AU2013203461B2 - Process for cell culturing by continuous perfusion and alternating tangential flow - Google Patents

Abstract

A method for limiting cell aggregation during cell culture to a level wherein no more than 5% of the animal cells in suspension in the culture comprise aggregates of at least 5 cells, comprising: carrying out continuous perfusion culturing of a cell culture comprising cell culture medium and animal cells that easily or inherently form aggregates during culturing, wherein the continuous cell culturing comprises: (a) adding cell culture medium to the cell culture, and (b) circulating the cell culture through a filter module comprising hollow fibers, in an alternating tangential flow resulting in an outflow of cell culture liquid through the pores of the filter module having a lower animal cell density per ml than the cell culture prior to circulating through the filter module, wherein no more than 5% of the animal cells in the culture form aggregates in suspension of at least 5 cells during the continuous perfusion culturing, and wherein the continuous perfusion culturing is continued until animal cells are present in the cell culture at a density of at least 80 x 106 viable animal cells/ml.

Description

1 PROCESS FOR CELL CULTURING BY CONTINUOUS PERFUSION This application is a divisional from Australian application 2005229359, the entire disclosure of which is incorporated herein by reference. The present invention relates to perfusion culturing of cells. 5 The present invention discloses a method for limiting cell aggregation during cell culture to a level wherein no more than 5% of the animal cells in suspension in the culture comprise aggregates of at least 5 cells, comprising: carrying out continuous perfusion culturing of a cell culture comprising cell culture medium and animal cells that easily or inherently form aggregates during culturing, wherein the 10 continuous cell culturing comprises: (a) adding cell culture medium to the cell culture, and (b) circulating the cell culture through a filter module comprising hollow fibers, in an alternating tangential flow resulting in an outflow of cell culture liquid through the pores of the filter module having a lower animal cell density per ml than the cell culture prior to circulating 15 through the filter module, wherein no more than 5% of the animal cells in the culture form aggregates in suspension of at least 5 cells during the continuous perfusion culturing, and wherein the continuous perfusion culturing is continued until animal cells are present in the cell culture at a density of at least 80 x 106 viable animal cells/ml. 20 The invention further discloses a method for the reduction of the degree of aggregation of cells with an inherent tendency to aggregate in a cell culture wherein a cell culture comprising cell culture medium and a suspension of cells with an inherent tendency to aggregate are maintained in a continuous perfusion culturing system, the cell culture medium is added to the cell culture, the cell culture is circulated over a filter module comprising hollow 25 fibers resulting in an outflow of liquid having a lower cell density than the cell culture, the flow within the filter module is an alternating tangential flow, and the culturing is continued while the desired cell density is reached. The invention further discloses a perfusion culture comprising a cell culture medium and a suspension of animal cells with an inherent tendency to aggregate, wherein said animal 30 cells are present in said perfusion culture at a density of at least 80x10 6 cells per ml, and where aggregates of at least 5 cells comprise at most 5% of the total amount of cells. It has surprisingly been found that by perfusion culturing of animal, in particular mammalian, cells or yeast cells according to the invention, extremely high viable cell densities can be obtained, whereas the cell culture further displays an extremely high cell viability. 35 Furthermore, it was found that the perfusion process of the invention leads to less cell aggregation in the culture, and even to a culture being a suspension of single cells without visible aggregates. This is a surprising finding because the use of low shear conditions, such 1a as in perfusion cell culturing, typically does not lead to disaggregation of cells. Cell aggregation during perfusion cell culturing is disadvantageous, because process control is more difficult, due to, for example, the heterogeneity in metabolic profiles of cells within the cell aggregates. This is especially troublesome if cells form aggregates of 5 cells or more and 5 when the aggregates comprise in total 5 % or more of the total amount of cells. A perfusion process is described in US 6,544,424. Although this document mentions that this process may be used for perfusion culturing of animal cells, it does neither disclose nor suggest the extremely high cell densities found in the present invention. Furthermore, US 6,544,424 B1 discloses that the perfusion process could diminish the attachment and growth 10 of an obstruction on the membrane surface of the hollow fibers, but it does neither disclose nor suggest that cells in the cell culture itself would aggregate less. Voisier et al. (Biotechnol. Bioeng. 82 (2003), 752-765) review various cell retention techniques in high-density perfusion culture of suspended mammalian cells. None of the reviewed cell retention systems is able to provide the extremely high viable cell densities 15 combined with the extremely high cell viability, of the present invention. Perfusion culturing of cells has its conventional meaning in the art, i.e. it means that during culturing cells are retained by a separation device in which there is an WO 2005/095578 PCT/EP2005/002374 -2 outflow of liquid having a lower cell density than prior to separation and in which there Is an Inflow of the cell culture medium. In the process of the present invention, the separation device is a filter module comprising hollow fibers. Perfusion culturing includes, but is not limited to continuous flow and semi 5 continuous flow, for example step-wise flow or staggered flow. With the term "hollow fiber" is meant a tubular membrane. The Internal diameter of the tube is preferably between 0.3 and 6.0 mm, more preferably between 0.5 and 3.0 mm, most preferably between 0.5 and 2.0 mm. Preferably, the mesh size in the membrane is chosen such that the size of the pores in the mesh is close to the 10 diameter of the cells, ensuring a high retention of cells while cell debris can pass the filter. Preferably, the mesh size is between 3-30 pm. Filter modules comprising hollow fibers are commercially available from for example General Electric (formerly Amersham). With "alternating tangential flow within the filter module" is meant that there is 15 one flow in the same direction as (i.e. tangential to) the membrane surfaces of the hollow fibers, which flow is going back and forth, and that there is another flow in a direction substantially perpendicular to said filter surface. Tangential flow can be achieved according to methods known to the person skilled in the art. For example, in US 6,544,424 it is described that alternating tangential flow can be achieved using one 20 pump to circulate the cell culture over a filter module comprising hollow fibers and another pump to remove the liquid having a lower cell density than prior to the filter separation. In the process of the invention, any type of cell culture medium suitable for the culturing of cells can in principle be used. Guidelines for choosing a cell culture 25 medium and cell culture conditions are well known in the art and are for instance provided in Chapter 8 and 9 of Freshney, R. 1. Culture of animal cells (a manual of basic techniques), 4th edition 2000, Wiley-Liss and in Doyle, A., Griffiths, J. B., Newell, D. G. Cell &Tissue culture: Laboratory Procedures 1993, John Wiley & Sons. Generally, a cell culture medium for mammalian cells comprises salts, amino 30 acids, vitamins, lipids, detergents, buffers, growth factors, hormones, cytokines, trace elements and carbohydrates. Examples of salts include magnesium salts, for example MgCl 2 .6H 2 0, MgSO 4 and MgSO 4 .7H 2 0 iron salts, for example FeSO 4 .7H 2 0, potassium salts, for example KH 2

PO

4 , KCI; sodium salts, for example NaH 2

PO

4 , Na 2

HPO

4 and calcium salts, for example CaC1 2 .2H 2 0. Examples of amino acids are all 20 known 35 proteinogenic amino acids, for example hystidine, glutamine, threonine, serine, methionine. Examples of vitamins include: ascorbate, biotin, choline.CI, myo-inositol, WO 2005/095578 PCT/EP2005/002374 -3 D-panthothenate, riboflavin. Examples of lipids include: fatty acids, for example linoleic acid and oleic acid; soy peptone and ethanol amine. Examples of detergents include Tween 80 and Pluronic F68. An example of a buffer is HEPES. Examples of growth factors/hormones/cytokines include IF, hydrocortisone and (recombinant) insulin. 5 Examples of trace elements are known to the person skilled in the art and include Zn, Mg and Se. Examples of carbohydrates include glucose, fructose, galactose and pyruvate. The pH, temperature, dissolved oxygen concentration and osmolarity of the cell culture medium are in principle not critical and depend on the type of cell chosen. 10 Preferably, the pH, temperature, dissolved oxygen concentration and osmolarity are chosen such that it is optimal for the growth and productivity of the cells. The person skilled in the art knows how to find the optimal pH, temperature, dissolved oxygen concentration and osmolarity for the perfusion culturing. Usually, the optimal pH is between 6.6 and 7.6, the optimal temperature between 30 and 39 0 C, the optimal 15 osmolarity between 260 and 400mOsm/kg. Cells that are advantageously subjected to the process of the invention may be any cell type benefiting from this process, i.e. culturing to an extremely high viable cell density and an extremely high cell viability. According to the process of the invention, an extremely high viable cell density 20 is a density of at least 80 x 106 cells per mL, preferably at least 100 x 106 cells per mL, more preferably at least 110 x 108 cells per mL, more preferably at least 120 x 106 cells per mL, more preferably at least 130 x 106 cells per mL, most preferably at least 140 x 100 cells per mL. Typically, a suitable upper limit in the cell density may lie around 500 x 106 cells per mL. 25 Surprisingly, the extremely high cell density of the process of the invention is accompanied by an extremly high cell viability. An extremely high cell viability is a viability of at least 90%, preferably at least 95%, more preferably at least 97%, most preferably at least 99%. It is to be understood that the very high viable cell density and very high cell 30 viability are reached after a certain period of perfusion culturing, generally when the cells have reached a steady state, for mammalian cells typically 12 to 25 days after the initiation of perfusion culturing. The process of the Invention Is suitable for culturing animal cells or yeast cells, especially for culturing mammalian cells. 35 The process of the Invention is further especially suitable for culturing cells that easily or inherently form aggregates during culturing, especially during perfusion 4 culturing (so-called aggregating cells). Surprisingly, the process of the invention not only diminishes aggregate disposal on the filter membrane, but also diminishes aggregation of cells during the perfusion culture process, even aggregation of cells with an inherent tendency to form aggregates. The culturing of cells with an inherent tendency to form 5 aggregates according to the invention results in a culture wherein aggregates of at least 5 fells comprise at the most 5% of the total amount of cells, preferably at the most 4%, more preferably at the most 3%, even more preferably at the most 2% of the total amount of cells. Especially preferably, the culturing of cells with an inherent tendency to form aggregates according to the invention results in a culture that is a real single cell suspension. 10 Cells with an inherent tendency to form aggregates (or aggregating cells) are cells that form aggregates of at least 5 cells, the aggregates comprising in total at least 5% of the total amount of cells. Preferably, the aggregates consist of at least 6, more preferably at least 7, even more preferably at least 8, even more preferably at least 9, even more preferably at least 10 cells. Preferably, the aggregates comprise in total at least 7%, more preferably at 15 least 10%, most preferably at least 15% of the total amount of cells. Examples of mammalian cells include: CHO (Chinese Hamster Ovary) cells, hybridomas, BHK (Baby Hamster Kidney) cells, myeloma cells, human cells, for example HEK-293 cells, human lymphoblastoid cells, PER.C6@ cells, mouse cells, for example NSO cells. Examples of yeast cells include Saccharomyces cerevisiae, Phaffia rhodozyma, 20 Kluyveromyces lactis, or yeast cells from the genus Pichia. Preferably, mammalian cells are used, more preferably CHO, NSO, PER.C6@ cells. Also preferably, cells known for their aggregating behaviour during culturing (aggregating cells) are used. Cell aggregation may for example be determined under a microscope. 25 The rate of addition of cell culture medium to the culture (the inflow rate or perfusion rate) influences the viability and the density of the cells. In one embodiment, the alternating tangential flow is achieved using one pump to circulate the cell culture over a filter module comprising hollow fibers and another pump to remove the liquid having a lower cell density than the cell culture prior to the filter separation. 30 In one embodiment, the cell culture is circulated inside a filter module comprising hollow fibers with an internal diameter of between 0.3 and 6.0mm. In one embodiment of the invention, the cell culture medium is added at a perfusion rate according to the following formula 1: Perfusion rate = SPR*total cell culture volume*viable cell density 35 wherein the perfusion rate is expressed in liters per day, wherein the SPR is the specific perfusion rate, i.e. the rate in which the cell culture medium is fed per time unit and wherein 4a the viable cell density is the number of viable cells per unit of volume. The number of viable cells can be determined by the person skilled in the art, for example 5 WO 2005/095578 PCT/EP2005/002374 -5 via the trypan blue exclusion method. The specific perfusion rate is preferably chosen between 0.01 and 0.3 nL/cell/day, more preferably between 0.01 and 0.2 nL/cell/day. It may be of advantage to take into account additional parameters when 5 adjusting the perfusion rate, for example the amount of glucose to be fed to the culture and/or the oxygen concentration. For example, for PER.C6@ the glucose perfusion rate Is preferably chosen between 3 and 20 mmoles/L, more preferably between 5 and 15 mmoles/L, 'as part of the medium perfusion rate. A person skilled in the art knows how to determine the outflow rate. The outflow 10 rate of the liquid is determined by the perfusion rate and is generally chosen at an equal value. In one embodiment of the invention, the outflow liquid is substantially devoid of viable cells. In another embodiment of the invention, biomass (i.e. cells in cell culture) is 15 removed at least once from the cell culture and additional cell culture medium is added to the cell culture to compensate for the biomass removal. Biomass removal may lead to higher cell densities. Biomass may be removed continuously or step-wise. In the step-wise approach, biomass is removed continuously for a defined time period. If a step-wise approach is used, biomass removal is preferably started just 20 before or just after the cells have reached a steady state. If a step-wise approach is used, a volume of biomass of preferably between 2 and 40 % of the working volume per day, more preferably between 5 and 30% of the working volume per day, even more preferably between 10 and 25% of the working volume per day is removed per biomass removal step. 25 With "working volume" is meant the total volume of the cell culture. With "biomass removal step" is meant the time from the start to the stop of the biomass removal. If a continuous approach is used, the biomass is removed continuously until the end of the cell culturing. Preferably, the continuous removal of biomass is started just before or just after the cells have reached a steady state. 30 Preferably, a volume of biomass is removed of between 2 and 40 % of the working volume per day, more preferably between 3 and 30% of the working volume per day, even more preferably between 4 and 15% of the working volume per day. The addition of the additional cell culture medium is done to compensate for the biomass removal. The feed wherein additional cell culture medium is added to the cell 35 culture may be merged into the perfusion feed, but may also be added in a separate feed. The person skilled in the art is aware how much additional cell culture medium is WO 2005/095578 PCTIEP2005/002374 -6 needed to compensate for the biomass removal. Generally, the rate of addition of the additional cell culture medium to the cell culture will be the same as the biomass removal rate. In yet another embodiment of the invention, a biological substance is produced 5 by the cells. The biological substances that can suitably be produced in the perfusion culturing of the cell are in principle all biological substances that can be produced by animal, especially mammalian, and yeast cells, for example therapeutic and diagnostic proteins, such as monoclonal antibodies, growth factors or peptide hormones, enzymes, polynucleotides, such as viral vectors used in gene therapy, vaccines, etc. 10 In the perfusion culturing process of the invention, the outflow liquid will have a lower cell density but the same concentration of the biological substance than the liquid prior to separation. Preferably, the process according to the invention is used for the production of a biopharmaceutical product, which is a biological substance with a medical 15 application. Examples of biopharmaceutical products are as follows (with examples of brand names of the corresponding biopharmaceutical product between brackets): Tenecteplase (TN Kase

TM

), (recombinant) antihemophilic factor (ReFactoTM), lymphoblastoid Interferon oc-n1 (Wellferon

TM

), (recombinant) Coagulation factor (NovoSevenTM), Etanercept, (Enbrel

TM

), Trastuzumab (Herceptin

TM

), Infliximab 20 (Remicaderm), Basiliximab (Simulect m ), Daclizumab (Zenapazi), (recombinant) Coagulation factor IX (Benefix

TM

), erythropoietin alpha (Epogen@), G-CSF (Neupogen@Filgrastim), Interferon alpha-2b (Infergen@), recombinant insulin (Humulin@), Interferon beta 1a (Avonex@), Factor VIII (KoGENate@), Glucocerebrosidase (Cerezymerm), Interferon beta lb (Betaseron@), TNF alpha 25 receptor (Enbrei@), Follicle stimulating hormone (Gonal-F@), Mab abcixmab (Synagls@, ReoPro@), Mab ritiximab (Rituxan@), tissue plasminogen activator (Activase @, Actilyase@), human growth hormone (Protropin@, Norditropin@, GenoTropinTM). Examples of polynucleotides with a possible medical application are gene therapeutic plasmid DNAs. Some gene therapeutic DNAs are presently tested in 30 clinical trials for their medical application. Examples of vaccines are live, oral, tetravalent Rotavirus vaccine (RotaShieldTM), rabies vaccine (RanAvert

TM

), Hepatitis B vaccin (RECOMBIVAX HB@, Engerix@) and inactivated hepatitis A vaccine

(VAQTA

T

m). The biological substance in the outflow may be further purified in so-called 35 downstream processing. Downstream processing usually comprises several purification steps in varying combinations and order. Examples of purification steps in -6a the downstream processing are separation steps (e.g. by affinity chromatography and/or ion exchange chromatography), steps for the concentration of the biological substance (e.g. by ultrafiltration or diafiltration), steps to exchange buffers and/or steps to remove or inactivate viruses (e.g. by 5 virusfiltration, pH shift or solvent detergent treatment). Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps. The discussion of documents, acts, materials, devices, articles and the 10 like is included in this specification solely for the purpose of providing a context N for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application. 15 The invention will now be elucidated by way of the following examples, without however being limited thereto. VVMants\FILESAU PMs\760O99Vetype pages-25 608 doc -7 Example 1: Process Optimisation of the Human Cell Line PER.C6@ for the production of Biopharmaceuticals 5 Introduction A number of expression platforms now exist for the production of biopharmaceuticals. Most of the new products must choose a mammalian system due in main part to the glycosylation machinery which these cells Nq contain and others lack. However to date the cell mass and resulting 10 productivity of these cells is a factor of 10 -100 times less than a corresponding microbial system if these cells had the machinery to make such products. A perfusion culture setup was developed for the PER.C6@ cell line, a human cell line that possesses a number of features that makes it favourable for the production of biopharmaceuticals. A perfusion setup involves the separation of 15 various components of the culture broth so that cells are retained, harvest is captured and medium refreshment occurs. The performance of a spinfilter, an acoustic device and an Alternating Tangential Flow (ATF) unit within a continuous perfusion culture of the PER.C6@ cell line was assessed. 20 Materials & Methods Cell line and maintenance : A PER.C6@ cell line was used in this study that produces a human IgG. Cells were maintained in a serum free commercial medium (EX-CELL TM VPRO medium, JRH Biosciences), supplemented with 6 mM L-glutamine (Gibco). The PER.C6@ cell line is a human embryonic cell-line 25 immortalised with adenovirus type-5 (ad5) El gene using a phosphoglyceratekinase promoter. Bioreactor Set-up: 1L and 4L working volume reactors (Applikon, Netherlands and 30 V:\Manta\ILESwU Pros\?80099'etype pagos.25 0 08 doc WO 2005/095578 PCTIEP2005/002374 -8 B.Braun, Germany) were used during this study. A Braun DCU3 controller (B. Braun, Germany) was used to operate the process at defined setpoints. Temperature was maintained at 36.5C (range 35.5-37.5 'C). Dissolved oxygen concentration was controlled at 50% (range 40-60%) of air saturation by automatic adjustment of inlet gas 5 composition through the headspace and intermittent sparging through a microporous sparger. The pH setpoint was 7.1 (range 6.7-7.5) and controlled by the flow of C02 via the headspace. Cells were inoculated in the fermenter with an inoculum viable cell density range of 0.2-0.5 *106 cells/mL. Perfusion started at a viable cell density in the range of 1-3*106 cells/mL. 10 Cell Retention: Cells were retained in the reactor using three different devices. First a spinfilter with a 10pm pore size (GKD, Duren Germany) was used. Secondly, a Biosep AD11015 cell retention system and controller (AppliSens, the Netherlands) was used. Finally an ATF-4 control unit and housing with associate hollow fiber membrane 15 module (Refine Technology, USA) was assessed. The hollow fiber filter used was model CFP-2-E-8SIP (0.2 micron, Area: 4600cm 2 , Amersham Bioscience obtained from Magellan instruments, USA). To maintain a constant culture volume a level sensor control loop was in operation. 20 Analytical methods: A cell count from the bioreactor was performed using the trypan blue exclusion method. The number of viable cells was determined as follows: An amount of cells stained with trypan blue was transferred to a Fuchs Rosenthal haemacytometer. The chamber of the haemacytometer was placed under a microscope and an appropriate number of boxes was counted. The viable cell density 25 was calculated using the following formula: Viable cell density (X1 05 cells/ml) = (A+B) x E/320 (2) Wherein 30 A = number of non-stained cells in square A B = number of non-stained cells in square B E = dilution factor Antibody concentration was determined by a analytical protein A column using a HPLC 35 with UV280nm absorption detection; the actual concentration was determined on basis of a calibration curve of a IgG1 reference standard.

WO 2005/095578 PCT[EP2005/002374 -.9 Results Perfusion Cultures 5 Results obtained with the above materials and methods are shown in Figures 1-6. Legends to the Figures: Figure 1: Viable cell density (x106 cells/ml) versus culture time (days) for two different continuous perfusion fermentations of an IgG1 producing PER.C6@ clone using a 10 spinfilter separation device. Stirrer speed setting of the IL Applikon fermenter was 100 150rpm. The perfusion runs were performed in 1L working volume. The specific perfusion rate (SPR) for both perfusion runs was 0.1-0.3 nL/celi/day. In both cases the perfusion runs had to be terminated because of spin filter clogging . Figure 2: Growth of IgG1 producing PER.C6@ cells in a continuous perfusion system 15 with an acoustic device as a cell retention system. Stirrer speed setting of the IL Applikon fermenter was 100-150rpm. The settings used for the run/stop cycle were 300s forward and 4.5s backwards. During the run this was adapted to a 300s/3s cycle (day 15). The specific perfusion rate (SPR) for the perfusion run was between 0.1-0.3 nL/cell/day. 20 Figure 3: Growth of IgG1 producing PER.C6@ cells in a continuous perfusion system with an ATF-4 unit as a cell retention system. The experiment was performed in a 4L Applikon fermenter. Setting for the stirrer speed was 125 rpm. The ATF-4 operated between 0.5 and 3 working volumes per day. The SPR was set at 0.03-0.08 nUcell/day. The inset shows the high cell density of the culture, being completely 25 devoid of aggregating cells. Figure 4: Productivity of igG1 versus culture time (days) for two different continuous perfusion fermentations of a IgG1 producing PER.C6@ clone using a spinfilter separation device. Figure 5: Productivity of IgG1 producing PER.C6TM cells in a continuous perfusion 30 system with an acoustic device as a cell retention system. Figure 6: Productivity of IgG1 producing PER.C6@ cells in a continuous perfusion system with an ATF unit as a cell retention system.

WO 2005/095578 PCTEP2005/002374 -10 Summary See Table 1 for an overview of the data obtained for the different types of perfusion. Table 1. Overview of the viable cell density, volumetric production rate (based on reactor volume) and the yield improvement of the perfusion runs using the three different retention devices. Batch and fed-batch results are added for comparison (data not shown). Process Max. Viable Cell Productivity Yield (total amount Density (106 cells/mL) of product produced) Improvement Factor Batch 8-10 0.5 g/L 1 Fed-Batch 8-10 1.2 g/L 2.4 Continuous Perfusion Spin filter retention 20-30 0.1-0.2 g/L/day 2.8-5.6 device Acoustic retention 20 0.6 g/L/day 16.8 device ATF retention device 100 . 0.9 g/1.day 25.2 5 It can be concluded that continuous perfusion experiments using the ATF unit show significant potential to achieve very high cell densities and product concentrations (100x106 cells/mL and 0.9 g/L/day), while no aggregation of the PER.C6@ cells was observed.. 10 Example 2: Culturing of PER.C6@ cells by perfusIon. Eauipment: B.Braun fermenter control unit (Braun, Germany), 7L Braun vessel and headplate with associated pH, dissolved oxygen (DO) and level sensor probes (Braun, 15 Germany), ATF-4 control unit and housing with associate hollow fiber membrane module (Refine Technology, USA). Filter Filter model: CFP-2-E-8SIP 20 Type: 0.2 micron Area: 4600 cm 2 Amersham Bioscience WO 2005/095578 PCT/EP2005/002374 Working vol ume Setpoint: 4.1 L Range: 3.8 - 4.7 L 5 ATF settings Parameter Setpoint Range Pressure rising setpoint (psi) Variable 2 - 4 Pressure rising flow (L/min) 3.2 2.5 - 4.0 Exhaust flow (Lmin) 3.2 2.5-4.0 Exhaust time (s) Variable 3 - 8 Pre-pressure (psi) Variable 5 - 9 Bleed rate No biomass removal was applied to this process. 10 Materials: 6 mM (final volume) L-glutamine (Gibco) in Ex-CELLm VPRO medium (JRH Bioscience, USA), 12% Na2CO 3 is used to control the pH. Cell Lines and Culture Conditions A PER.C6® cell line expressing a model IgG was investigated In this study. The 15 PER.C6@ cell line is generated from retina-derived primary human cells. The PER.C6® cell line is able to generate complete human monoclonal antibodies (including the glycans) (ref 1, ref 2). Cells were cultured in shaking Erlenymer flasks at 110 rpm and 36.5 0 C. The headspace of these flasks was controlled using a mixture of 5% C0 2 /Air. 20 Ref 1: Jones, D. H., van Berkel, P. H. C., Logtenberg, T. and Bout, A., 2002, 'PER.C6 cell line for human antibody production', Gen. Eng. News 22, 50-54. Ref 2: Jones, D. et al., 2003, 'High-level expression of recombinant IgG in the human cell line PER.C6', Biotechnol. Prog. 19, 163-168. 25 Operation of Fermenter Cells were cultured in a fermenter where dissolved oxygen tension, pH, temperature and agitation rate were controlled as detailed below.

WO 2005/095578 PCT/EP2005/002374 -12 Parameter Setpoint Range Temperature 36.5 0 C 35.5 - 37.5 pH > 6.7 7.5 -6.7 Active pH control using 12% Na 2

CO

3 if pH<6.7 DO 50% 40 -60% Agitation 100- 300 Staged increase as viable cell density (VCOD) increases; VCD (x10 5 cells/ml) Agitation (rpm) 0.3-10 120 10-30 150 30-50 170 50-80 200 80-100 230 100-120 260 >120 300 Process Description: Cells are inoculated in a fermenter with an inoculation viable cell density range of 0.2 0.5 x 106 cells/mi and a setpoint of 0.3 x 106 cells/mi. Perfusion is begun when the 5 viable cell density > 2 x 106 cells /ml or at day 5 of the culture whichever is achieved first. The perfusion rate is dependent on the cell density of the culture and the rates used are described in the table below. Both the flow rate and the dilution rate are adjusted as the cell density in the fermenter increases. 10 Perfusion rates utilized for culture of PER.C6@ cells Viable cell density Specific perfusion rate Setpoint of specific (x10 6 cells/ml) (nl/cell/day) perfusion rate (nl/cell/day) Day 1 of perfusion 0.15-0.25 0.2 3 - 50 0.03 -0.06 0.04 50 -80 0.025 - 0.035 0.03 >80 0.01 - 0.03 0.02 The actual data and results from this example (amongst others flow rates and specific 15 perfusion rates used in this example) are shown in Table 2 below and in Figures 7 and 8. Figure 7: Culture time (days) versus flow (L/day) and specific perfusion rate (SPR in nl/cell/day) for PER.C6@ cells cultured using a perfusion process. 20 Figure 8: Viable cell density and cell viability using the procedure described in example 2.

WO 2005/095578 PCT/EP2005/002374 -13 Table 2. Raw data obtained for example 2 flow rate dilution rate Specific Viable Specific Volumetric o tln perfusion count Viability product production production E(erf(D cnetrationpr (FR) (D) rate (SPR) (VC) concentration rate of IgG1 rate day L/day orukin nL/cell.day 10 6 /mL % g/L g/L.day __ ___volume/day day) _____ 0 0.00 0.00 0.00 0.6 90 0.012 NA NA 1 0.00 0.00 0.00 0.3 77 0.008 NA 0.000 2 0.00 0.00 0.00 0.3 73 0.008 0.0 0.000 3 0.00 0.00 0.00 0.5 80 0.013 12.1 0.000 4 0.00 0.00 0.00 0.9 87 0.019 9.3 0.000 5 0.00 0.00 0.00 1.4 92 0.033 12.0 0.000 6 2.39 0.52 0.20 2.6 95 0.035 5.5 0.009 7 1.06 0.24 0.05 4.9 95 0.054 9.5 0.017 8 2.70 0.57 0.08 7.3 97 0.073 7.2 0.026 9 2.60 0.57 0.05 12.3 97 0.067 3.5 0.040 10 4.29 0.95 0.05 18.6 97 0.115 7.8 0.069 11 5.40 1.20 0.04 26.9 97 0.140 7.0 0.137 12 6.80 1.48 0.05 31.8 96 0.127 5.6 0.179 13 7.39 1.68 0.04 41.4 99 0.129 5.6 0.202 14 8.28 1.88 0.04 44.3 98 0.139 5.8 0.238 15 10.26 2.33 0.03 68.3 98 0.116 4.4 0.269 16 10.70 2.43 0.03 86.1 99 0.151 4.6 0.318 17 12.10 2.63 0.03 80.3 98 0.163 4.9 0.397 18 11.83 2.57 0.02 112.3 98 0.292 7.6 0.592 19 12.50 2.78 0.02 123.0 99 0.291 6.6 0.780 20 12.09 2.57 0.02 126.0 99 0.293 6.3 0.781 21 11.91 2.59 0.02 135.0 98 0.332 6.5 0.806 22 13.70 2.98 0.02 127.5 97 0.395 8.2 1.012 23 10.00 2.17 0.02 128.5 95 0.470 9.3 1.114

Claims (4)

1. 3. A method according to claim 1, wherein the animal cells are CHO cells, hybridomas, BHK cells, myeloma cells, human cells or mouse cells, 4, A method according to any one of the preceding claims, wherein biomass is removed at least once from the cell culture and additional cell culture medium is added to the cell culture. 5, A method according to claim 4, wherein the biomass removal is started just before or just after the cells have reached a steady state. 6, A method according to any one of the preceding claims, wherein the alternating tangential flow is achieved using one pump to circulate the cell culture over a filter module comprising hollow fibers and using another pump to remove the liquid having a lower cell density than the cell culture prior to the filter separation.
2. 7. A method according to any one of the preceding claims, wherein the cells produce a biological substance.
3. 8. A method according to claim 8, wherein the biological substance is a therapeutic or diagnostic protein, such as a monoclonal antibody, a growth factor or a peptide hormone, an enzyme, a polynucleotide, such as a viral vector used in gene therapy, or a vaccine, preferably a monoclonal antibody.
4. 9. A method according to claim 8 or 9, wherein the biological substance is further purified in downstream processing. 15 10 A method according to any of the preceding claims, wherein the cell culture medium is added to the cell culture at a perfusion rate of between 0.01 and 3 nL/cell/day. 11 A method according to any of the preceding claims, wherein the cell culture is maintained in a continuous perfusion culturing system at a pH between 6.6 and 7.6, a temperature between 30* and 390 C, and an osmolarity between 260 and 400 mOsm/kg; 12 A method according to any of the preceding claims, wherein the cell culture is circulated inside a filter module comprising hollow fibers with an internal diameter of between 0.3 and 6.0 mm. 13, A method according to claim one substantially as hereinbefore described with reference to any one of the Examples.