[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2020035050A1 - 用于调节哺乳动物细胞乳酸代谢的培养基及其应用 - Google Patents

用于调节哺乳动物细胞乳酸代谢的培养基及其应用 Download PDF

Info

Publication number
WO2020035050A1
WO2020035050A1 PCT/CN2019/100980 CN2019100980W WO2020035050A1 WO 2020035050 A1 WO2020035050 A1 WO 2020035050A1 CN 2019100980 W CN2019100980 W CN 2019100980W WO 2020035050 A1 WO2020035050 A1 WO 2020035050A1
Authority
WO
WIPO (PCT)
Prior art keywords
medium
ata
cell
culture
ammonium salt
Prior art date
Application number
PCT/CN2019/100980
Other languages
English (en)
French (fr)
Inventor
孙瑞强
蒋媛媛
傅亦德
曹云
唐亮
Original Assignee
上海药明生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海药明生物技术有限公司 filed Critical 上海药明生物技术有限公司
Publication of WO2020035050A1 publication Critical patent/WO2020035050A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components

Definitions

  • the invention relates to the field of culture media, and in particular, to a culture medium for regulating lactic acid metabolism of mammalian cells and application thereof.
  • CHO cells Choinese Hamster Ovary Cells
  • CHO cells are one of the most important and extensive expression systems currently used in the development and production of biotechnology drugs including engineered antibodies and recombinant proteins. Compared with other expression systems, it has the following advantages: continuous cell lines, rapid growth, uniform cell types, can be passaged for more than one hundred generations, and facilitate large-scale production; belongs to fibroblast, a non-secretory cell, It rarely secretes CHO endogenous proteins, so it is very beneficial for the isolation and purification of target proteins; it has good expression and modification capabilities, such as glycosylation, and is an ideal host for the expression of complex biological macromolecules. Important recombinant proteins and their expression production mainly come from the expression system of CHO cells.
  • the medium used to culture CHO cells is rich in nutrients, and some nutrients will be converted into cytotoxic metabolic byproducts.
  • lactic acid is the main metabolic by-product during cell culture.
  • CHO cells usually undergo rapid accumulation of lactic acid during the logarithmic growth phase of strong metabolism. Some cell lines can consume these lactic acid metabolism, but some cell lines lack this ability.
  • the accumulation of a large amount of lactic acid will seriously affect protein expression and protein quality.
  • the accumulation of lactic acid will cause the pH of the culture system to decrease.
  • the lye added to increase the pH will often increase the osmotic pressure of the system, thereby affecting the normal growth and metabolism of cells. And if the pH is not adjusted by adding lye, the low pH environment will affect the enzyme activity and thus affect the cell growth.
  • reducing lactic acid accumulation has always been a great challenge in the cell culture process.
  • the methods of reducing lactic acid accumulation in the prior art include: (1) genetic modification of cells can reduce lactic acid accumulation, such as knocking out the lactate dehydrogenase (LDH) gene, overexpressing the pyruvate carboxylase (PYC2) gene, etc., but The operability is not strong. (2) Lactic acid regulation can also be optimized by cell culture strategies. Replacing glucose as a carbon source with slowly metabolizing sugars can reduce the accumulation of lactic acid, such as the use of galactose, fucose, and maltose.
  • LDH lactate dehydrogenase
  • PYC2 pyruvate carboxylase
  • Aurintricarboxylic acid has a molecular formula of C 22 H 14 O 9 .
  • Aurintricarboxylic acid ammonium salt also known as Aluminon, Ammonium aurintricarboxylate, ATA ammonium
  • the ammonium tricarboxylic acid ammonium salt may also be referred to as an ATA ammonium salt in the present disclosure.
  • the medium supplement has the following advantages: it is a single substance with a clear composition; it is easy to remove in the downstream purification process; the cost is low; and the preparation and use are convenient.
  • ATA is mentioned in other literatures and patents related to the formulation of the culture medium, its use manner, action effect, and principle of exerting biological functions are obviously different from the present invention.
  • the literature disclosed in the prior art mainly has the following problems: ATA is only used as a substitute for insulin, IGF-1, or transferrin, and is not added to a commercial medium in a separate form.
  • US Pat. No. 5,045,454 discloses a serum-free growth medium.
  • the invention chooses to use iron chelate (including citrate / citric acid and Fe / EDTA), ATA, and alkali metal EDTA.
  • the combination serves as a substitute for transferrin in the culture medium.
  • US 6,338,964 B1 discloses a culture medium suitable for culturing mammalian cells under conditions of low dissolved CO 2 concentration, the culture medium comprising: an organic buffer with a concentration higher than 2 mM Liquid, metal complexing agent with a concentration higher than 5 mM, and NaHCO 3 with a concentration less than 1 g / L, wherein the metal complexing agent comprises histidine, iminodiacetic acid, citrate, phosphoserine, ATA and hydroxylysine. It can be seen that the ATA described in this document is not added to the medium in the form of a single component to play a role and solve its claimed technical problem.
  • the invention provides a culture medium for regulating lactic acid metabolism in mammalian cells.
  • FSP fast stable cell pool
  • stable pool stable cell pool
  • clones with lactic acid metabolism problems without optimization and screening.
  • it is to provide a culture medium capable of simultaneously effectively increasing the living cell density and cell viability of mammalian cells, reducing the accumulation of lactic acid in the culture solution, and increasing the yield of mammalian cells expressing foreign proteins.
  • the present invention completes the present invention by adding an optimized concentration of ATA or an ATA ammonium salt to the basic culture at the beginning of the cell culture. That is, the present invention is as follows:
  • the present invention provides a culture medium for regulating lactic acid metabolism in mammalian cells, characterized in that the culture medium contains the following components: (1) a basal medium, and (2) ATA or ATA ammonium salt.
  • the basal medium is free of serum, animal proteins and plant proteins. All the components in the basic medium described herein are clear, and can also be called chemical medium (CDM), and no animal-derived components are added to the medium; the animal proteins include But not limited to bovine serum albumin (Bovine serum albumin, BSA), the plant protein includes but is not limited to soy protein.
  • the basal medium is ActiPro medium, HyCell CHO medium, CDM4MAB medium or Dynamis medium.
  • the basic medium is ActiPro medium
  • the medium can effectively increase the living cell density of mammalian cells, reduce the accumulation of lactic acid in the culture solution, and increase the protein expression of mammalian cells.
  • the concentration of ATA or ATA ammonium salt in the medium is about 0.05-0.8 mM, more preferably the concentration is about 0.1-0.4 mM, and the most preferred concentration is about 0.2 mM.
  • the concentration of the ATA or ATA ammonium salt in the basic culture is higher than 0.8 mM, cell growth will be inhibited; when the concentration of the ATA or ATA ammonium salt in the basic culture is less than 0.05 mM, it will not be effective Regulates cell lactic acid accumulation.
  • the present invention also provides a method for regulating lactic acid metabolism in mammalian cells, which is characterized in that mammalian cells expressing foreign proteins are cultured in the above-mentioned medium, preferably, the mammalian cells are cultured. Culture is initially performed in the medium.
  • the mammalian cell is a CHO cell
  • the foreign protein is an Fc fusion protein, an antibody, or an enzyme
  • the CHO mammalian cell is a CHO-K1 cell
  • the foreign protein is a monoclonal antibody.
  • the present invention further provides the use of the above-mentioned medium for expressing foreign proteins in mammalian cells.
  • the medium is selected for cultivation at the beginning of the mammalian cell culture.
  • the mammalian cell is a CHO cell
  • the foreign protein is an Fc fusion protein, an antibody, or an enzyme
  • the mammalian cell is a CHO-K1 cell
  • the external The source protein is a monoclonal antibody.
  • the Fc fusion protein refers to a new type of protein produced by fusing a biologically active functional protein molecule with an Fc fragment by using technologies such as genetic engineering.
  • the functional protein may be capable of binding an endogenous receptor (or ligand).
  • the monoclonal antibody can be any monoclonal antibody suitable for exogenous expression using CHO-K1 cells, including but not Limited to IgG, IgA monoclonal antibodies.
  • Figure 1-1 and Figure 1-2 are the effects of ATA and ATA ammonium salts on cell growth (Figure 1-1 is the change curve of living cell density with time; Figure 1-2 is the change curve of cell viability with time Figure);
  • Figure 1-3 and Figure 1-4 are the effects of ATA and ATA ammonium salt on the inhibition of apoptosis (Figure 1-3 is the change curve of living cell density with time; Figure 1-4 is the cell viability with time Graph of change);
  • Figures 1-5 to 1-7 are the effects of ATA ammonium salts on ActiPro, HyCell, CHO, and Dynamis media on the lactic acid metabolism of cells (Figure 1-5 is the change curve of living cell density with time; Figure 1-6 It is a graph of the change of cell viability with time; Figure 1-7 is a graph of the change of lactic acid concentration with time);
  • Figure 1-8 to Figure 1-10 are the effects of ATA ammonium salt on the lactic acid metabolism of cells in CDM4MAB medium (Figure 1-8 is the change curve of living cell density with time; Figure 1-9 is the cell viability with Time change curve; Figure 1-10 is the change curve of lactic acid concentration with time);
  • Figure 2-1 to Figure 2-3 are the research diagrams of the concentration of ATA ammonium salt (Figure 2-1 is the change curve of living cell density with time; Figure 2-2 is the change curve of cell viability with time; Figure 2-3 is a graph of the change of lactic acid concentration with time);
  • Figures 2-4 to 2-6 are the study diagrams of the addition time of ATA ammonium salt (Figure 2-4 is the change curve of living cell density with time; Figure 2-5 is the change curve of cell viability with time; Figure 2-6 shows the change curve of lactic acid concentration with time).
  • Media refers to the liquid environment in which the cells are in direct contact with them, and is a collection of various nutrients and buffer systems.
  • Base medium refers to the medium selected for initial inoculation under various operating modes of cell culture, wherein the basic medium is free of serum, animal proteins and plant proteins, and has a clear composition, Good repeatability between batches, easy to detect and analyze.
  • the present invention provides a medium for regulating lactic acid metabolism in mammalian cells, characterized in that the medium contains the following components: (1) a basal medium, and (2) ATA Or ATA ammonium salt.
  • the basal medium is free of serum, animal proteins and plant proteins. All the components in the basic medium described herein are clear, and can also be called chemical medium (CDM), and no animal-derived components are added to the medium; the animal proteins include But not limited to bovine serum albumin (Bovine serum albumin, BSA), the plant protein includes but is not limited to soy protein.
  • ATA or ATA ammonium salt is added as a single component to several of the most important conventional commercial culture media on the market, which effectively improves the lactic acid metabolism level of mammalian cells and reduces the process risk caused by lactic acid accumulation.
  • the use of the culture medium of the present invention can effectively increase the living cell density and cell viability of FSP, stable pool, and cloned cells, and simultaneously increase the yield and quality of foreign proteins expressed by mammalian cells.
  • the culture medium of the present invention serves as a beneficial supplement to a commercial culture medium and improves the applicable range of the commercial culture medium.
  • Aurintricarboxylic acid (ATA) has a molecular formula of C 22 H 14 O 9 .
  • Aurintricarboxylic acid ammonium salt also known as Aluminon, Ammonium aurintricarboxylate, ATA ammonium
  • Agentine tricarboxylic acid ammonium salt refers to an ionic compound composed of three ammonium ions and one amine tricarboxylic acid, which is easily soluble in water and convenient to prepare.
  • the ATA or ATA ammonium salt can be obtained from commercial sources.
  • ATA or ATA ammonium salt can be added as a single component to several of the most important conventional commercial culture media on the market.
  • ATA or ATA ammonium salt has the following advantages: It is a single substance with clear composition; it is easy to remove in the downstream purification process; low cost; easy to prepare and use.
  • the medium containing aurin tricarboxylic acid or ammonium tricarboxylic acid ammonium salt is used for regulating lactic acid metabolism in mammalian cells.
  • the medium contains the following components: (1) a basal medium, and (2) ATA or an ATA ammonium salt.
  • the basal medium is free of serum, animal proteins and plant proteins. All the components in the basic medium described herein are clear, and can also be called chemical medium (CDM), and no animal-derived components are added to the medium; the animal proteins include But not limited to bovine serum albumin (Bovine serum albumin, BSA), the plant protein includes but is not limited to soy protein.
  • the basal medium is ActiPro medium, HyCell, CHO medium, CDM4MAB medium or Dynamis medium.
  • the basic medium is ActiPro medium
  • the medium can effectively increase the living cell density of mammalian cells, reduce the accumulation of lactic acid in the culture solution, and increase the protein expression of mammalian cells.
  • the concentration of ATA or ATA ammonium salt in the culture medium is about 0.05-0.8 mM, more preferred is about 0.1-0.4 mM, and most preferred is about 0.2 mM.
  • a storage solution of aurin tricarboxylic acid or ammonium tricarboxylic acid ammonium salt is prepared; the storage solution is preheated at 37 ° C; a certain volume of the preheated storage solution is added to ActiPro medium, HyCell, CHO medium, CDM4MAB medium or Dynamis medium, mix well to obtain ATA or ATA ammonium salt medium with specific concentration.
  • the composition of the 100 ⁇ stock solution is shown in Table 1.
  • the method for preparing the storage solution described in Table 1 is as follows:
  • Step 1) Select a suitable size container for the quality of water for injection described in Table 1;
  • Step 2 Add ATA or ATA ammonium salt powder of the quality described in Table 1 to the container described in Step 1);
  • Step 3) ATA needs to be dissolved under alkaline conditions: After adding an appropriate amount of NaOH to adjust to an appropriate pH value, mix it uniformly at room temperature; the ATA ammonium salt can be dissolved at room temperature and in water. After it is fully dissolved, add water for injection to the final quality and mix again;
  • Step 4) filtering the solution using a filter membrane with an appropriate pore size
  • Step 5 Store at about 4-8 ° C.
  • the pH value is about 12.2 ⁇ 0.2, 12.2 ⁇ 0.1, or 12.2.
  • the pore size of the filter membrane is about 0.1 ⁇ m, 0.2 ⁇ m, or 0.3 ⁇ m.
  • ATA or ATA ammonium salt is added as a single component to several of the most important conventional commercial culture media on the market, which effectively improves the lactic acid metabolism level of mammalian cells and reduces the effects of lactic acid accumulation. Process risk.
  • using the medium can effectively increase the live cell density and cell viability of FSP, stable pool, and cloned cells, and simultaneously increase the yield and quality of foreign proteins expressed by mammalian cells.
  • the culture medium serves as a beneficial supplement to a commercial culture medium, improving the applicable range of the commercial culture medium.
  • mammalian cell refers to a mammalian cell line that is selected and domesticated for the production of biological agents in accordance with production conditions. Post-translational modifications of the expressed protein are maintained in the protein organism. It has advantages in chemical activity, stability and antigenicity. Many cell lines are available from commercial sources, such as the American Center for Standards and Biological Collection (ATCC). Non-limiting examples of mammalian cells that can be used in the present invention include CHO, BHK, HEK293 and other cells used in the production of biological products. In at least one embodiment, wherein the mammalian cell is a CHO cell, and more preferably, the mammalian cell is a CHO-K1 cell.
  • the mammalian cell is a CHO cell, and optionally, the mammalian cell is a CHO-K1 cell.
  • the cells used are cell lines capable of stably expressing foreign proteins constructed by transfecting CHO-K1 cells with a dual expression vector containing a foreign-encoded gene.
  • the foreign protein is an Fc fusion protein, an antibody, or an enzyme.
  • the antibody is a monoclonal antibody.
  • the cell used is a cell line capable of stably expressing an IgG1 monoclonal antibody constructed by transfecting CHO-K1 cells with a dual expression vector containing an IgG1 encoding gene.
  • Exogenous protein in this article refers to genes that encode protein of interest other than host cells in the field of genetic engineering. Using recombinant DNA technology, efficient amplification and expression of host cells produces proteins of practical value.
  • the exogenous protein is known in the art and can be obtained from commercial sources or by methods known in the art.
  • the exogenous protein is an Fc fusion protein, an antibody, or an enzyme; optionally, the antibody is a monoclonal antibody or a secondary antibody, and the monoclonal antibody may be any suitable CHO-K1 cell
  • Monoclonal antibodies for exogenous expression include, but are not limited to, IgG, IgA monoclonal antibodies.
  • the monoclonal antibody is an IgG1 monoclonal antibody.
  • the IgG1 monoclonal antibody includes, but is not limited to, targets in the tumor field and the autoimmune field, such as CD19 / HER2, CD20 , PD-1, LAG3 and so on.
  • the Fc fusion protein refers to a new type of protein produced by fusing a biologically active functional protein molecule with an Fc fragment using technologies such as genetic engineering.
  • the functional protein may be capable of binding endogenous receptors.
  • the enzyme refers to an enzyme used in a therapeutic drug, that is, a medicinal enzyme, including but not limited to glucosidase, lipase, and the like.
  • mammalian cells expressing a foreign protein are cultured in the medium, and optionally, the mammalian cell culture is initiated in the medium.
  • the mammalian cell is a CHO cell, and more preferably, the mammalian cell is a CHO-K1 cell.
  • the foreign protein is an Fc fusion protein, an antibody, or an enzyme, preferably, the antibody is a monoclonal antibody.
  • ActiPro medium (Hyclone, Cat No .: SH31037), HyCell, CHO medium (Hyclone, Cat No .: SH30933.04), and CDM4MAB medium (Hyclone, Cat No .: SH30800.04): Dynamis medium (Gibco, Cat No .: A26175-03): purchased from Gibco;
  • ATA (Sigma, Cat. No: A1895), ATA ammonium salt (Sigma, Cat. No: A36883), insulin (Sigma, Cat. No: 91077C), sodium butyrate (Sigma, Cat. No: 303410), insulinoid Like growth factor (Repligen, Cat. No: 6082-229).
  • Example 1 Preparation of medium containing aurin tricarboxylic acid or ammonium tricarboxylic acid
  • Step 1) Select a suitable size container for the quality of water for injection described in Table 1;
  • Step 2 Add ATA or ATA ammonium salt powder of the quality described in Table 1 to the container described in Step 1);
  • Step 3) ATA needs to be dissolved under alkaline conditions: add an appropriate amount of 10N NaOH to adjust the pH to 12.2 ⁇ 0.2, and mix well at room temperature; the ATA ammonium salt can be dissolved at room temperature and in water. After it is fully dissolved, add water for injection to the final quality and mix again;
  • Step 4) filtering the solution by using a 0.1 ⁇ m or 0.2 ⁇ m pore size filter membrane;
  • Step 5 Store at 4-8 ° C.
  • # 1 The amount of water for injection is the original amount added, and the final amount of water for injection is not shown.
  • ActiPro medium was selected as the basic medium, and the following five groups of cell culture medium were prepared: (1) control group (Ctrl), that is, no additional substances were added to ActiPro medium; (2) insulin group (Insulin), that is, in ActiPro medium was supplemented with 0.2 mg / L of insulin; (3) Insulin-like growth factor group (IGF-1), that is, 20 ⁇ g / L of IGF-1 was added to ActiPro culture medium; (4) Trigonal tricarboxylic acid Group (ATA), that is, ActiPro medium containing 0.2 mM ATA was prepared according to the method described in Example 1; (5) ATA Ammonium group, that is, 0.2 mM ATA ammonium salt described in Example 1 ActiPro medium.
  • control group that is, no additional substances were added to ActiPro medium
  • insulin group Insulin
  • IGF-1 Insulin-like growth factor group
  • ATA Trigonal tricarboxylic acid Group
  • Cells were inoculated into 250 mL shake flasks containing the above five groups of cell culture medium at a density of 0.4 ⁇ 10 6 cells / mL, cultured in a volume of 50 mL, and placed in a shaker at 36.5 ° C. and 125 rpm for batch culture. During the culture, the density and viability of live cells were monitored.
  • the ATA and ATA ammonium salt groups can significantly increase the viable cell density of the cells; while the insulin group and insulin-like growth factor group have lower viable cell density than the control group ( Figure 1-1 As shown).
  • the culture medium of ATA and ATA ammonium salt group can also effectively increase the cell viability of the cells (as shown in Figure 1-2).
  • ATA and ATA ammonium salts show different growth promoting effects from insulin and insulin-like growth factors during cell culture.
  • ATA ammonium salt Because the structure and function of ATA ammonium salt are similar to ATA, and ATA ammonium salt is easier to prepare, in the following embodiments, only ATA ammonium salt is used for testing.
  • ActiPro control group that is, no additional substances were added to ActiPro culture medium
  • ATA ammonium group ATA Ammonium
  • Methods ActiPro medium containing 0.2mM ATA ammonium salt was prepared; (3) Sodium butyrate group (NaBu), that is, 2mM sodium butyrate was added to ActiPro medium; (4) ATA ammonium salt / sodium butyrate group (ATA ammonium Salt / NaBu), that is, based on the 0.2 mM ATA ammonium salt group, 2 mM sodium butyrate was added on the fifth day of cell culture.
  • Cells were seeded at a density of 0.4 ⁇ 10 6 cells / mL into 250 mL shake flasks containing the above-mentioned four groups of cell culture medium, the culture volume was 50 mL, and the cells were cultured in a shaker at 36.5 ° C and 125 rpm. The day was moved to a shaker at 31.0 ° C, 125 rpm. During the culture process, the density of living cells, cell viability, and antibody expression were monitored.
  • ATA ammonium salt is more suitable than sodium butyrate as a single component to increase the expression of foreign protein in CHO cells into the basal medium; combined with the results of Example 2.1, we can see: ATA and ATA ammonium salts can effectively maintain cell viability and significantly inhibit apoptosis.
  • ATA ammonium salts on lactic acid metabolism of CHO cells in different commercial media were investigated here, including basic media such as ActiPro, HyCell, CHO, CDM4MAB, and Dynamis.
  • basic media such as ActiPro, HyCell, CHO, CDM4MAB, and Dynamis.
  • the CHO-K1 cells cultured in the media expressed Fc fusion Proteins, monoclonal antibodies, monoclonal antibodies and pharmaceutical enzymes.
  • HyCell CHO and Dynamis medium a fed-batch culture process is used.
  • the culture process is based on the culture conditions described in 2.2.
  • the culture process in CDM4MAB is perfusion culture, and the culture process is as follows: cells are seeded at a density of 0.4 ⁇ 10 6 cells / mL into a microreactor containing CDM4MAB medium containing 0.2mM ATA ammonium salt and cultured.
  • the culture volume was 15 mL
  • the pH was 7.0 ⁇ 0.2
  • the stirring speed was 500 rpm
  • the culture temperature was 35.0 ° C.
  • the liquid exchange ratio on day 3-5 was 2: 1 (V culture supernatant : V fresh medium ), and the liquid exchange ratio thereafter was 1: 1.
  • the viable cell density, viability, and lactic acid concentration of the cells were monitored.
  • the addition of ATA ammonium salts to ActiPro, HyCell, CHO, and CDM4MAB media can significantly increase the viable cell density and viability of cells.
  • Example 1 the following four groups of culture medium were prepared: (1) ActiPro control group (ActiPro), that is, no additional substances were added to ActiPro culture medium; (2) 0.1mM ATA ammonium salt group, That is, ActiPro medium containing 0.1 mM ATA ammonium salt was prepared by referring to the method described in Example 1. (3) 0.2 mM ATA ammonium salt group, that is, ActiPro culture containing 0.2 mM ATA ammonium salt was prepared according to the method described in Example 1. (4) 0.4 mM ATA ammonium salt group, that is, the ActiPro medium containing 0.4 mM ATA ammonium salt was prepared according to the method described in Example 1.
  • Culture was performed with reference to the inoculation and culture conditions described in 2.2, and live cell density, cell viability, lactic acid concentration, and antibody expression were monitored.
  • ATA ammonium salts at different concentrations can significantly increase the expression of antibodies, and the effects are even more effective when added at concentrations of 0.1 mM and 0.2 mM.
  • 0.2 mM ATA ammonium salt is the most preferred addition concentration.
  • ActiPro medium was selected as the basal medium and set as the control group (ActiPro), respectively at the initial stage of cell culture (ie, day 0, referred to as D0) and the middle stage of culture (ie, day 6, referred to as D6) ) Add 0.2 mM ATA ammonium salt to the basal medium.
  • Culture was performed with reference to the inoculation and culture conditions described in 2.2, and live cell density, cell viability, lactic acid concentration, and antibody expression were monitored.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Reproductive Health (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

细胞培养技术领域中的一种用于调节哺乳动物细胞乳酸代谢的培养基及其应用。所述培养基包含以下组分：(1)基础培养基，和(2)金精三羧酸或金精三羧酸铵盐，其中所述基础培养基无血清、无动物性蛋白和植物性蛋白，且组成成分明确。上述培养基能够改善哺乳动物细胞的乳酸代谢水平，同时能够提高哺乳动物细胞的活细胞密度、细胞活率以及外源蛋白表达。

Description

用于调节哺乳动物细胞乳酸代谢的培养基及其应用
优先权和相关申请
本申请要求2018年8月16日提交的名称为用于调节哺乳动物细胞乳酸代谢的培养基及其应用的中国专利申请201810933714.6的优先权，该申请包括附录在内的全部内容作为参考并入本申请。
技术领域
本发明涉及培养基领域，尤其涉及一种用于调节哺乳动物细胞乳酸代谢的培养基及其应用。
背景技术
CHO细胞(Chinese hamster ovary cell，中国仓鼠卵巢细胞)是目前用于包括工程抗体和重组蛋白在内的生物技术药物研发和生产中最重要、最广泛的表达系统之一。与其他表达系统相比，它具有以下优势：连续细胞系，生长快速，细胞类型均一，可以传代百代以上，便于更大规模生产；属于成纤维细胞(fibroblast)，是一种非分泌型细胞，本身很少分泌CHO内源蛋白，因此对目标蛋白分离纯化工作十分有利；具有较好的表达好修饰能力，如糖基化的功能，是表达复杂生物大分子的一种理想宿主，至今批准的重要重组蛋白及其表达生产主要来自于CHO细胞的表达系统。
用以培养CHO细胞的培养基含有丰富的营养物质，部分营养物质会被转化为具有细胞毒性的代谢副产物。其中，乳酸是细胞培养过程中的主要代谢 副产物。CHO细胞在代谢旺盛的对数生长期通常会进行乳酸的快速累积，部分细胞株可以将这些乳酸代谢消耗掉，但部分细胞株却缺乏此能力。大量乳酸的累积将严重影响蛋白表达及蛋白质量。乳酸的累积会导致培养体系的pH降低，为使pH回升添加的碱液往往会增加体系的渗透压，从而影响细胞正常生长代谢。而如果不通过添加碱液调节pH，低pH环境会影响酶活，从而影响细胞生长。
为了改善细胞的生长，提高蛋白产量及质量，获得稳定持续的培养过程，减少乳酸积累一直是细胞培养过程中的一个巨大挑战。现有技术中减少乳酸积累的方法包括：(1)对细胞进行基因改造可以降低乳酸积累，如敲除乳酸脱氢酶(LDH)基因、过表达丙酮酸羧化酶(PYC2)基因等，但操作性不强。(2)也可优化细胞培养策略进行乳酸调控。用代谢缓慢的糖类代替葡萄糖作为碳源可以降低乳酸的积累，如半乳糖、岩藻糖和麦芽糖的使用。而当乳酸有一定积累时，可以通过葡萄糖的浓度来改变细胞的代谢路径，从而减少乳酸累积。但这些乳酸调控策略会对细胞的生长及蛋白质量产生不利影响。(3)此外，铜离子(Cu 2+)的添加曾在多种CHO细胞中被证实可以减少乳酸积累。Cu 2+的加入可扩大线粒体的氧化容量，从而降低乳酸的积累。但Cu 2+的添加会影响到蛋白的质量，如增加碱性峰的比例、降低高甘露糖型水平等。因此，目前市场急需开发一种能够调控乳酸代谢的更适宜的方法。
培养基是影响细胞生长代谢乃至生存的最直接、最重要的环境因素。在悬浮培养和无血清培养已成为动物细胞培养的理想模式和动物细胞表达产品工业化生产的首要选择的现阶段，本发明的发明人提供一种可显著降低乳酸累积的支持CHO细胞悬浮培养的无血清培养基。Hyclone公司生产的 ActiPro，HyCell CHO和CDM4MAB培养基，以及Gibco公司生产的Dynamis培养基是针对CHO细胞悬浮培养较常用的无血清、无蛋白且化学成分限定的商品化培养基。以添加适宜浓度的金精三羧酸或金精三羧酸铵盐的此类基础培养基进行细胞培养，可有效调控乳酸的堆积问题。
金精三羧酸(Aurintricarboxylic acid，ATA)，分子式为C 22H 14O 9。金精三羧酸铵盐(Aurintricarboxylic acid ammonium salt，又称为Aluminon、Ammonium aurintricarboxylate、ATA ammonium)，分子式为C 22H 14O 9·3NH 3。所述金精三羧酸铵盐在本公开中又可称为ATA铵盐。该培养基添加物具有以下优点：为成分明确的单一物质；易在下游纯化过程中去除；成本低廉；配制和使用方便等。虽然ATA在其它文献和涉及培养基配方的专利中有所提及，但是其使用方式、作用效果以及发挥生物学功能的原理均与本发明存在明显不同。现有技术公开的文献主要存在以下问题：ATA仅仅是作为胰岛素、IGF-1或者转铁蛋白(transferrin)的替代物，且不是以单独形式添加至商品化的培养基中。例如，公开号为US5,045,454的专利文献中公开了一种无血清生长培养基，该发明选择用铁螯合物(包括柠檬酸盐/柠檬酸和Fe/EDTA)、ATA以及碱金属EDTA的组合作为培养基中转铁蛋白的替代物。也就是说，在该专利文献中ATA并不是以单一组分的形式添加到无血清生长培养基中发挥作用的，而是需要铁螯合物和碱金属EDTA的配合才能解决其声称的技术问题。另外，公开号为US6,338,964B1的专利文献中公开了一种适用于在低溶解CO 2浓度的条件下培养哺乳动物细胞的培养基，所述培养基中包含：浓度高于2mM的有机缓冲液、浓度高于5mM的金属络合剂(metal complexing agent)以及浓度小于1g/L的NaHCO 3，其中所述金属络合剂由组 氨酸、亚氨基二乙酸、柠檬酸盐、磷酸丝氨酸、ATA和羟基赖氨酸组成。由此可见，该文献中所述ATA也不是以单一组分的形式添加到所述培养基中发挥作用并解决其声称的技术问题。
发明内容
在一个方面，本发明提供了一种用于调节哺乳动物细胞乳酸代谢的培养基。使用本发明所述的培养基能够同时对未经优化和筛选的快速稳定细胞群(fast stable pool，FSP)、稳定细胞群(stable pool)及存在乳酸代谢问题的克隆(clone)具有良好的调控乳酸代谢水平的作用。
在另一个方面，在于提供一种能够同时有效提高哺乳动物细胞的活细胞密度、细胞活率，降低培养液中的乳酸堆积，提高哺乳动物细胞表达外源蛋白产量的培养基。
在一个或多个实施方式中，本发明通过在细胞培养之初添加浓度优化的ATA或ATA铵盐于基础培养中，从而完成了本发明。即，本发明如下所述：
在至少一个实施例中，本发明提供了一种用于调节哺乳动物细胞乳酸代谢的培养基，其特征在于，所述培养基中包含以下组分：(1)基础培养基，和(2)ATA或ATA铵盐。其中所述基础培养基中无血清、无动物性蛋白和植物性蛋白。其中所述的基础培养基中的所有成分都是明确的，又可称为成分培养基(Chemical defined medium，CDM)，在所述培养基中不添加任何动物源成分；所述动物性蛋白包括但不限于牛血清白蛋白(Bovine serum albumin，BSA)，所述植物性蛋白包括但不限于大豆蛋白。
优选的，所述基础培养基为ActiPro培养基，HyCell CHO培养基， CDM4MAB培养基或Dynamis培养基。当所述基础培养基为ActiPro培养基时，所述培养基能够同时有效提高哺乳动物细胞的活细胞密度，降低培养液中的乳酸堆积，提高哺乳动物细胞的蛋白表达。
优选的，所述培养基中的ATA或ATA铵盐的浓度为约0.05～0.8mM，更优选的浓度为约0.1～0.4mM，最优选的浓度为约0.2mM。
当所述ATA或ATA铵盐在基础培养中的浓度高于0.8mM时，会对细胞生长产生抑制；当所述ATA或ATA铵盐在基础培养中的浓度低于0.05mM时，将无法有效调节细胞乳酸堆积问题。
本发明还提供一种调节哺乳动物细胞乳酸代谢的方法，其特征在于：将表达外源蛋白的哺乳动物细胞在上文所述的培养基中进行培养，优选的，在所述哺乳动物细胞培养起始在所述培养基中进行培养。
在所述方法中，优选的，所述哺乳动物细胞为CHO细胞，所述外源蛋白为Fc融合蛋白、抗体或酶；更优选的，所述CHO哺乳动物细胞为CHO-K1细胞，所述外源蛋白为单克隆抗体。
本发明进一步提供上文所述的培养基在哺乳动物细胞表达外源蛋白中的应用，优选的，在所述哺乳动物细胞培养起始选择所述培养基进行培养。
在所述应用中，优选的，所述哺乳动物细胞为CHO细胞，所述外源蛋白为Fc融合蛋白、抗体或酶；更优选的，所述哺乳动物细胞为CHO-K1细胞，所述外源蛋白为单克隆抗体。所述Fc融合蛋白是指利用基因工程等技术将某种具有生物学活性的功能蛋白分子与Fc片段融合而产生的新型蛋白，功能蛋白可以是能结合内源性受体(或配体)的可溶性配体(或受体)分子或其他需要延长半衰期的活性物质(如细胞因子)；所述单克隆抗体可以是任何适合利用 CHO-K1细胞进行外源表达的单克隆抗体，其包括但不限于IgG、IgA单克隆抗体。
附图说明
图1-1和图1-2为ATA及ATA铵盐对细胞生长的影响图(图1-1为活细胞密度随时间的变化曲线图；图1-2为细胞活率随时间的变化曲线图)；
图1-3和图1-4为ATA及ATA铵盐对抑制细胞凋亡的影响图(图1-3为活细胞密度随时间的变化曲线图；图1-4为细胞活率随时间的变化曲线图)；
图1-5至图1-7为ATA铵盐在ActiPro，HyCell CHO和Dynamis培养基中对细胞乳酸代谢的影响图(图1-5为活细胞密度随时间的变化曲线图；图1-6为细胞活率随时间的变化曲线图；图1-7为乳酸浓度随时间的变化曲线图)；
图1-8至图1-10为ATA铵盐在CDM4MAB培养基中对细胞乳酸代谢的影响图(图1-8为活细胞密度随时间的变化曲线图；图1-9为细胞活率随时间的变化曲线图；图1-10为乳酸浓度随时间的变化曲线图)；
图2-1至图2-3分别为ATA铵盐添加浓度的研究图(图2-1为活细胞密度随时间的变化曲线图；图2-2为细胞活率随时间的变化曲线图；图2-3为乳酸浓度随时间的变化曲线图)；
图2-4至图2-6分别为ATA铵盐添加时间的研究图(图2-4为活细胞密度随时间的变化曲线图；图2-5为细胞活率随时间的变化曲线图；图2-6为乳酸浓度随时间的变化曲线图)。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。应该强调的是，本发明不限于所举例说明的具体实施方案。此外，本文使用的任何章节标题仅用于组织目的，并不被解释为限制所描述的主题。
除非在此另外定义，否则与本发明结合使用的科学和技术术语将具有本领域普通技术人员通常理解的含义。此外，除非上下文另有要求，单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非上下文另外明确指出，否则单数形式“一”，“一个”和“该”包括复数指示物。因此，例如，提及“一种蛋白质”包括多种蛋白质；提及“一个细胞”包括细胞的混合物等。在本申请中，除非另有说明，否则使用“或”意指“和/或”。此外，术语“包含”以及其他形式(诸如“包括”和“含有”)的使用不是限制性的。此外，说明书和所附权利要求中提供的范围包括端点和断点之间的所有值。
定义
“约”、“大约”：当此处所用的术语“约”和“大约”用于一种或多种具有细胞培养条件时，是指与所述培养条件下指定的参考值相似的一系列值。在某些实施例中，术语“约”是指处在所述培养条件下指定参考值的百分之25、20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5、4、3、2、1或更小范围的系列值。
“培养基”是指与细胞直接接触的，细胞赖以生存的液体环境，是各种营养成分以及缓冲体系的集合。
“基础培养基”是指在在各种细胞培养的操作方式下，初始接种时所选用的培养基，其中所述基础培养基无血清、无动物性蛋白和植物性蛋白，且 组成成分明确，批次间重复性好，易于检测分析。
详细说明
在一些实施例中，本发明提供了一种用于调节哺乳动物细胞乳酸代谢的培养基，其特征在于，所述培养基中包含以下组分：(1)基础培养基，和(2)ATA或ATA铵盐。其中所述基础培养基中无血清、无动物性蛋白和植物性蛋白。其中所述的基础培养基中的所有成分都是明确的，又可称为成分培养基(Chemical defined medium，CDM)，在所述培养基中不添加任何动物源成分；所述动物性蛋白包括但不限于牛血清白蛋白(Bovine serum albumin，BSA)，所述植物性蛋白包括但不限于大豆蛋白。本发明将ATA或ATA铵盐作为单一组分添加到目前市场上最重要的几种常规商用培养基，有效改善了哺乳动物细胞的乳酸代谢水平，降低了由于乳酸堆积带来的工艺风险。使用本发明所述的培养基能够有效提高FSP、stable pool和克隆细胞的活细胞密度、细胞活率，同时提升哺乳动物细胞表达的外源蛋白的产量和质量。本发明的培养基作为商用培养基的有益补充，改善了商用培养基的适用范围。
ATA或ATA铵盐
“金精三羧酸(Aurintricarboxylic acid，ATA)”，分子式为C 22H 14O 9。金精三羧酸铵盐(Aurintricarboxylic acid ammonium salt，又称为Aluminon、Ammonium aurintricarboxylate、ATA ammonium)，分子式为C 22H 14O 9·3NH 3。
“金精三羧酸铵盐”是指三个铵根离子与一个金精三羧酸构成的离子化合物，易溶于水，配制便利。
所述ATA或ATA铵盐可从商业来源获得，此外，可将ATA或ATA铵盐作 为单一组分添加到目前市场上最重要的几种常规商用培养基，ATA或ATA铵盐具有以下优点：为成分明确的单一物质；易在下游纯化过程中去除；成本低廉；配制和使用方便等。
含金精三羧酸或金精三羧酸铵盐培养基
本文中含金精三羧酸或金精三羧酸铵盐培养基用于调节哺乳动物细胞乳酸代谢。在至少一个实施例中，所述培养基中包含以下组分：(1)基础培养基，和(2)ATA或ATA铵盐。其中所述基础培养基中无血清、无动物性蛋白和植物性蛋白。其中所述的基础培养基中的所有成分都是明确的，又可称为成分培养基(Chemical defined medium，CDM)，在所述培养基中不添加任何动物源成分；所述动物性蛋白包括但不限于牛血清白蛋白(Bovine serum albumin，BSA)，所述植物性蛋白包括但不限于大豆蛋白。
在至少一个实施例中，所述基础培养基为ActiPro培养基，HyCell CHO培养基，CDM4MAB培养基或Dynamis培养基。当所述基础培养基为ActiPro培养基时，所述培养基能够同时有效提高哺乳动物细胞的活细胞密度，降低培养液中的乳酸堆积，提高哺乳动物细胞的蛋白表达。
在至少一个实施例中，所述培养基中的ATA或ATA铵盐的浓度为约0.05～0.8mM，更优选的浓度为约0.1～0.4mM，最优选的浓度为约0.2mM。
含金精三羧酸或金精三羧酸铵盐培养基的制备
在至少一个实施例中，首先制备获得金精三羧酸或金精三羧酸铵盐贮存液；所述的贮存液置于37℃进行预热；取一定体积预热后的贮存液加入到ActiPro培养基，HyCell CHO培养基，CDM4MAB培养基或Dynamis培养基中，混合均匀，从而得到含有特定浓度的ATA或ATA铵盐培养基。100×贮存液的 组成成分如表1所示。
在至少一个实施例中，表1中所述贮存液的制备方法如下所示：
步骤1)选择合适大小的容器，盛装表1中所述质量的注射用水；
步骤2)将表1中所述质量的ATA或ATA铵盐粉末加入到步骤1)中所述的容器中；
步骤3)ATA需在碱性条件下溶解：加入适量NaOH调节至适当pH值后，在室温下混合均匀；ATA铵盐在室温、水中即可溶解。待充分溶解后，添加注射用水至最终质量并再次混合均匀；
步骤4)使用合适孔径的滤膜对所述溶液进行过滤；
步骤5)置于约4～8℃保存。
在至少一个实施例中，所述PH值为约12.2±0.2、12.2±0.1或12.2。
在至少一个实施例中，所述滤膜的孔径为约0.1μm、0.2μm或0.3μm。
金精三羧酸及金精三羧酸铵盐的功能
在至少一个实施例中，将ATA或ATA铵盐作为单一组分添加到目前市场上最重要的几种常规商用培养基，有效改善了哺乳动物细胞的乳酸代谢水平，降低了由于乳酸堆积带来的工艺风险。
在另一些实施例中，使用所述的培养基能够有效提高FSP、stable pool和克隆细胞的活细胞密度、细胞活率，同时提升哺乳动物细胞表达的外源蛋白的产量和质量。
在至少一个实施例中，所述培养基作为商用培养基的有益补充，改善了商用培养基的适用范围。
哺乳动物细胞
本文中“哺乳动物细胞”是在制药工业生产中，指按照生产条件选择和驯化的用于生产制备生物制剂的，源于哺乳动物的细胞系，其表达的蛋白的翻译后修饰在保持蛋白生物学活性，稳定性及抗原性具有优势。许多细胞系可从商业来源获得，例如美国标准生物品收藏中心(ATCC)。可用于本发明的哺乳动物细胞的非限制性实例，包括CHO，BHK，HEK293等用于生物制品生产的细胞。在至少一个实施例中，其中所述哺乳动物细胞为CHO细胞，更优选的，所述哺乳动物细胞为CHO-K1细胞。
在至少一个实施例中，所述哺乳动物细胞为CHO细胞，可选的，所述哺乳动物细胞为CHO-K1细胞。
在至少一个实施例中，所使用的细胞是由包含外源编码基因的双表达载体转染CHO-K1细胞所构建的能够稳定表达外源蛋白的细胞株。
在至少一个实施例中，所述外源蛋白为Fc融合蛋白、抗体或酶，可选的，所述抗体为单克隆抗体。
在至少一个实施例中，所使用的细胞是由包含IgG1编码基因的双表达载体转染CHO-K1细胞所构建的能够稳定表达IgG1单克隆抗体的细胞株。
外源蛋白
本文中的“外源蛋白”是指基因工程范畴内，编码宿主细胞以外的目的蛋白基因，利用DNA重组技术，通过宿主细胞有效的扩增与表达，生产出具有实用价值的蛋白。所述外源蛋白是本领域已知的，并可从商业来源获得，或者通过本领域已知的方法获得。在至少一个实施例中，所述外源蛋白为Fc融合蛋白、抗体或酶；可选的，所述抗体为单克隆抗体或双抗，所述单克隆抗体可以是任何适合利用CHO-K1细胞进行外源表达的单克隆抗体，其包括 但不限于IgG、IgA单克隆抗体。在至少一个实施例中，所述单克隆抗体为IgG1单克隆抗体，在至少一个实施例中，所述IgG1单克隆抗体包括但不限于肿瘤领域和自身免疫领域靶点，如CD19/HER2，CD20，PD-1，LAG3等。
在至少一个实施例中，所述Fc融合蛋白是指利用基因工程等技术将某种具有生物学活性的功能蛋白分子与Fc片段融合而产生的新型蛋白，功能蛋白可以是能结合内源性受体(或配体)的可溶性配体(或受体)分子或其他需要延长半衰期的活性物质(如细胞因子)，在至少一个实施例中，所述Fc融合蛋包括但不限于类IL-2/Fc，GLP-1/Fc等。
在至少一个实施例中，所述酶是指治疗药物应用的酶品，即药用酶，包括但不限于葡萄糖苷酶，脂肪酶等。
调节哺乳动物细胞乳酸代谢的方法
在至少一个实施例中，将表达外源蛋白的哺乳动物细胞在所述的培养基中进行培养，可选的，在所述哺乳动物细胞培养起始在所述培养基中进行培养。
在至少一个实施例中，其中所述哺乳动物细胞为CHO细胞，更优选的，所述哺乳动物细胞为CHO-K1细胞。
在至少一个实施例中，，其中所述外源蛋白为Fc融合蛋白、抗体或酶，优选的，所述抗体为单克隆抗体。
实施例
本发明实施例中所使用的培养基和培养基添加物均可市购获得：
ActiPro培养基(Hyclone，Cat No.：SH31037)，HyCell CHO培养基(Hyclone，Cat No.：SH30933.04)和CDM4MAB培养基(Hyclone，Cat No.： SH30800.04)：Dynamis培养基(Gibco，Cat No.：A26175-03)：购自Gibco公司；
ATA(Sigma，Cat.No：A1895)、ATA铵盐(Sigma，Cat.No：A36883)、胰岛素(Sigma，Cat.No：91077C)、丁酸钠(Sigma，Cat.No：303410)、类胰岛素样生长因子(Repligen，Cat.No：6082-229)。
实施例1：含金精三羧酸或金精三羧酸铵盐培养基的制备
1.1金精三羧酸或金精三羧酸铵盐贮存液的制备方法
选择注射用水(water for injection，WFI)来制备所有溶液。100×贮存液的组成成分如表1所示。
表1中所述贮存液的制备方法如下所示：
步骤1)选择合适大小的容器，盛装表1中所述质量的注射用水；
步骤2)将表1中所述质量的ATA或ATA铵盐粉末加入到步骤1)中所述的容器中；
步骤3)ATA需在碱性条件下溶解：加入适量10N NaOH调节pH值至12.2±0.2后，在室温下混合均匀；ATA铵盐在室温、水中即可溶解。待充分溶解后，添加注射用水至最终质量并再次混合均匀；
步骤4)使用0.1μm或者0.2μm孔径的滤膜对所述溶液进行过滤；
步骤5)置于4～8℃保存。
表1 20mM ATA及ATA铵盐贮存液的成分(1.0kg)
成分 期望含量(g) 最小含量(g) 最大含量(g) 可接受误差
注射用水 #1 800.00 784.00 816.00 ±2％
金精三羧酸 8.45 8.28 8.62 ±2％
金精三羧酸铵盐 9.47 9.28 9.66 ±2％
最终质量(g) 1000.0 980.0 1020.0 ±2％
#1：所述注射用水的量为原始添加量，未示出注射用水的最终添加量。
1.2用于细胞培养的含有金精三羧酸或金精三羧酸铵盐培养基的制备方法
1)将本实施例1.1节中所述的贮存液置于37℃进行预热；
2)取一定体积预热后的贮存液加入到ActiPro培养基，HyCell CHO培养基，CDM4MAB培养基或Dynamis培养基中，混合均匀，从而得到含有特定浓度的ATA或ATA铵盐培养基。
实施例2：金精三羧酸及金精三羧酸铵盐的功能研究
2.1金精三羧酸及金精三羧酸铵盐对细胞生长的影响
采用分批培养(batch culture)的方法，研究ATA及ATA铵盐在ActiPro细胞培养基中对CHO-K1细胞生长的影响，所述细胞外源表达单克隆抗体。
选择ActiPro培养基作为基础培养基，分别配制以下五组细胞培养基：(1)对照组(Ctrl)，即在ActiPro培养基中不额外添加其它物质；(2)胰岛素组(Insulin)，即在ActiPro培养基中添加0.2mg/L的胰岛素；(3)类胰岛素样生长因子组(IGF-1)，即在ActiPro培养基中添加20μg/L的IGF-1；(4)金精三羧酸组(ATA)，即根据实施例1所述的方法配制含有0.2mM ATA的ActiPro培养基；(5)ATA铵盐组(ATA Ammonium)，即实施例1中所述的含有0.2mM ATA铵盐的ActiPro培养基。
将细胞按照0.4×10 6cells/mL的密度分别接种到装有上述五组细胞培养基的250mL摇瓶中，培养体积50mL，置于36.5℃，125rpm的摇床中进行分批培养。在培养的过程中，对活细胞密度及细胞活率进行监测。
试验结果：和对照组相比，ATA和ATA铵盐组能显著提高细胞的活细胞密度；而胰岛素组和类胰岛素样生长因子组中的活细胞密度均低于对照组 (如图1-1所示)。另外，ATA和ATA铵盐组的培养基还能有效提高细胞的细胞活率(如图1-2所示)。
由此可见，ATA和ATA铵盐在细胞培养过程中显示出了不同于胰岛素和类胰岛素生长因子的促生长作用。
2.2金精三羧酸及金精三羧酸铵盐对抑制细胞凋亡的影响
由于ATA铵盐与ATA结构及功能相似，且ATA铵盐更易制备，因此在以下的实施案例中仅使用ATA铵盐进行试验。
相关研究表明，丁酸钠可以提高多种外源蛋白在CHO细胞中的表达量，但同时会抑制细胞生长、阻断细胞周期，诱导细胞凋亡。此处试验采用流加培养(fed-batch culture)的方法，通过添加丁酸钠诱导细胞凋亡，验证ATA铵盐的抑制细胞凋亡作用。
分别配制以下四组培养基：(1)ActiPro对照组(Ctrl)，即在ActiPro培养基中不额外添加其它物质；(2)ATA铵盐组(ATA Ammonium)，即参照实施例1所述的方法配制含0.2mM ATA铵盐的ActiPro培养基；(3)丁酸钠组(NaBu)，即在ActiPro培养基中添加2mM丁酸钠；(4)ATA铵盐/丁酸钠组(ATA铵盐/NaBu)，即在0.2mMATA铵盐组的基础上，在细胞培养的第五天添加2mM丁酸钠。
将细胞按照0.4×10 6cells/mL的密度分别接种到装有上述四组细胞培养基的250mL摇瓶中，培养体积50mL，置于36.5℃，125rpm的摇床中进行培养，并于第5天移至31.0℃，125rpm的摇床中。在培养的过程中，对活细胞密度、细胞活率及抗体表达量进行监测。
试验结果：(1)四组不同培养基对于活细胞密度的影响程度依次为：ATA 铵盐组＞ATA铵盐/丁酸钠组＞ActiPro对照组＞丁酸钠组。由此可见，在基础培养基中添加ATA铵盐能够提高细胞的活细胞密度，而添加丁酸钠会降低细胞的活细胞密度(如图1-3所示)。
(2)在基础培养基中添加ATA铵盐能够有效提高细胞的细胞活率，而在基础培养基中添加NaBu会影响细胞的活率(如图1-4所示)。
(3)四组不同培养基对于抗体表达量的影响程度依次为：ATA铵盐组＞ATA铵盐/丁酸钠组＞ActiPro对照组＞丁酸钠组。由此可见，在基础培养基中添加ATA铵盐能够提高细胞抗体表达的能力，而添加丁酸钠会降低细胞表达抗体的能力(如表2所示)。
(4)根据本实施案例的结果可以看出：ATA铵盐比丁酸钠更适合作为提高CHO细胞外源蛋白表达量的单一组分添加到基础培养基中；结合实施案例2.1的结果可见：ATA及ATA铵盐具有有效保持细胞活率、明显抑制细胞凋亡的作用。
表2不同ATA铵盐添加时间的抗体表达对比
Figure PCTCN2019100980-appb-000001
2.3金精三羧酸铵盐对细胞乳酸代谢的影响
此处实验考察了ATA铵盐在不同商业培养基中对CHO细胞乳酸代谢的影响，包括ActiPro，HyCell CHO、CDM4MAB和Dynamis等基础培养基，所述培养基培养的CHO-K1细胞分别表达Fc融合蛋白、单克隆抗体、单克隆抗体和药用酶。
分别配制以下八组细胞培养基：(1)ActiPro对照组(ActiPro)，即在ActiPro培养基中不额外添加其它物质；(2)ActiPro+ATA铵盐试验组(ActiPro+ATA Ammonium)，即根据实施例1所述的方法配制含0.2mM ATA铵盐的ActiPro培养基；(3)HyCell CHO对照组(HyCell CHO)，即在HyCell CHO培养基中不额外添加其它物质；(4)HyCell CHO+ATA铵盐试验组(HyCell CHO+ATA Ammonium)，即根据实施例1所述的方法配制含0.2mM ATA铵盐的HyCell CHO培养基；(5)Dynamis对照组(Dynamis)，即在Dynamis培养基中不额外添加其它物质；(6)Dynamis+ATA铵盐试验组(Dynamis+ATA Ammonium)，即根据实施例1所述的方法配制含0.2mM ATA铵盐的Dynamis培养基；(7)CDM4MAB对照组(CDM4MAB)，即在CDM4MAB培养基中不额外添加其它物质；(8)CDM4MAB+ATA铵盐试验组(CDM4MAB+ATA Ammonium)，即根据实施例1所述的方法配制含0.2mM ATA铵盐的CDM4MAB培养基。
在ActiPro，HyCell CHO和Dynamis培养基中采用流加培养工艺，培养过程参照2.2中所述的培养条件。在CDM4MAB中的培养工艺为灌注培养，培养过程为：将细胞按照0.4×10 6cells/mL的密度接种到装有含0.2mM ATA铵盐的CDM4MAB培养基的微型反应器中进行培养。培养体积为15mL，pH为7.0±0.2，搅拌转速为500rpm，培养温度为35.0℃。从第3天开始，将培养液沉降一定时间后，用新鲜培养基对培养上清液进行换液操作。第3-5天的换液比为2∶1(V 培养上清液∶V 新鲜培养基)，此后的换液比为1∶1。在培养过程中，对细胞的活细胞密度、细胞活率和乳酸浓度进行监测。
试验结果：如图1-5至1-10所示，ATA铵盐的添加可有效解决细胞代谢 过程中的乳酸堆积问题。此外，在ActiPro，HyCell CHO和CDM4MAB培养基中添加ATA铵盐，可显著提高细胞的活细胞密度及细胞活率。
实施例3：金精三羧酸铵盐添加浓度及添加时间的研究
3.1金精三羧酸铵盐添加浓度的研究
采用流加培养(fed-batch culture)的方法，研究不同浓度的ATA铵盐对表达单克隆抗体的CHO-K1细胞的活细胞密度、细胞活率、乳酸浓度和抗体表达的影响。
参照实施例1中所述的方法，分别配制以下四组培养基：(1)ActiPro对照组(ActiPro)，即在ActiPro培养基中不额外添加其它物质；(2)0.1mM ATA铵盐组，即参照实施例1所述的方法配制含0.1mM ATA铵盐的ActiPro培养基；(3)0.2mM ATA铵盐组，即按照实施例1所述的方法配制含0.2mM ATA铵盐的ActiPro培养基；(4)0.4mM ATA铵盐组，即参照实施例1所述的方法配制含0.4mM ATA铵盐的ActiPro培养基。
参照2.2中所述的接种及培养条件进行培养，对活细胞密度、细胞活率、乳酸浓度和抗体表达量进行监测。
试验结果：(1)如图2-1中所示的曲线图可以看出，和对照组相比，0.1～0.4mM浓度的ATA铵盐组均能显著提高细胞的活细胞密度。浓度为0.1mM及0.2mM相对于0.4mM更能够提高活细胞密度。
(2)如图2-2所示的曲线图可以看出，在ActiPro培养基中添加0.1～0.4mM浓度的ATA铵盐能够显著维持细胞的细胞活率。浓度为0.2mM及0.4mM相对于0.1mM能够在经历较长的培养时间后依然维持较高的细胞活率。
(3)如图2-3中所示的曲线图可以看出，不同浓度的ATA铵盐均能够有 效降低培养液中的乳酸堆积。浓度为0.1mM及0.2mM相较于0.4mM的效果更加。
(4)如表3所示，不同浓度的ATA铵盐均能显著提升抗体表达量，以0.1mM及0.2mM的浓度添加效果更加。
表3不同ATA铵盐添加浓度的抗体表达量对比
Figure PCTCN2019100980-appb-000002
(5)综上结果，0.2mM ATA铵盐为最优选的添加浓度。
3.2金精三羧酸铵盐添加时间的研究
为了确定ATA铵盐的添加时间，设计以下试验：
采用流加培养(fed-batch culture)的方法进行培养。选择ActiPro培养基作为基础培养基并将其设置为对照组(ActiPro)，分别于细胞培养的起始阶段(即第0天，简称为D0)以及培养的中期(即第6天，简称为D6)在所述基础培养基中添加0.2mM ATA铵盐。
参照2.2中所述的接种及培养条件进行培养，对活细胞密度、细胞活率、乳酸浓度和抗体表达量进行监测。
试验结果：(1)如图2-4至2-6及表4所示，不同的ATA铵盐添加时间未对活细胞密度、细胞活率以及抗体表达量产生影响，但乳酸浓度曲线之间存在差异。虽然不同的添加时间均可抑制培养后期的乳酸生成，但在培养初始阶段加入ATA铵盐可降低培养前期的乳酸积累。
(2)综上结果，最为优选的ATA铵盐的添加时间为培养的初始阶段。
表4不同ATA铵盐添加时间的抗体表达量对比
Figure PCTCN2019100980-appb-000003
以上对本发明的具体实施例进行了详细描述，但其只作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对该实用进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。

Claims (10)

  1. 一种用于调节哺乳动物细胞乳酸代谢的培养基，其特征在于，所述培养基包含以下组分：(1)基础培养基，和(2)金精三羧酸或金精三羧酸铵盐。
  2. 如权利要求1所述的培养基，其中所述基础培养基为ActiPro，HyCell CHO，CDM4MAB或Dynamis等商用培养基，优选的，所述基础培养基为ActiPro培养基。
  3. 如权利要求1或2所述的培养基，其中所述金精三羧酸或金精三羧酸铵盐的浓度为约0.05～0.8mM，更优选的浓度为约0.1～0.4mM，最优选的浓度为约0.2mM。
  4. 一种调节哺乳动物细胞乳酸代谢的方法，其特征在于：将表达外源蛋白的哺乳动物细胞在权利要求1～3中任一项所述的培养基中进行培养，优选的，在所述哺乳动物细胞培养起始在所述培养基中进行培养。
  5. 如权利要求4所述的方法，优选的，其中所述哺乳动物细胞为CHO细胞，更优选的，所述哺乳动物细胞为CHO-K1细胞。
  6. 如权利要求4或5所述的方法，其中所述外源蛋白为Fc融合蛋白、抗体或酶，优选的，所述抗体为单克隆抗体。
  7. 权利要求1～3任一项所述的培养基在哺乳动物细胞表达外源蛋白中的应用。
  8. 如权利要求7所述的应用，其特征在于，在所述哺乳动物细胞培养起始选择所述培养基进行培养。
  9. 如权利要求7或8所述的应用，优选的，其中所述哺乳动物细胞为CHO细胞，更优选的，所述哺乳动物细胞为CHO-K1细胞。
  10. 如权利要求7或8所述的应用，其中所述外源蛋白为Fc融合蛋白、抗体或酶，优选的，所述抗体为单克隆抗体。
PCT/CN2019/100980 2018-08-16 2019-08-16 用于调节哺乳动物细胞乳酸代谢的培养基及其应用 WO2020035050A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810933714.6 2018-08-16
CN201810933714.6A CN110835622B (zh) 2018-08-16 2018-08-16 用于调节哺乳动物细胞乳酸代谢的培养基及其应用

Publications (1)

Publication Number Publication Date
WO2020035050A1 true WO2020035050A1 (zh) 2020-02-20

Family

ID=69524693

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/100980 WO2020035050A1 (zh) 2018-08-16 2019-08-16 用于调节哺乳动物细胞乳酸代谢的培养基及其应用

Country Status (2)

Country Link
CN (1) CN110835622B (zh)
WO (1) WO2020035050A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113862217A (zh) * 2021-10-12 2021-12-31 上海药明生物技术有限公司 培养哺乳动物细胞的方法
CN114164166A (zh) * 2021-12-16 2022-03-11 无锡多宁生物科技有限公司 一种ata在用来培养cho细胞系细胞的基础培养基当中的新应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1772884A (zh) * 2005-10-18 2006-05-17 中国人民解放军军事医学科学院生物工程研究所 一种支持hek293细胞贴附培养的无动物来源成分无血清培养基
CN101864393A (zh) * 2009-05-06 2010-10-20 中国人民解放军军事医学科学院生物工程研究所 一种用于Vero细胞微载体培养的无动物来源成分无血清培养基
CN103484426A (zh) * 2013-10-15 2014-01-01 齐鲁制药有限公司 一种无动物源的低蛋白培养基
WO2014110053A1 (en) * 2013-01-08 2014-07-17 Genzyme Corporation Use of inos inhibitors to increase viral yield in culture

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NO162160C (no) * 1987-01-09 1989-11-15 Medi Cult As Serumfritt vekstmedium, samt anvendelse derav.
US6338964B1 (en) * 1999-05-07 2002-01-15 Bayer Corporation Process and medium for mammalian cell culture under low dissolved carbon dioxide concentration
CN107012115A (zh) * 2017-04-18 2017-08-04 广东顺德工业设计研究院(广东顺德创新设计研究院) 支持细胞高密度悬浮培养的培养基及其制备方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1772884A (zh) * 2005-10-18 2006-05-17 中国人民解放军军事医学科学院生物工程研究所 一种支持hek293细胞贴附培养的无动物来源成分无血清培养基
CN101864393A (zh) * 2009-05-06 2010-10-20 中国人民解放军军事医学科学院生物工程研究所 一种用于Vero细胞微载体培养的无动物来源成分无血清培养基
WO2014110053A1 (en) * 2013-01-08 2014-07-17 Genzyme Corporation Use of inos inhibitors to increase viral yield in culture
CN103484426A (zh) * 2013-10-15 2014-01-01 齐鲁制药有限公司 一种无动物源的低蛋白培养基

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN ZHAOLIE ET AL: "A low-cost chemically defined protein free medium for a recombinant CHO cell line producing prothrombin", BIOTECHNOLOGY LETTERS, KLUWER ACADEMIC PUBLISHERS, DORDRECHT, vol. 22, no. 10, 1 May 2000 (2000-05-01), Dordrecht, pages 837 - 841, XP002509008, ISSN: 0141-5492, DOI: 10.1023/A:1005665530028 *
CHEN, Z. L.: "High-density culture of recombinant Chinese hamster ovary cells producing prothrombin in protein-free medium", BIOTECHNOLOGY LETTERS, vol. 23, 31 December 2001 (2001-12-31), pages 767 - 770, XP055686868, DOI: 10.1023/A:1010373527375 *
LIU, XINGMAO ET AL.: "Serum-free Medium for Suspension Culture of Recombinant Chinese Hamster Ovary (11G-S) Cells", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 26, no. 8, 25 August 2010 (2010-08-25), pages 1116 - 1122 *

Also Published As

Publication number Publication date
CN110835622A (zh) 2020-02-25
CN110835622B (zh) 2021-04-27

Similar Documents

Publication Publication Date Title
JP7034991B2 (ja) 改良型細胞培養培地
JP6982578B2 (ja) 細胞培養培地
KR101848945B1 (ko) 개선된 세포 배양 배지
AU632065B2 (en) Cell culture medium for enhanced cell growth, culture longevity and product expression
JP6177260B2 (ja) 連続細胞培養で対象となるポリペプチドまたはウイルスを生成する方法
JPWO2008136398A1 (ja) 高濃度アミノ酸含有培地を用いた細胞の培養方法
CN107629124A (zh) 动物细胞的培养方法
WO2020035050A1 (zh) 用于调节哺乳动物细胞乳酸代谢的培养基及其应用
CN111454877B (zh) 一种cho细胞培养方法
JP6108170B2 (ja) 細胞培養用培地
CN108823267B (zh) 调节cho-k1表达系统所分泌抗体的酸性峰含量的方法
WO2018018613A1 (zh) 一种提高抗体纯度的细胞培养基和培养方法
KR20160113710A (ko) 관류 배지
CN111849863B (zh) 支持cho细胞高效生产单克隆抗体的培养基添加剂及其制备方法和应用
CN113862217A (zh) 培养哺乳动物细胞的方法
KR101318498B1 (ko) 재조합 단백질의 생산성 향상을 위한 배양용 배지 첨가용 조성물
KR101625595B1 (ko) 재조합 단백질 생산 증가를 위한 염화리튬을 포함하는 세포 배양용 배지 조성물 및 이의 용도
CN115340984B (zh) 一种cho细胞培养方法
JP7051820B2 (ja) 組換えタンパク質の生産プロファイルを改変するための方法
RU2798069C2 (ru) Улучшенная среда для культивирования клеток
CN117143946A (zh) 表达重组蛋白的cho工程细胞株的低代谢高表达方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19849972

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 02/06/2021)

122 Ep: pct application non-entry in european phase

Ref document number: 19849972

Country of ref document: EP

Kind code of ref document: A1