 20 mg, about 5 mg to about 25 mg, about 5 mg to about 30 mg, About 5 mg to about 35 mg, about 5 mg to about 40 mg, about 5 mg to about 45 mg, about 5 mg to about 50 mg, about 5 mg to about 55 mg, about 5 mg to about 60 mg, about 5 mg ~ about 65 mg, about 5 mg ~ about 70 mg, about 5 mg ~ about 75 mg, about 5 mg ~ about 80 mg, about 10 mg ~ about 15 mg, about 10 mg ~ about 20 mg, about 10 mg ~ About 25 mg, about 10 mg to about 30 mg, about 10 mg to about 35 mg, about 10 mg to about 40 mg, about 10 mg to about 45 mg, about 10 mg to about 50 mg, about 10 mg to about 55 mg, about 10 mg to about 60 mg, about 10 mg to about 65 mg, about 10 mg to about 70 mg, about 10 mg to about 75 mg, about 10 mg to about 80 mg, about 15 mg to about 20 mg, About 15 mg to about 20 mg, about 15 mg to about 25 mg, about 15 mg to about 30 mg, about 15 mg to about 35 mg, about 15 mg to about 40 mg, about 15 mg to about 45 mg, about 15 mg ~ about 50 mg, about 15 mg ~ about 55 mg, about 15 mg ~ about 60 mg, about 15 mg ~ about 65 mg, about 15 mg ~ about 70 mg, about 15 mg ~ about 75 mg, about 15 mg ~ About 80 mg, about 20 mg to about 25 mg, about 20 mg to about 30 mg, about 20 mg to about 35 mg, about 20 mg to about 40 mg, about 20 mg to about 45 mg, about 20 mg ~ about 50 mg, about 20 mg ~ about 55 mg, about 20 mg ~ about 60 mg, about 20 mg ~ about 65 mg, about 20 mg ~ about 70 mg, about 20 mg ~ about 75 mg, about 20 mg ~ About 80 mg, about 25 mg to about 30 mg, about 25 mg to about 35 mg, about 25 mg to about 40 mg, about 25 mg to about 45 mg, about 25 mg to about 50 mg, about 25 mg to about 55 mg, about 25 mg to about 60 mg, about 25 mg to about 65 mg, about 25 mg to about 70 mg, about 25 mg to about 75 mg, about 25 mg to about 80 mg, about 30 mg to about 35 mg, About 30 mg to about 40 mg, about 30 mg to about 45 mg, about 30 mg to about 50 mg, about 30 mg to about 55 mg, about 30 mg to about 60 mg, about 30 mg to about 65 mg, about 30 mg ~ about 70 mg, about 30 mg ~ about 75 mg, about 30 mg ~ about 80 mg, about 35 mg ~ about 40 mg, about 35 mg ~ about 45 mg, about 35 mg ~ about 50 mg, about 35 mg ~ About 55 mg, about 35 mg to about 60 mg, about 35 mg to about 65 mg, about 35 mg to about 70 mg, about 35 mg to about 75 mg, about 35 mg to about 80 mg, about 40 mg to about 45 mg, about 40 mg to about 50 mg, about 45 mg to about 55 mg, about 45 mg to about 60 mg, about 45 mg to about 65 mg, about 45 mg to about 70 mg, about 45 mg to about 75 mg, About 45 mg to about 80 mg, about 50 mg to about 55 mg, about 50 mg to about 60 mg, about 50 mg to about 65 mg, about 50 mg to about 70 mg, about 50 mg to about 75 mg, about 50 mg ~ about 80 mg, about 55 mg ~ about 60 mg, about 55 mg ~ about 65 mg, about 55 mg ~ about 70 mg, about 55 mg ~ about 75 mg, about 55 mg ~ about 80 mg, about 60 mg ~ About 65 mg, about 60 mg to about 70 mg, about 60 mg to about 75 mg, about 60 mg to about 80 mg, about 65 mg to about 70 mg, about 65 mg to about 75 mg, about 65 mg to about 80 mg, about 70 mg to about 75 mg, about 70 mg to about 80 mg, about 75 mg to about 80 mg).

One or more additional unit operations In some embodiments of any of the methods provided herein, one or more additional unit operations may be performed on the recombinant protein. In some embodiments, the one or more additional unit operations include virus inactivation, virus filtration, polishing, size exclusion chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, mixing mode. It is selected from the group consisting of chromatography, reverse phase chromatography, and ultrafiltration / diafiltration. In some embodiments, the one or more additional unit operations include cleaning in place (CIP) from which precipitated and / or denatured material is removed.

In some embodiments of any of the methods described herein, the method may further comprise the step of formulating the recombinant protein. In some embodiments, the recombinant protein is formulated into a recombinant protein formulation.

The present invention will be further described in the following examples. These examples do not limit the scope of the invention described in the claims.

Example 1. Comparison test experiment setup between conventional purification method and "capture purification" method using clarified supernatant Expression of construct C26, a conventional IgG antibody, in HEK 293 suspended cells on a 1 L cell culture scale for 5 days. I let you. Upon harvesting of construct C26, the supernatant was clarified by centrifugation and filtration through a filter with a pore size of 0.22 μm. The clarified supernatant was divided into 4 x 250 mL aliquots and stored at 4 ° C until required for testing. Three different production methods were tested using 150 mL of clarified supernatant (or 185 mL for AKTA purification). All three purification methods were performed using the same elution buffer and the same neutralization buffer. The AKTA method uses PBS washing and at least one citrate washing. The first purification method, AKTA, is a programmed purification method that uses an immobilized resin in a prepacked column (ie, a commercially available column). The AKTA purification method is based on directional flow. The second purification method is the conventional batch capture method. This conventional batch capture method involves overnight shaking with the resin free-floating in the clarified supernatant. A third purification method, called "capture purification," involves the use of chromatographic filtration pods as described herein. The capture and purification method also includes the same overnight shaking as the conventional batch capture method.

Results Experiments revealed that the yield and purity of construct C26 were comparable for all three methods tested (Table 1).

* 純度は分析的サイズ排除クロマトグラフィ（SEC）により決定した。図20および21のクロマトグラフも参照されたい。 (Table 1) Summary table of yield and purity comparing the three purification methods

* Purity was determined by analytical size exclusion chromatography (SEC). See also the chromatographs in Figures 20 and 21.

The AKTA purification method used 5 mL of resin, while the conventional batch capture and capture purification methods both used 3 mL. Nevertheless, 3 mL of resin was sufficient to capture the equivalent amount of IgG antibody in the supernatant, based on the observed binding capacity of the resin. Next, SDS is used to analyze 20 μL flow-through (FT) samples, 20 μL wash samples (W), and 1 μg elution samples (E) collected during conventional batch and capture purification methods. -PAGE was performed (Fig. 5). Reduced and non-reduced samples from both purification methods were run onto Life Tech 4-12% gels. The protein of interest was not lost during the flow-through or wash phase in either method, suggesting that protein capture was complete in both methods.

Figure 6 shows an outline of the experimental timeline of the three methods. As shown in Figure 6, the AKTA purification method can take up to 7 days after transfection to complete, with 5-6 hours or more devoted to clarification and experimental equipment setup. In addition, the AKTA purification method is flow rate constrained and therefore can dramatically increase the time to completion for larger volumes (> 1 L), sometimes longer than shown in Figure 6. It may take. In addition, the AKTA purification method can produce only one protein at a time. In contrast, conventional batch and capture purification methods can simultaneously capture and purify multiple proteins in a time frame as shown in FIG. Traditional batch purification methods can take up to 7 days after transfection to complete and are the most labor-intensive of the three methods tested. Both the AKTA purification method and the conventional batch method require far more consumables than the capture and purification methods described herein. The capture and purification methods of the present invention are much less labor intensive and require very little user interaction. The capture and purification methods of the invention can be completed by day 6 after transfection.

Example 2. Test experiment setup of "capture purification" method with pods containing a porous mesh with an average pore size of 20 μm or 40 μm Harvest and harvest 750 mL cultures of HIS-tagged human protein (C27) expressed as in Example 1. Divided into two groups for post-processing. The supernatant labeled "filtered" in Table 2 was clarified by centrifugation and then passed through a filter with a pore size of 0.22 microns. The other group did not perform any treatment and was marked as "unfiltered" in Table 2. Two 125 mL aliquots of "filtered" and "unfiltered" medium were added to 250 mL bottles. A capture and purification device was added to each bottle with 1 mL of NI-NTA affinity resin and either a 20 micron mesh or a 40 micron mesh covering the bottom of the device (the only opening in this case). Conventional batch capture conditions were not included in this experiment. The experimental conditions and yields are shown in detail in Table 2 below.

: コンストラクトC27に対する従来のバッチ捕捉の平均収率は約46 mg/mLである。
& 濃度を測定する前に除去された、濃縮処理中の凝集が、この条件の収率を低下させた。 (Table 2) Experimental conditions

: The average yield of conventional batch capture for construct C27 is about 46 mg / mL.
Aggregation during the concentration process, which was removed before measuring the & concentration, reduced the yield in this condition.

As shown in Figure 7, high purity was seen across all four conditions (approximately 92%). These data were not compromised in purity compared to a 20 μm porous mesh using a 40 μm porous mesh; and HIS affinity tag purification with the capture and purification device of the present invention. It is shown that the yield equivalent to that of the conventional batch capture method using the above can be obtained.

Example 3. Test Experimental Setup for Purification Method Using Pods Containing 40 μm Porous Mesh Different experimental setups were tested for the ability to capture transiently expressed human IgG. In the first setup (Conditions # 2 and 3), the pod contained a porous mesh only at the bottom edge of the pod. In the second setup (Condition # 4), the pod had porous meshes on both the top and bottom edges of the pod and had vents on the walls of the pod (Figure 4).

Both setups are performed with approximately 175 mL medium in a 150 mL bottle, by positioning the pod so that its vertical axis is approximately vertical (in filtered or unfiltered liquid cell culture). Placed in post-filtered or unfiltered liquid cell culture (as described in Examples 1 and 2). After removing the pods from the liquid cell culture, the columns were washed and then eluted with PBS and citrate buffer, respectively.

(Table 3) Experimental conditions for capture purification for IgG purification

The yields in mg / L for all four conditions are shown in Table 3. The purity by HPLC was the same across all four conditions (Figure 8), but the third and fourth setups yielded higher yields compared to conventional batch capture cultures, while the second setup had lower yields. It was.

Example 4. Test of purification method using pod with 20 μm porous mesh In this test, the conventional batch capture culture method using the filtered supernatant was applied only to the unfiltered supernatant and the lower end of the pod. A comparison was made with a method using a pod having a porous mesh as described in Example 3 (Table 4).

(Table 4) Experimental conditions

The yields in mg / L for both conditions are shown in Table 4. The purity by HPLC was the same over the test conditions of both methods (Fig. 9), but the yield of the capture purification method was higher than that of the conventional batch capture culture.

Example 5. Test of purification method using a pod with a 20 μm or 40 μm porous mesh and adding a stopcock to control the flow rate after absorption In this test, transient expression of IgG was 0.9 L and it. With subsequent purification, conventional batch capture methods were compared to methods using pods and unfiltered medium. The pod, in this case, had openings in the vertical sides and base covered with either a 20 micron or 40 micron mesh. The 0.9 L culture was divided into 5 180 mL aliquots. One of these aliquots was clarified by centrifugation and filtered through a filter with a pore size of 0.22 microns; in the other four aliquots, the cell culture medium was untreated and was called "unfiltered". All aliquots were added to 250 mL bottles. A pod as described above with 1.5 mL of protein A resin added internally was added to the unfiltered condition, and 1.5 mL of protein A resin was added to the filtered condition. All 250 mL bottles were then incubated overnight with shaking. Addition of a stopcock to control the flow rate was performed after removing the pod from the medium after overnight incubation. See Table 5 for experimental conditions and results.

(Table 5) Experimental conditions and results

Yields are shown for all conditions. Purity was similar under all conditions, but pods sometimes showed similar yields to conventional batch capture methods.

Example 6. Test Experiment Setup of Purification Method Using Pods Containing 40 μm Porous Mesh For the ability to capture antibodies from antibody-containing cell culture, either post-filtered or unfiltered but otherwise identical. Different experimental setups were tested. For setups with devices such as those shown in Figures 27-28, the supernatant was not filtered. For the positive control setup, the supernatant was filtered before adding the free-floating resin.

The first setup used 2 mL of free-floating protein A chromatographic resin exposed to the filtered supernatant in a free-floating batch capture environment with stirring at 150 RPM. Subsequent purification used a gravity flow column to capture the resin from the supernatant and then wash and elute the antibody from the resin. The gravity column was washed with 25 mL PBS and eluted with 20 mL elution buffer. The chromatography column was loaded with the same filtered antibody-containing cell culture medium used for contact with the device / pod used in the third setup.

The second setup used 2.5 mL of free-floating protein A chromatographic resin exposed to the filtered supernatant in a free-floating batch capture environment with stirring at 150 RPM. Subsequent purification used a gravity flow column to capture the resin from the supernatant and then wash and elute the antibody from the resin. The gravity column was washed with 37.5 mL PBS and eluted with 30 mL elution buffer. The chromatographic column was loaded with the same filtered cell culture medium used for contact with the device / pod used in the fourth setup.

The third setup used the devices shown in Figures 27 and 28 loaded with 2 mL of Protein A chromatographic resin. The resin in the pod / device was washed with 75 mL PBS and eluted with 30 mL elution buffer. The elution buffer was first contacted with the resin in the pod / device, followed by a 5-minute rest, and then an additional elution buffer was run through the resin in the pod / device. Chromatography was performed using the AKTA system. The resin in the pod / device was loaded with the same filtered cell culture medium used for contact with the device / pod used in the first setup. More specifically, the pods were contacted with 350 mL of filtered cell culture medium in a 500 mL bottle.

The fourth setup used the devices shown in Figures 27 and 28 loaded with 3 mL of Protein A chromatographic resin. The resin in the pod / device was washed with 65 mL PBS and eluted with 25 mL elution buffer (flow rate 3 mL / min). Chromatography was performed using the AKTA system. The resin in the pod / device was loaded with the same filtered cell culture medium used for contact with the device / pod used in the second setup. More specifically, the pods were contacted with 350 mL of filtered cell culture medium in a 500 mL bottle.

The resulting data are shown in Table 6 below. Chromatographs obtained from device / pod elution by the third and fourth setups are shown in Figures 32 and 33, respectively.

(Table 6) Experimental conditions for capture purification for IgG purification

The above data show that the pods / devices provided herein provide similar recombinant antibody yields as compared to the use of standard chromatographic columns.

Other Aspects Although the present invention has been described with its detailed description, it is understood that the above description is intended to illustrate, but not limit, the scope of the invention as defined by the appended claims. It should be. Other aspects, advantages, and modifications are within the claims below.

Claims (106)

It comprises a container formed by a wall and having an upper end and a lower end,
The wall forms a seal with the lower end, and one or more of the wall, the upper end, and the lower end allow liquid to reversibly flow into the pod from outside the pod or semi-porous or Includes at least one area covered by a porous material,
Filtration and chromatography pods. One of (a) that the semi-porous or porous material does not allow cells to flow into the pod from outside the pod, and (b) that the resin is retained or substantially retained inside the pod. The pod according to claim 1, characterized by or both. The pod according to claim 2, wherein the cell is a mammalian cell. The pod according to claim 2, wherein the cells are insect cells or yeast cells. The pod according to claim 2, wherein the cell is a bacterial cell. The pod according to any one of claims 1 to 5, wherein the material is a semipermeable membrane or a porous membrane. The pod according to any one of claims 1 to 5, wherein the material is a semipermeable or porous mesh. The pod according to claim 7, wherein the mesh is made of nylon. The pod according to any one of claims 1 to 8, wherein the material has a pore size of about 50 nm to about 50 μm. 9. The pod of claim 9, wherein the material has a pore size of about 100 nm to about 30 μm. 10. The pod of claim 10, wherein the material has a pore size of about 500 nm to about 20 μm. 11. The pod of claim 11, wherein the material has a pore size of about 1 μm to about 10 μm. 12. The pod of claim 12, wherein the material has a pore size of about 2 μm to about 5 μm. The pod according to any one of claims 1 to 13, wherein the container has an internal volume of about 1 mL to about 500 mL. 14. The pod according to claim 14, wherein the container has an internal volume of about 1 mL to about 250 mL. 15. The pod according to claim 15, wherein the container has an internal volume of about 1 mL to about 100 mL. The pod according to any one of claims 1 to 16, wherein the lower end comprises at least one area covered with the material. The pod according to any one of claims 1 to 16, wherein the wall comprises at least one area covered with the material. The pod according to any one of claims 1 to 16, wherein the lower end and the wall each include at least one area covered with the material. The pod according to any one of claims 1 to 16, wherein the upper end comprises at least one area covered with the material. The pod according to any one of claims 1 to 16, wherein each of the upper end and the lower end comprises at least one area covered with the material. The pod according to any one of claims 1 to 21, wherein each of the at least one area has a surface area of about 1 mm ² to about 25 cm ² . 22. The pod according to claim 22, wherein each of the at least one area has a surface area of about 1 mm ² to about 20 cm ² . 23. The pod according to claim 23, wherein each of the at least one area has a surface area of about 1 mm ² to about 10 cm ² . 24. The pod according to claim 24, wherein each of the at least one area has a surface area of about 1 mm ² to about 5 cm ² . 25. The pod according to claim 25, wherein each of the at least one area has a surface area of about 1 mm ² and 2.5 cm ² . 26. The pod according to claim 26, wherein each of the at least one area has a surface area of about 1 mm ² and about 1 cm ² . The pod according to any one of claims 1 to 27, which has a flat lower end. The pod according to any one of claims 1 to 27, which has a hemispherical lower end. The pod according to any one of claims 1 to 27, which has a conical lower end. The pod according to any one of claims 1 to 30, wherein the wall has a cylindrical shape. The pod according to any one of claims 1 to 30, wherein the wall has a conical shape. The pod according to any one of claims 1 to 30, wherein the wall has a spherical shape. The pod according to any one of claims 1 to 30, wherein the wall has a hemispherical shape. The pod according to any one of claims 1 to 30, wherein the wall has an hourglass shape. The pod according to any one of claims 1 to 35, wherein the pod further comprises a chromatographic resin arranged inside the container. 36. The pod according to claim 36, wherein the chromatographic resin is a commercially available chromatographic resin. The pod according to claim 36 or 37, wherein the chromatography resin is an affinity chromatography resin. 38. The pod according to claim 38, wherein the affinity chromatography resin is a protein A resin, a protein G resin, a protein L resin, a nickel resin, or a magnetic bead resin. The pod according to any one of claims 36 to 39, wherein the chromatographic resin has a volume of about 1 mL to about 500 mL. 40. The pod of claim 40, wherein the chromatographic resin has a volume of about 1 mL to about 250 mL. 41. The pod of claim 41, wherein the chromatographic resin has a volume of about 1 mL to about 100 mL. 42. The pod of claim 42, wherein the chromatographic resin has a volume of about 1 mL to about 50 mL. The pod according to any one of claims 36 to 43, wherein the chromatographic resin has a sterility assurance level of about 1 x 10 ^-6 to about 1 x 10 ^-10 . A kit comprising any of the pods according to claims 1-44. The kit according to claim 45, further comprising means for stably positioning the pod inside the culture vessel. 46. The kit of claim 46, wherein the culture vessel is a shaking flask, shaking tube, bioreactor, or multi-well plate. 47. The kit of claim 47, wherein the bioreactor is a perfusion bioreactor. 47. The kit of claim 47, wherein the bioreactor is a feed batch bioreactor. (A) With at least one pod according to any one of claims 18, 19, and 22-44;
(B) One or more panels that can be securely anchored over each of the one or more areas in the wall to prevent fluid from leaking through the wall from inside the pod to outside the pod. A kit that includes and. (A) With the pod according to any one of claims 18, 19, and 22-44;
(B) Securely anchored onto the pod to cover the at least one area of the wall so that fluid is prevented from leaking from the inside of the pod to the outside of the pod through the at least one area of the wall. A kit with an outer sleeve. 51. The kit of claim 51, wherein the sleeve is an empty chromatography column. (A) With the pod according to any one of claims 1-44;
(B) A kit comprising an elution outlet that can be stably fixed on the lower end and one or both of a buffer reservoir that can be stably fixed on the upper end. 53. The kit of claim 53, comprising a pod and an elution outlet. 53. The kit of claim 53, comprising a pod and a buffer reservoir. 53. The kit of claim 53, comprising a pod, an elution outlet, and a buffer reservoir. The kit according to any one of claims 53, 54, and 56, wherein the elution outlet is a funnel. The kit of any one of claims 53, 54, and 56, wherein the elution outlet comprises a flat surface having a port. The kit of any one of claims 53, 54, and 56-58, wherein the elution outlet is configured to regulate the flow of fluid through the elution outlet. The kit of any one of claims 53, 54, and 56-58, wherein the elution outlet is not configured to regulate the flow of fluid through the elution outlet. The kit of any one of claims 53, 54, and 56-60, wherein the elution outlet can be screwed or clicked onto the pod. (A) With the pod according to any one of claims 18, 19, and 22-44;
(B) Rotation of the movable outer sleeve may cover the at least one area of the wall so that fluid does not leak from the inside of the pod to the outside of the pod through the at least one area of the wall. A kit comprising a movable outer sleeve so connected to the wall. The kit according to any one of claims 50 to 62, further comprising means for stably positioning the pod inside the culture vessel. (A) With the pod according to any one of claims 17 and 20-44;
(B) A kit provided with means for stably positioning the long axis of the pod in a horizontal position inside the culture vessel. (A) With the pod according to any one of claims 1-19, 22-28, 31, and 36-44;
(B) An inlet system having an upper end, an elongated cylindrical body with a fluid conduit, and a lower end, the upper end of the inlet system having a fluid inlet and stable at the upper end of the pod. It is configured to be attached to the pod; and when stably connected to the pod, allows the flow of fluid through the fluid inlet and the fluid conduit and out of the lower end of the inlet system and into the pod; And with the inlet system, the elongated cylindrical body of the inlet system is configured to fit inside the wall of the pod;
(C) An outlet system comprising a fluid outlet, configured to be stably attached to the lower end of the pod, and when stably connected to the lower end of the pod, through the fluid outlet. A kit with an outlet system that allows the flow of fluid out of the pod. 65. The kit of claim 65, wherein the top edge of the inlet system and the top edge of the pod include threading that allows the top edge of the pod to be stably attached to the top edge of the inlet system. The kit of claim 65 or 66, wherein the thread at the top of the inlet system, or the thread at the top of the pod, comprises an O-ring. A claim in which the inlet system and the pod are configured such that the pod contains a chromatographic resin; and when the inlet system and the pod are stably attached, the lower end of the inlet system is close to the chromatography resin. The kit according to any one of items 65 to 67. The kit of claim 68, wherein the inner surface of the pod and the lower end of the inlet system are configured to allow stable attachment of the pod to the lower end of the inlet system. 22. The kit of claim 69, wherein the inner surface of the pod and the lower end of the inlet system are threaded to allow stable attachment of the pod to the lower end of the inlet system. The kit of claim 70, wherein the threading on the inner surface of the pod, or threading on the lower end of the inlet system, comprises an O-ring. The kit according to any one of claims 65 to 71, wherein the outlet system and the lower end of the pod are threaded to allow the outlet system to be stably attached to the lower end of the pod. 72. The kit of claim 72, wherein the threading in the outlet system, or threading at the lower end of the pod, comprises an O-ring. The kit according to any one of claims 65 to 73, wherein the fluid inlet can be attached to the tubing. The kit according to any one of claims 65 to 74, wherein the fluid outlet can be attached to the tubing. (D) The kit according to any one of claims 65 to 75, further comprising means for stably positioning the long axis of the pod in a horizontal position inside the culture vessel. A method of capturing recombinant protein produced by liquid cell culture, comprising contacting any of the pods of claims 36-44 with the liquid cell culture for a period of time.
The contact step results in the contact of at least a portion of the at least one area with the liquid cell culture medium, and the contact results in the capture of the recombinant protein by a chromatographic resin. The way it is done. The method of claim 77, wherein the liquid cell culture medium is placed in a culture vessel. A method of purifying recombinant protein produced by liquid cell culture, comprising the following steps:
(A) The step of contacting any of the pods of claims 36-44 with the liquid cell culture medium over a period of time, the result of which at least a portion of the at least one area is in contact with the liquid cell culture solution. The contact is carried out under conditions that result in the capture of the recombinant protein by the chromatographic resin; and (b) elution of the recombinant protein from the chromatographic resin after the period. The step of purifying the recombinant protein. 49. The method of claim 79, further comprising (b) followed by (c) one or more additional unit operations on the recombinant protein. 38. The method of claim 80, further comprising the step of formulating the recombinant protein into a recombinant protein formulation, after (c). The method according to any one of claims 77 to 81, further comprising washing the chromatographic resin between steps (a) and step (b). The method of any one of claims 77-82, wherein the contact step is performed under sterile conditions. The method according to any one of claims 77 to 83, wherein the period is about 5 minutes to about 2 weeks. The method of claim 84, wherein the period is from about 5 minutes to about 168 hours. 85. The method of claim 85, wherein the period is from about 5 minutes to about 96 hours. 86. The method of claim 86, wherein the period is from about 5 minutes to about 48 hours. 87. The method of claim 87, wherein the period is from about 5 minutes to about 24 hours. 88. The method of claim 88, wherein the period is from about 5 minutes to about 12 hours. The method of any one of claims 77-89, wherein the period occurs when the liquid cell culture is in an exponential growth phase. The method of any one of claims 77-89, wherein the period occurs when the liquid cell culture is in a steady growth phase. The method of any one of claims 77-91, wherein at least 30% of the total surface area of the at least one area is in contact with the liquid cell culture medium over the period of step (a). 92. The method of claim 92, wherein at least 50% of the total surface area of the at least one area is in contact with the liquid cell culture medium over the period of step (a). 39. The method of claim 93, wherein at least 75% of the total surface area of the at least one area is in contact with the liquid cell culture medium over the period of step (a). The method of claim 94, wherein at least 90% of the total surface area of the at least one area is in contact with the liquid cell culture medium over the period of step (a). The method according to any one of claims 77 to 95, wherein the liquid cell culture solution is arranged in a culture vessel. 96. The method of claim 96, wherein the culture vessel is a shaking tube, shaking flask, or bioreactor. The method of claim 97, wherein the bioreactor is a perfusion bioreactor. 97. The method of claim 97, wherein the bioreactor is a fed batch bioreactor. The method according to any one of claims 77 to 99, wherein the liquid cell culture medium comprises bacterial cells, yeast cells, plant cells, or mammalian cells. The method of claim 100, wherein the mammalian cell is a human cell. 10. The method of claim 101, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell. The one or more additional unit operations described above include virus inactivation, virus filtration, polishing, size exclusion chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, mixed mode chromatography, and ultrafiltration. 80. The method of claim 80, selected from the group consisting of / diafiltration. The method according to any one of claims 77 to 103, wherein the recombinant protein is a secretory protein. The method of any one of claims 77-104, wherein the recombinant protein is any protein with or without a tag. The method according to any one of claims 77 to 104, wherein the recombinant protein is an antibody, an antigen-binding antibody fragment, an enzyme, or a fusion protein.

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