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CN101603026B - Animal-source-free low-protein medium suitable for production of animal cell products - Google Patents

Animal-source-free low-protein medium suitable for production of animal cell products Download PDF

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CN101603026B
CN101603026B CN2009100535068A CN200910053506A CN101603026B CN 101603026 B CN101603026 B CN 101603026B CN 2009100535068 A CN2009100535068 A CN 2009100535068A CN 200910053506 A CN200910053506 A CN 200910053506A CN 101603026 B CN101603026 B CN 101603026B
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sodium
acid
culture medium
vitamin
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CN101603026A (en
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谭文松
赵亮
周燕
陈飞
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East China University of Science and Technology
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Abstract

The invention relates to an animal origin-free low-protein culture medium suitable for animal cell product production, comprising 24 basic metabolism nutrients, 11 vitamins, 3 transferrin substitute compounds, 5 lipid compounds, 2 nucleic acid compounds, 4 hormones and growth factors, 3 antioxidants, 1 shear-resistant protective agent, 1 pH indicator, 2 pH buffers, 9 other inorganic salts, soy hydrolysates adopted to substitute animal origin component, and composition of ferrous sulfate, ferric nitrate and EDTA-2Na adopted to substitute transferrin. The culture medium can be made by dissolving the aforementioned components in triply distilled water. The positive effects of the culture medium are as follows: the culture medium contains no animal origin component, the total protein content is lower than 10mg/L, which helps separate and purify products and is suitable for production of recombinant protein medicaments; the culture medium supports normal growth and long-term subculturing of animal cells; the culture medium can be used without adaptation, is easily prepared and is suitable for massive production of animal cell products.

Description

适于动物细胞产品生产的无动物来源低蛋白培养基 Animal-source-free low-protein medium suitable for production of animal cell products

【技术领域】【Technical field】

本发明涉及现代生物技术之培养基研发技术领域，具体地说，是一种适于动物细胞大规模培养及抗体、融合蛋白等重组蛋白药物产品生产的无动物来源低蛋白培养基。The invention relates to the technical field of culture medium research and development of modern biotechnology, in particular, it is an animal-free low-protein culture medium suitable for large-scale cultivation of animal cells and production of recombinant protein pharmaceutical products such as antibodies and fusion proteins.

【技术背景】【technical background】

动物细胞培养技术是当今生物工程领域中的前沿课题之一，无论是基础研究还是生产实践方面都得到生物技术界的重视。2000年以来，FDA批准的所有创新生物技术药物中，用酵母表达的只有2种，用大肠杆菌表达的只有6种，而用哺乳动物细胞表达或生产的药物达到55种，占70％以上。2003年6月～2004年6月全球销售额最高的10个生物技术药物中，哺乳动物细胞表达的产品占8个，并且年销售额超过20亿美元的前6位的生物技术药物全部是哺乳动物细胞表达的产品。2005年全球生物技术药物销售额超过500亿美元，哺乳动物细胞表达产品占70％。至2007年末，仅单克隆抗体药物的销售额就达到260亿美元，预计至2013年其销售额将达到490亿美元。应用无血清培养技术进行动物细胞的规模化培养及重组蛋白表达生产已成为一种趋势。Animal cell culture technology is one of the frontier topics in the field of bioengineering today, and has been valued by the biotechnology community in both basic research and production practice. Since 2000, among all the innovative biotechnology drugs approved by the FDA, only 2 were expressed in yeast, only 6 were expressed in Escherichia coli, and 55 were expressed or produced in mammalian cells, accounting for more than 70%. From June 2003 to June 2004, among the top 10 biotechnological drugs with the highest global sales, 8 products expressed in mammalian cells accounted for 8, and the top 6 biotechnological drugs with annual sales of more than 2 billion US dollars were all breast-feeding Products expressed in animal cells. In 2005, the global biotechnology drug sales exceeded 50 billion US dollars, and mammalian cell expression products accounted for 70%. By the end of 2007, the sales of monoclonal antibody drugs alone reached 26 billion U.S. dollars, and its sales are expected to reach 49 billion U.S. dollars by 2013. It has become a trend to apply serum-free culture technology to large-scale culture of animal cells and expression and production of recombinant proteins.

随着哺乳动物细胞培养规模的扩大和生物药物需求的增长，基于细胞及产品特性的无血清培养基的研制已成为细胞工程领域的重要课题。在疫苗生产、单克隆抗体和各种生物活性蛋白等生物制品的应用领域中，优化无血清培养基的成分，使工程细胞在最有利于其生长和目的产物表达的培养环境中维持较高的活细胞密度和较长的培养时间，是降低生产成本的有效方法。With the expansion of the scale of mammalian cell culture and the growth of demand for biopharmaceuticals, the development of serum-free medium based on the characteristics of cells and products has become an important topic in the field of cell engineering. In the field of application of biological products such as vaccine production, monoclonal antibodies and various biologically active proteins, optimize the composition of serum-free medium, so that engineered cells can maintain a high level in the culture environment that is most conducive to their growth and target product expression Viable cell density and longer culture time are effective ways to reduce production costs.

与传统的有血清培养基相比，无血清培养基具有诸多优势：第一，无批次差异、无不明血清组分对细胞的影响；第二，避免了血清中外源性污染及对细胞的毒害作用；第三，使产品易于纯化，提高回收率；第四，成分明确，有利于研究细胞的生理状态，可根据不同细胞株设计和优化能支持其高密度培养和产物高水平表达的培养基。此外，无血清培养技术也是进行细胞生长、增殖、分化及基因表达调控等基础研究的关键技术之一。Compared with traditional serum-containing medium, serum-free medium has many advantages: first, there is no batch difference and no influence of unknown serum components on cells; second, exogenous contamination in serum and damage to cells are avoided. Toxic effect; third, it makes the product easy to purify and improves the recovery rate; fourth, the composition is clear, which is conducive to the study of the physiological state of the cell, and can design and optimize the culture that can support its high-density culture and high-level expression of the product according to different cell lines base. In addition, serum-free culture technology is also one of the key technologies for basic research on cell growth, proliferation, differentiation and gene expression regulation.

无血清培养基的开发与优化，主要有以下两个发展方向：一是不添加任何动物来源的成分；二是降低培养基的蛋白质含量。据此，无血清培养基可以分为以下三种：The development and optimization of serum-free medium mainly has the following two development directions: one is not to add any animal-derived components; the other is to reduce the protein content of the medium. Accordingly, serum-free media can be divided into the following three types:

(一)传统的无血清培养基，即用各类可替代血清功能的组分配制而成，如添加牛血清白蛋白(BSA)、转铁蛋白、胰岛素、从血清中提取的混合脂类及水解蛋白等组分，其特点是蛋白含量高，含有大量动物来源蛋白。(1) Traditional serum-free medium, which is formulated with various components that can replace serum functions, such as adding bovine serum albumin (BSA), transferrin, insulin, mixed lipids extracted from serum, and Components such as hydrolyzed protein are characterized by high protein content and contain a large amount of animal-derived protein.

(二)无动物来源成分的培养基，即祛除培养基中的动物来源成分。由于动物来源成分中可能含有潜在的朊蛋白和病毒污染的危险，因此，在培养基中必须慎用动物来源成分，这样才可确保生产的重组蛋白安全可靠。(2) Medium without animal-derived components, that is, to eliminate animal-derived components in the medium. Due to the potential risk of prions and viral contamination in animal-derived components, animal-derived components must be used with caution in culture media to ensure safe and reliable production of recombinant proteins.

(三)低蛋白或无蛋白培养基，即减少或祛除培养基中蛋白质成分。降低培养基中的蛋白质含量，可减少产物的分离纯化步骤，提高目标蛋白的回收率和品质，保证批次间的一致性，从而实现整个生产过程的高效率和经济性。(3) Low-protein or protein-free medium, that is, to reduce or eliminate protein components in the medium. Reducing the protein content in the medium can reduce the separation and purification steps of the product, improve the recovery rate and quality of the target protein, and ensure the consistency between batches, so as to achieve high efficiency and economy in the entire production process.

综上所述，动物细胞无血清培养基的开发与优化是朝着“无动物来源、无蛋白”的方向发展的。To sum up, the development and optimization of animal cell serum-free medium is moving in the direction of "no animal source, no protein".

经过几十年的发展，哺乳动物细胞〔如杂交瘤、中国仓鼠卵巢细胞(CHO)、NSO等〕无血清培养基技术已取得了可喜的进展：After decades of development, mammalian cell (such as hybridoma, Chinese hamster ovary cell (CHO), NSO, etc.) serum-free medium technology has made gratifying progress:

Darfler等人开发了CITTL无血清培养基，以DMEM/F12(1∶1)为基础培养基，添加了5mg/L的过氧化氢酶、1.5mg/L的胰岛素、1.5～3.0mg/L的转铁蛋白、2nmol/L睾酮、0.5mg/L的二亚油酰磷脂胆碱和1.5mg/L的β-甘油磷酸，可支持sf9细胞的克隆化生长，也可用于杂交瘤细胞的培养。Darfler et al. developed the CITTL serum-free medium, which was based on DMEM/F12 (1:1), added 5 mg/L catalase, 1.5 mg/L insulin, 1.5-3.0 mg/L insulin Transferrin, 2nmol/L testosterone, 0.5mg/L dilinoleoylphosphatidylcholine and 1.5mg/L β-glycerophosphate can support the clonal growth of sf9 cells and can also be used for the culture of hybridoma cells.

Murakami等人开发了无血清培养基，以DMEM/F12(1∶1)为基础培养基，添加了5mg/L胰岛素、2～35mg/L转铁蛋白、20μmol/L乙醇胺和1nmol/L亚硒酸钠等物质，这就是现被广泛使用的DMEM/F12-ITES配方。Murakami et al. developed a serum-free medium based on DMEM/F12 (1:1), supplemented with 5 mg/L insulin, 2-35 mg/L transferrin, 20 μmol/L ethanolamine and 1 nmol/L selenite Sodium acid and other substances, this is the widely used DMEM/F12-ITES formula.

Kover和Frank开发了无血清培养基，以RPMI1640为基础培养基，并添加10mg/L胰岛素、5mg/L转铁蛋白、20μmol/L乙醇胺、5mg/L亚油酸、1g/L牛血清蛋白、3mg/L抗坏血酸、2μg氢化可的松和12种微量元素，可支持几种细胞的生长。Kover and Frank developed a serum-free medium based on RPMI1640, and added 10mg/L insulin, 5mg/L transferrin, 20μmol/L ethanolamine, 5mg/L linoleic acid, 1g/L bovine serum albumin, 3mg/L ascorbic acid, 2μg hydrocortisone and 12 kinds of trace elements can support the growth of several kinds of cells.

美国专利U.S.P.4927762公开了一种含有抗氧化剂的培养基，阐述了细胞在还原性介质中能更好地生长，同时指出，N-乙酰半胱氨酸、硫乳酸、巯基乙醇等物质的添加可以起到抗氧化的作用。U.S. Patent U.S.P.4927762 discloses a culture medium containing antioxidants, which states that cells can grow better in reducing media, and points out that the addition of substances such as N-acetylcysteine, sulfur lactic acid, and mercaptoethanol can Play an antioxidant role.

美国专利U.S.P.4767704主要就微量元素的添加进行了研究，通过对铜、铁、镁、锌、硅、锰、钒、镍、锡、铝、钙、溴、氯、碘、硒、锗等元素和孕酮、腐胺、油酸、嘧啶等物质的添加，开发出了一种无蛋白培养基，使人们认识到微量元素的重要性。U.S. Patent U.S.P.4767704 mainly studies the addition of trace elements, through the analysis of copper, iron, magnesium, zinc, silicon, manganese, vanadium, nickel, tin, aluminum, calcium, bromine, chlorine, iodine, selenium, germanium and other elements and The addition of progesterone, putrescine, oleic acid, pyrimidine and other substances has developed a protein-free medium, making people aware of the importance of trace elements.

Jinyou Zhang等人以Fe-Na2SeO4混合物替代转铁蛋白，研制出的卵巢细胞(CHO)无蛋白培养基能支持较高的活细胞密度。但Fe-Na2SeO4混合物的配制十分复杂，条件苛刻，不适用于规模化生产。Jinyou Zhang et al. replaced transferrin with Fe-Na 2 SeO 4 mixture, and developed a protein-free medium for ovarian cells (CHO) that can support a higher density of viable cells. However, the preparation of the Fe-Na 2 SeO 4 mixture is very complicated and the conditions are harsh, so it is not suitable for large-scale production.

目前，以Gibico，Hyclone、JRH等为代表的商业无血清、无蛋白培养基也已相继问世。在已有的无血清培养基中，有些无法实现高密度培养过程；有些虽能很好地支持细胞生长，但可能导致细胞表达产物能力降低，甚至丧失；此外，商业无血清培养基的配方常属商业机密，且组分复杂、价格昂贵，既不利于特定细胞株培养技术的研究，也无法直接用于细胞培养过程的研究、开发和优化。At present, commercial serum-free and protein-free media represented by Gibico, Hyclone, JRH, etc. have also come out one after another. In the existing serum-free medium, some cannot achieve high-density culture process; although some can support cell growth well, it may lead to the reduction or even loss of the ability of cells to express products; in addition, the formula of commercial serum-free medium is often It is a commercial secret, and its components are complex and expensive, which is not conducive to the research of specific cell line culture technology, nor can it be directly used in the research, development and optimization of cell culture process.

【发明内容】【Content of invention】

本发明的目的在于克服现有技术的不足，提供一种适于动物细胞产品生产的无动物来源低蛋白培养基，以适应动物细胞大规模培养及工业生产重组蛋白药物产品的需要。The purpose of the present invention is to overcome the deficiencies of the prior art and provide an animal-free low-protein medium suitable for the production of animal cell products to meet the needs of large-scale cultivation of animal cells and industrial production of recombinant protein pharmaceutical products.

为实现上述目的，本发明采取的技术方案为：In order to achieve the above object, the technical scheme that the present invention takes is:

一种适于动物细胞产品生产的无动物来源低蛋白培养基，其特征是，采用大豆水解物替代动物来源成分(如，牛血清白蛋白等)，采用硫酸亚铁、硝酸铁和EDTA·2Na的组合物替代转铁蛋白，其成分包括基础代谢营养物类、维生素类、转铁蛋白替代物、脂类、核酸类、激素及生长因子类、抗氧化剂类、抗剪切保护剂、pH指示剂、pH缓冲剂和其他无机盐类；A kind of non-animal-derived low-protein medium suitable for the production of animal cell products is characterized in that soybean hydrolyzate is used to replace animal-derived components (such as bovine serum albumin, etc.), and ferrous sulfate, ferric nitrate and EDTA·2Na are used The composition replaces transferrin, and its composition includes basic metabolic nutrients, vitamins, transferrin substitutes, lipids, nucleic acids, hormones and growth factors, antioxidants, anti-shear protection agents, pH indicators Agents, pH buffers and other inorganic salts;

——所述的基础代谢营养物类包括：葡萄糖、大豆水解物、丙酮酸钠、谷氨酰胺、丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、胱氨酸、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸；——The basal metabolic nutrients include: glucose, soybean hydrolyzate, sodium pyruvate, glutamine, alanine, arginine, asparagine, aspartic acid, cysteine, cystine Acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine amino acid, valine;

——所述的维生素类包括：生物素、维生素、氯化胆碱、叶酸、肌醇、烟酰胺、泛酸钙、维生素B6、核黄素、维生素B1、硫辛酸；——The vitamins include: biotin, vitamins, choline chloride, folic acid, inositol, nicotinamide, calcium pantothenate, vitamin B6, riboflavin, vitamin B1, lipoic acid;

——所述的转铁蛋白替代物类包括：硫酸亚铁、硝酸铁、EDTA·2Na；——The transferrin substitutes include: ferrous sulfate, ferric nitrate, EDTA·2Na;

——所述的脂类包括：乙醇胺、吐温80、鱼肝油、胆固醇、亚油酸；——The lipids include: ethanolamine, Tween 80, cod liver oil, cholesterol, linoleic acid;

——所述的核酸类包括：胸苷、次黄嘌呤；——The nucleic acids include: thymidine, hypoxanthine;

——所述的激素及生长因子类包括：孕酮、氢化可的松、重组胰岛素、腐胺；——The hormones and growth factors include: progesterone, hydrocortisone, recombinant insulin, putrescine;

——所述的抗氧化剂类包括：亚硒酸钠、生育酚、牛磺酸；- the antioxidants include: sodium selenite, tocopherol, taurine;

——所述的抗剪切保护剂为嵌段式聚醚F68；——The anti-shear protection agent is block polyether F68;

——所述的pH指示剂为酚红；- the pH indicator is phenol red;

——所述的pH缓冲剂包括碳酸氢钠、Hepes；- the pH buffer comprises sodium bicarbonate, Hepes;

——所述的其他无机盐类包括：氯化钙、氯化镁、硫酸镁、氯化钾、磷酸二氢钠、氯化钠、磷酸氢二钠、硫酸锌、硫酸铜。——The other inorganic salts mentioned include: calcium chloride, magnesium chloride, magnesium sulfate, potassium chloride, sodium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate, zinc sulfate, copper sulfate.

一种适于动物细胞产品生产的无动物来源低蛋白培养基，其各种成分的含量为(毫克/升)：A kind of animal-source-free low-protein medium suitable for the production of animal cell products, the content of its various components is (mg/L):

葡萄糖(D-Glucose)                  2000～7000Glucose ( D -Glucose) 2000～7000

大豆水解物(Lucratone Soy)          500～1000Soybean hydrolyzate (Lucratone Soy) 500～1000

丙酮酸钠(Sodium Pyruvate)          55～220Sodium Pyruvate (Sodium Pyruvate) 55～220

谷氨酰胺(L-Glutamine)              200～900Glutamine ( L -Glutamine) 200～900

丙氨酸(L-Alanine)                  4～15Alanine ( L -Alanine) 4～15

精氨酸(L-Arginine·HCl)            100～300Arginine ( L -Arginine·HCl) 100～300

天冬酰胺(L-Asparagine·H2O)        6～15Asparagine ( L -Asparagine·H 2 O) 6～15

天冬氨酸(L-Aspartic Acid)          6～15Aspartic acid ( L -Aspartic Acid) 6～15

半胱氨酸(L-Cysteine·H2O)            15～50Cysteine ( L -Cysteine·H 2 O) 15～50

胱氨酸(L-Cystine·2HCl)              30～65Cystine ( L -Cystine·2HCl) 30～65

谷氨酸(L-Glutamic Acid)              5～20Glutamic acid ( L -Glutamic Acid) 5～20

甘氨酸(Glycine)                      15～40Glycine 15～40

组氨酸(L-Histidine·HCl·H2O)        15～40Histidine ( L -Histidine·HCl·H 2 O) 15～40

异亮氨酸(L-Isoleucine)               50～100Isoleucine ( L -Isoleucine) 50～100

亮氨酸(L-Leucine)                    50～150Leucine ( L -Leucine) 50～150

赖氨酸(L-Lysine·HCl)                80～185Lysine ( L -Lysine·HCl) 80～185

甲硫氨酸(L-Methionine)               15～35Methionine ( L -Methionine) 15～35

苯丙氨酸(L-Phenyalanine)             30～70Phenylalanine ( L -Phenyalanine) 30～70

脯氨酸(L-Proline)                    10～34.5Proline ( L -Proline) 10～34.5

丝氨酸(L-Serine)                     20～52.5Serine ( L -Serine) 20～52.5

苏氨酸(L-Threonine)                  50～107Threonine ( L -Threonine) 50～107

色氨酸(L-Tryptophan)                 9～18Tryptophan ( L -Tryptophan) 9～18

酪氨酸(L-Tyrosine·2Na·2H2O)        55～120Tyrosine ( L -Tyrosine·2Na·2H 2 O) 55～120

缬氨酸(L-Valine)                     50～120Valine ( L -Valine) 50～120

生物素(Biotin)                       0.0035～0.0105Biotin 0.0035～0.0105

维生素B12(Vitamin B12)               0.50～2.50Vitamin B12 (Vitamin B12) 0.50～2.50

氯化胆碱(Choline Chloride)           8.50～30Choline Chloride 8.50～30

叶酸(Folic acid)                     2.50～8Folic acid 2.50～8

肌醇(I-Nositol)                      12～30Inositol ( I -Nositol) 12～30

烟酰胺(Niacinamide)                  2～7Niacinamide 2～7

泛酸钙(D-Ca·Pantothenate)           2～8Calcium pantothenate ( D -Ca·Pantothenate) 2～8

维生素B6(Pyridoxine·HCl)            2～8Vitamin B6 (Pyridoxine·HCl) 2～8

核黄素(Riboflavin)                   0.2～0.7Riboflavin 0.2～0.7

维生素B1(Thiamine·HCl)              2～8Vitamin B1 (Thiamine·HCl) 2～8

硫辛酸(Lipoic acid)                  0.08～0.50Lipoic acid 0.08～0.50

硫酸亚铁(FeSO4·7H2O)                0.417～5Ferrous sulfate (FeSO 4 7H 2 O) 0.417～5

硝酸铁(Fe(NO3)3·9H2O)               0.05～0.8Iron nitrate (Fe(NO 3 ) 3 9H 2 O) 0.05～0.8

EDTA·2Na                          2～10EDTA·2Na 2～10

乙醇胺(Ethanolamine)               3～10Ethanolamine 3～10

吐温80(Tween 80)                   0.24～0.5Tween 80 (Tween 80) 0.24～0.5

鱼肝油(Cod liver oil)              0.1～0.2Cod liver oil 0.1～0.2

胆固醇(Cholesterol)                0.045～0.1Cholesterol 0.045～0.1

亚油酸(Linoleic acid)              0.042～0.084Linoleic acid (Linoleic acid) 0.042～0.084

胸苷(Thymidine)                    0.4～2.5Thymidine 0.4～2.5

次黄嘌呤(Hypoxanthine)             2～20Hypoxanthine 2～20

孕酮(Progesterone)                 0.003～0.012Progesterone 0.003～0.012

氢化可的松(Hydrocortisone·HCl)    0.036～0.072Hydrocortisone (Hydrocortisone·HCl) 0.036～0.072

重组胰岛素(Insulin)                3～8Recombinant insulin (Insulin) 3～8

腐胺(Sodium Putrescine·HCl)       0.08～20Putrescine (Sodium Putrescine·HCl) 0.08～20

亚硒酸钠(Sodium Selenate)          0.02～0.08Sodium Selenite (Sodium Selenate) 0.02～0.08

生育酚(α-Tocohcrol acetate)       1～4Tocopherol (α-Tocohcrol acetate) 1～4

牛磺酸(Taurin)                     20～50Taurine 20～50

嵌段式聚醚F68(Pluronic-F68)        500～2000Block polyether F68 (Pluronic-F68) 500～2000

酚红(Phenol Red)                   8.1Phenol Red 8.1

碳酸氢钠(NaHCO3)                   2450Sodium bicarbonate (NaHCO 3 ) 2450

Hepes                              2000～5000Hepes 2000～5000

氯化钙(CaCl2)                      110～150Calcium chloride (CaCl 2 ) 110～150

氯化镁(MgCl2)                      40～75Magnesium chloride (MgCl 2 ) 40～75

硫酸镁(MgSO4)                      45～65Magnesium sulfate (MgSO 4 ) 45～65

氯化钾(KCl)                        310～400Potassium chloride (KCl) 310～400

磷酸二氢钠(NaH2PO4·H2O)           60～120Sodium dihydrogen phosphate (NaH 2 PO 4 ·H 2 O) 60～120

氯化钠(NaCl)                       6995.5Sodium chloride (NaCl) 6995.5

磷酸氢二钠(Na2HPO4)                55～120Disodium hydrogen phosphate (Na 2 HPO 4 ) 55～120

硫酸锌(ZnSO4·7H2O)                0.432～1.25Zinc sulfate (ZnSO 4 7H 2 O) 0.432～1.25

硫酸铜(CuSO4·5H2O)                0.0013～0.0065。Copper sulfate (CuSO 4 ·5H 2 O) 0.0013～0.0065.

本发明的积极效果是：The positive effect of the present invention is:

(1)不含动物来源成分，总蛋白含量低于10毫克/升，有利于产物的分离纯化，适于动物细胞表达的重组蛋白类药物产品生产，安全可靠。(1) It does not contain animal-derived components, and the total protein content is less than 10 mg/L, which is conducive to the separation and purification of products, and is suitable for the production of recombinant protein pharmaceutical products expressed in animal cells, which is safe and reliable.

(2)支持动物细胞的正常生长、重组蛋白表达和长期传代培养，无需适应即可使用，其最高活细胞密度及重组蛋白产量均超过已知的商业培养基。(2) It supports normal growth of animal cells, recombinant protein expression and long-term subculture, and can be used without adaptation, and its highest living cell density and recombinant protein production exceed known commercial media.

(3)成分简单，配制方便，适合于动物细胞产品的大规模生产。(3) The composition is simple, the preparation is convenient, and it is suitable for large-scale production of animal cell products.

【附图说明】【Description of drawings】

附图为CHO细胞在本发明的无动物来源低蛋白培养基中批培养生长和产物表达曲线的坐标图。The accompanying drawing is a coordinate diagram of the growth and product expression curves of batch culture of CHO cells in the animal-source-free low-protein medium of the present invention.

【具体实施方式】【Detailed ways】

以下对本发明适于动物细胞产品生产的无动物来源低蛋白培养基的实施例进行具体说明，但本发明不限于以下的实施方式。The examples of the animal-source-free low-protein medium suitable for the production of animal cell products of the present invention are described in detail below, but the present invention is not limited to the following embodiments.

一种适于动物细胞产品生产的无动物来源低蛋白培养基，包括24种基础代谢营养物、11种维生素、3种转铁蛋白替代物类化合物、5种脂类化合物、2种核酸类化合物、4种激素及生长因子、3种抗氧化剂、1种抗剪切保护剂、1种pH指示剂、2种pH缓冲剂、9种其他无机盐，各种成分的含量为(毫克/升)：An animal-source-free low-protein medium suitable for the production of animal cell products, including 24 basic metabolic nutrients, 11 vitamins, 3 transferrin substitute compounds, 5 lipid compounds, and 2 nucleic acid compounds , 4 kinds of hormones and growth factors, 3 kinds of antioxidants, 1 kind of anti-shear protection agent, 1 kind of pH indicator, 2 kinds of pH buffering agents, 9 kinds of other inorganic salts, the content of each component is (mg/L) :

葡萄糖(D-Glucose)                    4500Glucose ( D -Glucose) 4500

大豆水解物(Lucratone Soy)            1000Soybean hydrolyzate (Lucratone Soy) 1000

丙酮酸钠(Sodium Pyruvate)            55Sodium Pyruvate 55

谷氨酰胺(L-Glutamine)                584.6Glutamine ( L -Glutamine) 584.6

丙氨酸(L-Alanine)                    5Alanine ( L -Alanine) 5

精氨酸(L-Arginine·HCl)              155Arginine ( L -Arginine·HCl) 155

天冬酰胺(L-Asparagine·H2O)          7.5Asparagine ( L -Asparagine·H 2 O) 7.5

天冬氨酸(L-Aspartic Acid)            6.65Aspartic acid ( L -Aspartic Acid) 6.65

半胱氨酸(L-Cysteine·H2O)            17.56Cysteine ( L -Cysteine·H 2 O) 17.56

胱氨酸(L-Cystine·2HCl)          31.29Cystine ( L -Cystine·2HCl) 31.29

谷氨酸(L-Glutamic Acid)          7.35Glutamic Acid ( L -Glutamic Acid) 7.35

甘氨酸(Glycine)                  18.75Glycine 18.75

组氨酸(L-Histidine·HCl·H2O)    18.75Histidine ( L -Histidine·HCl·H 2 O) 18.75

异亮氨酸(L-Isoleucine)           54.47Isoleucine ( L -Isoleucine) 54.47

亮氨酸(L-Leucine)                59.05Leucine ( L -Leucine) 59.05

赖氨酸(L-Lysine·HCl)            91.25Lysine ( L -Lysine·HCl) 91.25

甲硫氨酸(L-Methionine)           17.24Methionine ( L -Methionine) 17.24

苯丙氨酸(L-Phenyalanine)         35.48Phenylalanine ( L -Phenyalanine) 35.48

脯氨酸(L-Proline)                17.25Proline ( L -Proline) 17.25

丝氨酸(L-Serine)                 26.25Serine ( L -Serine) 26.25

苏氨酸(L-Threonine)              53.45Threonine ( L -Threonine) 53.45

色氨酸(L-Tryptophan)             9.02Tryptophan ( L -Tryptophan) 9.02

酪氨酸(L-Tyrosine·2Na·2H2O)    55.79Tyrosine ( L -Tyrosine·2Na·2H 2 O) 55.79

缬氨酸(L-Valine)                 52.85Valine ( L -Valine) 52.85

生物素(Biotin)                   0.0105Biotin 0.0105

维生素B12(Vitamin B12)           2.04Vitamin B12 (Vitamin B12) 2.04

氯化胆碱(Choline Chloride)       26.94Choline Chloride 26.94

叶酸(Folic acid)                 7.95Folic acid 7.95

肌醇(I-Nositol)                  12.6Inositol ( I -Nositol) 12.6

烟酰胺(Niacinamide)              6.06Niacinamide 6.06

泛酸钙(D-Ca·Pantothenate)       6.72Calcium pantothenate ( D -Ca Pantothenate) 6.72

维生素B6(Pyridoxine·HCl)        6.093Vitamin B6 (Pyridoxine·HCl) 6.093

核黄素(Riboflavin)               0.657Riboflavin 0.657

维生素B1(Thiamine·HCl)          6.51Vitamin B1 (Thiamine·HCl) 6.51

硫辛酸(Lipoic acid)              0.305Lipoic acid 0.305

硫酸亚铁(FeSO4·7H2O)            1.217Ferrous sulfate (FeSO 4 7H 2 O) 1.217

硝酸铁(Fe(NO3)3·9H2O)           0.65Iron nitrate (Fe(NO 3 ) 3 9H 2 O) 0.65

EDTA·2Na                          5EDTA·2Na 5

乙醇胺(Ethanolamine)               9Ethanolamine 9

吐温80(Tween 80)                   0.24Tween 80 (Tween 80) 0.24

鱼肝油(Cod liver oil)              0.1Cod liver oil 0.1

胆固醇(Cholesterol)                0.045Cholesterol 0.045

亚油酸(Linoleic acid)              0.042Linoleic acid 0.042

胸苷(Thymidine)                    2.365Thymidine 2.365

次黄嘌呤(Hypoxanthine)             10Hypoxanthine 10

孕酮(Progesterone)                 0.006Progesterone 0.006

氢化可的松(Hydrocortisone·HCl)    0.036Hydrocortisone (Hydrocortisone·HCl) 0.036

胰岛素(Insulin)                    8Insulin 8

腐胺(Sodium Putrescine·HCl)       0.081Putrescine (Sodium Putrescine·HCl) 0.081

亚硒酸钠(Sodium Selenate)          0.0519Sodium Selenite 0.0519

黄体酮(α-Tocohcrol acetate)       2Progesterone (α-Tocohcrol acetate) 2

牛磺酸(Taurin)                     30Taurine 30

嵌段式聚醚F68(Pluronic-F68)        1000Block polyether F68 (Pluronic-F68) 1000

酚红(Phenol Red)                   8.1Phenol Red 8.1

碳酸氢钠(NaHCO3)                   2450Sodium bicarbonate (NaHCO 3 ) 2450

Hepes                              3500Hepes 3500

氯化钙(CaCl2)                      116.6Calcium chloride (CaCl 2 ) 116.6

氯化镁(MgCl2)                      40.4Magnesium chloride (MgCl 2 ) 40.4

硫酸镁(MgSO4)                      48.84Magnesium sulfate (MgSO 4 ) 48.84

氯化钾(KCl)                        311.8Potassium chloride (KCl) 311.8

磷酸二氢钠(NaH2PO4·H2O)           120Sodium dihydrogen phosphate (NaH 2 PO 4 ·H 2 O) 120

氯化钠(NaCl)                       6995.5Sodium chloride (NaCl) 6995.5

磷酸氢二钠(Na2HPO4)                110Disodium hydrogen phosphate (Na 2 HPO 4 ) 110

硫酸铜(CuSO4·5H2O)                0.006Copper sulfate (CuSO 4 5H 2 O) 0.006

硫酸锌(ZnSO4·7H2O)                0.432。Zinc sulfate (ZnSO 4 ·7H 2 O) 0.432.

将上述组分溶解于三次蒸馏水中，即可配制成适于动物细胞产品生产的无动物来源低蛋白培养基。Dissolving the above components in triple distilled water can be prepared into an animal-source-free low-protein medium suitable for the production of animal cell products.

使用本发明所述的无动物来源低蛋白培养基，将表达抗CD20嵌合单克隆抗体的中国仓鼠卵巢细胞(CHO)接种于2升生物反应器中，接种密度为2.39×105细胞/毫升，接种时细胞活性为98.45％；细胞在此2升反应器中进行155小时的批培养，对数生长期的平均比生长速率为0.652天-1，最大活细胞密度达3.75×106细胞/毫升，细胞扩增15.7倍，最大单抗浓度为203毫克/升，活细胞密度对时间的积分值为13.41×109细胞·天，对数生长期单位细胞每天的抗体产量达15.88毫克/109细胞·天。Chinese hamster ovary cells (CHO) expressing anti-CD20 chimeric monoclonal antibody were inoculated in a 2-liter bioreactor with an inoculation density of 2.39×10 5 cells/ml using the animal-derived low-protein medium of the present invention , the cell viability was 98.45% at the time of inoculation; the cells were batch cultured in this 2-liter reactor for 155 hours, the average specific growth rate in the logarithmic growth phase was 0.652 days -1 , and the maximum viable cell density reached 3.75×10 6 cells/ ml, cell expansion was 15.7 times, the maximum monoclonal antibody concentration was 203 mg/L, the integral value of viable cell density versus time was 13.41×10 9 cells per day, and the daily antibody output per unit cell in the logarithmic growth phase reached 15.88 mg/10 9 cells · day.

附图为CHO细胞在本发明的无动物来源低蛋白培养基中批培养生长和产物表达曲线的坐标图，附中，Accompanying drawing is the coordinate diagram of CHO cell batch culture growth and product expression curve in the animal-free low-protein medium of the present invention, attached middle,

◆活细胞曲线；◆Live cell curve;

■死细胞曲线；■ dead cell curve;

▲总细胞曲线；▲Total cell curve;

□产物曲线。□ Product curve.

从附图的曲线可以得知，本发明适于动物细胞产品生产的无动物来源低蛋白培养基能良好地支持细胞的正常生长和产物表达，其最高活细胞密度及产物浓度均超过已知的商业培养基。As can be seen from the curve of the accompanying drawing, the animal-free low-protein medium suitable for the production of animal cell products of the present invention can well support the normal growth of cells and product expression, and its highest viable cell density and product concentration all exceed known commercial media.

Claims (2)

1. animal origin-free low-protein culture medium that is suitable for the zooblast products production, be applicable to the cultivation of Chinese hamster ovary celI, it is characterized in that, adopt the soybean hydrolyzate to substitute animal derived components, adopt the composition of ferrous sulfate, iron nitrate and EDTA2Na to substitute Transferrins,iron complexes, its composition comprises basal metabolism nutrition class, vitamins, Transferrins,iron complexes surrogate, lipid, nucleic acid class, hormone and somatomedin class, antioxidant, anti-shearing protective material, pH indicator, pH buffer reagent and other inorganic salts;
---described basal metabolism nutrition class comprises: glucose, soybean hydrolyzate, Sodium.alpha.-ketopropionate, glutamine, L-Ala, arginine, l-asparagine, aspartic acid, halfcystine, Gelucystine, L-glutamic acid, glycine, Histidine, Isoleucine, leucine, Methionin, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine, Xie Ansuan;
---described vitamins comprises: vitamin H, vitamin B12, choline chloride 60, folic acid, inositol, niacinamide, calcium pantothenate, vitamin B6, riboflavin, VITMAIN B1, Thioctic Acid;
---described Transferrins,iron complexes surrogate class comprises: ferrous sulfate, iron nitrate, EDTA2Na;
---described lipid comprises: thanomin, tween 80, Oils,glyceridic,cod-liver, cholesterol, linolic acid;
---described nucleic acid class comprises: thymidine, xanthoglobulin;
---described hormone and somatomedin class comprise: progesterone, hydrocortisone, Recombulin, putrescine;
---described antioxidant comprises: Sodium Selenite, tocopherol, taurine;
---described anti-shearing protective material is blocked polyethers F68;
---described pH indicator is phenol red;
---described pH buffer reagent comprises sodium bicarbonate, Hepes;
---described other inorganic salts comprise: calcium chloride, magnesium chloride, sal epsom, Repone K, SODIUM PHOSPHATE, MONOBASIC, sodium-chlor, Sodium phosphate dibasic, zinc sulfate, copper sulfate.
2. the animal origin-free low-protein culture medium that is suitable for the zooblast products production according to claim 1 is characterized in that, dissolves following component in three distilled water, and its various compositions are as follows, and unit is a mg/litre:
Glucose 2000～7000
Soybean hydrolyzate 500～1000
Sodium.alpha.-ketopropionate 55～220
Glutamine 200～900
L-Ala 4～15
Arginine 100～300
L-asparagine 6～15
Aspartic acid 6～15
Halfcystine 15～50
Gelucystine 30～65
L-glutamic acid 5～20
Glycine 15～40
Histidine 15～40
Isoleucine 50～100
Leucine 50～150
Methionin 80～185
Methionine(Met) 15～35
Phenylalanine-3,4-quinone 0～70
Proline(Pro) 10～34.5
Serine 20～52.5
Threonine 50～107
Tryptophane 9～18
Tyrosinase 15 5～120
Xie Ansuan 50～120
Vitamin H 0.0035～0.0105
Vitamin B12 0.50～2.50
Choline chloride 60 8.50～30
Folic acid 2.50～8
Inositol 12～30
Niacinamide 2～7
Calcium pantothenate 2～8
Vitamin B6 2～8
Riboflavin 0.2～0.7
VITMAIN B1 2～8
Thioctic Acid 0.08～0.50
Ferrous sulfate 0.417～5
Iron nitrate 0.05～0.8
EDTA·2Na 2～10
Thanomin 3～10
Tween 80 0.24～0.5
Oils,glyceridic,cod-liver 0.1～0.2
Cholesterol 0.045～0.1
Linolic acid 0.042～0.084
Thymidine 0.4～2.5
Xanthoglobulin 2～20
Progesterone 0.003～0.012
Hydrocortisone 0.036～0.072
Recombulin 3～8
Putrescine 0.08～20
Sodium Selenite 0.02～0.08
Tocopherol 1～4
Taurine 20～50
Blocked polyethers F68 500～2000
Phenol red 8.1
Sodium bicarbonate 2450
Hepes 2000～5000
Calcium chloride 110～150
Magnesium chloride 40～75
Sal epsom 45～65
Repone K 310～400
SODIUM PHOSPHATE, MONOBASIC 60～120
Sodium-chlor 6995.5
Sodium phosphate dibasic 55～120
Zinc sulfate 0.432～1.25
Copper sulfate 0.0013～0.0065.
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