WO2011110073A1 - 细胞培养混合物的分离纯化方法 - Google Patents

细胞培养混合物的分离纯化方法 Download PDF

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WO2011110073A1
WO2011110073A1 PCT/CN2011/071472 CN2011071472W WO2011110073A1 WO 2011110073 A1 WO2011110073 A1 WO 2011110073A1 CN 2011071472 W CN2011071472 W CN 2011071472W WO 2011110073 A1 WO2011110073 A1 WO 2011110073A1
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culture mixture
cell culture
membrane
filter
virus
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PCT/CN2011/071472
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French (fr)
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陈文庆
王建超
刘俊生
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北京清大天一科技有限公司
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    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/00051—Methods of production or purification of viral material

Definitions

  • the present invention relates to a method for separating and purifying a biological product, and more particularly to a method for separating and purifying a cell culture mixture for obtaining a biological product of interest.
  • Separation and purification of the culture mixture of the target biological product is one of the most critical steps in the production of biological products. It is a pivotal step between the harvesting culture mixture and further downstream processing, and has direct production, quality and reproducibility of biological products. The impact also determines the efficiency and cost of the overall production process of biological products.
  • the separation and purification methods of the target culture mixture commonly used in the production process of existing biological products are mostly combined with several methods such as centrifugation, gel chromatography, filtration, etc., and there are many problems.
  • the equipment used is less processed in a single batch, and it is not easy to expand the production scale. Again, repeated use of the equipment used requires repeated cleaning, which increases the labor and production costs.
  • existing processes often fail to completely separate the purification culture mixture of interest, resulting in a relatively large amount of heterologous material in the final biological product.
  • the existing technical means are used to separate and purify the cell culture mixture. Due to the above problems, the separation and purification process is complicated, and the cell culture mixture cannot be separated and purified in a large amount, quickly and with high purity, resulting in high cost of separation and purification, long production time and the like. In addition, the final biological products have more heterogeneous substances, and the side reactions are relatively large when used. It can be seen that the existing separation and purification methods have been unable to meet the needs of actual production and life.
  • the present invention specifically studies the separation and purification of cell culture mixtures, and finds that the cell culture mixture has complex components, the content of the target product is generally low, the initial amount is large, and the different virus particle sizes have certain differences. .
  • the inventors have pioneered a new method for separating and purifying them, which overcomes the above drawbacks.
  • the invention provides a method for separating and purifying a cell culture mixture, 1.
  • a method for separating and purifying a cell culture mixture comprising filtering a cell culture mixture through a filter, the filter having at least two filter membranes having different pore sizes, filtering
  • the nominal pore size of each membrane of the membrane is gradually reduced in the direction of flow of the filtrate, and the nominal pore size of the first filtration membrane is about 2 ⁇ ⁇ , and the nominal pore size of the last filtration membrane is about 0.1 to 0.2 ⁇ m.
  • the method for separating and purifying a cell culture mixture according to the invention wherein the cell culture mixture is selected from the group consisting of an animal virus cell culture mixture, a human virus cell culture mixture or an antibody-secreting hybridoma cell culture mixture.
  • the folded filter membrane divides the interior of the housing into a central zone and an edge zone.
  • the cell culture mixture is selected from the group consisting of: a foot-and-mouth disease virus culture mixture, a porcine reproductive and respiratory syndrome virus culture mixture, and a swine fever virus culture. Mixture, rabies virus culture mixture, hepatitis B virus culture mixture, epidemic encephalitis virus culture mixture, hepatitis A virus culture mixture or poliovirus culture mixture.
  • the method for separating and purifying a cell culture mixture according to the first aspect, wherein
  • the cell culture mixture is a foot-and-mouth disease virus culture mixture
  • the filter membrane is a two-layer filter membrane.
  • the first membrane membrane has a nominal pore size of about 2 to 10 ⁇ m
  • the second membrane membrane has a nominal pore diameter of about 0.1 to 0 ⁇ 2 ⁇ m.
  • the filtration membrane is a three-layer filtration membrane, and the first filtration membrane
  • the nominal pore size is about 2 ⁇ 10 ⁇
  • the nominal pore size of the second filter membrane is about 0.2 ⁇ 2 ⁇
  • the nominal pore size of the third filter membrane is about 0.1 ⁇ 0.2 ⁇ .
  • the method for separating and purifying a cell culture mixture of the invention has the advantages of strong pertinence, large processing amount, single separation and purification step, stable product quality, low cost, and the like, and is particularly suitable for separating and purifying the foot-and-mouth disease virus culture mixture or pig breeding.
  • a mixture of virus vaccines, etc. is cultured with a respiratory syndrome virus to obtain a mixture of virus vaccines of higher purity.
  • a large single treatment amount can be achieved up to several hundred liters, thereby easily expanding the production scale; and the separation and purification method of the cell culture mixture of the present invention separates the target substance.
  • the purification is more thorough, and the cell debris and other larger substances in the product are trapped, thereby reducing the side effects of immunization of the biological product, reducing the transfer exposure of the target culture, thereby reducing the probability of contamination of the target.
  • the method for separating and purifying the cell culture mixture of the invention is easy to control, and the filter element can be used for single-use products without cleaning, cleaning and other processes, saving labor and production costs, and improving production efficiency;
  • the method for separating and purifying the cell culture mixture of the invention is easy to control, and the filter elements of different sizes and combinations thereof can be selected according to different separation and purification objects, and the utility model has the advantages of flexible use and wide application range. detailed description
  • animal refers to various animals other than humans, and particularly preferred are livestock or poultry such as pigs, dogs, cows, sheep, chickens, ducks and/or geese.
  • the term "cell culture mixture” in the present invention means a cell culture mixture obtained by culturing a virus, an antibody or the like under the existing conditions.
  • the cell culture mixture includes an animal virus cell culture mixture, a human virus cell culture mixture, and an antibody-secreting hybridoma cell culture mixture.
  • the cell culture mixture mainly contains virus particles (or antibodies), and cultured cells, cultured cell debris, proteins, nucleic acids, polysaccharides and the like, if used cells
  • the medium is a serum-containing cell culture medium, and the resulting mixture also contains residual serum.
  • the filtrate obtained is required to have a higher content of the target virus, and impurities such as host proteins, host nucleic acids and the like are controlled as low as possible, aseptic contamination.
  • the virus is usually between 20 nm and 200 nm in diameter, and different cells are cultured for different viruses.
  • the inventors have carefully studied the cell culture mixture of different viruses and antibodies.
  • the virus cell culture mixture of the animal is as follows: Foot and mouth disease
  • the virus culture mixture has a virus diameter of about 20-25 nm.
  • the commonly used cultured cells are baby hamster kidney passage cells (BHK21 cells); the porcine reproductive and respiratory syndrome virus culture mixture, the virus diameter is about 50-60 nm, and the commonly used cultured cells are Marcl45 cells;
  • the prion culture mixture has a virus diameter of about 70 nm.
  • the commonly used culture cells are bovine testicular primary cells (BT cells), porcine kidney cells (PK cells) and porcine testicular cells (ST cells); rabies virus culture mixture, virus length 170- 180 nm, about 75-80 nm wide, the commonly used cultured cell is BHK21.
  • the human virus cell culture mixture is as follows: a rabies virus culture mixture, the virus is about 170-180 nm long, about 75-80 nm wide, and the commonly used cultured cells are African green monkey kidney cells (Vera cells); hepatitis B virus culture mixture, The virus is about 22nm in diameter.
  • the commonly used cultured cells are Chinese hamster ovary cells (CHO cells); the epidemic encephalitis virus culture mixture, the virus diameter is about 40nm, the commonly used cultured cells are Vera cells; Hepatitis virus culture mixture, virus The diameter of the culture is about 27-32 nm.
  • the commonly used cultured cells are human diploid cells KMB 17 or 2BS; the poliovirus culture mixture has a virus diameter of about 27-30 nm, and the commonly used cultured cells are human diploid cells 2BS.
  • viruses have a diameter of about 20-100 nm, and individual viruses have a diameter of about 100-200 nm.
  • the target mixture obtained by separating the cell culture mixture is required to be free of contaminants, and the content of the impurities should be controlled to a minimum level, such as cells, bacteria, molds and/or mycoplasmas, etc., such as impurities, Different biological products have different requirements and restrictions on nucleic acids and/or bovine serum.
  • the inventors have found that the diameters of various contaminants are quite different from the diameter of the virus.
  • the diameter of the animal cells is usually about 10-100 ⁇ m, and the diameter of the bacterial cells is usually about 0.5-2.0 ⁇ m.
  • the fungal cell diameter is usually about 10-40 ⁇ m, and the mycoplasma diameter is usually about 0.2-0.3 ⁇ m. Therefore, by selecting the appropriate filtration conditions, the filtration efficiency can be greatly improved, thereby reducing the steps of obtaining the final product, increasing the production efficiency, and reducing the cost.
  • the present invention provides a method for separating and purifying a cell culture mixture, which comprises filtering a cell culture mixture through a double-layer or multi-layer filtration membrane to obtain a separated and purified filtrate.
  • the nominal pore size of each layer of the two-layer or multi-layered filtration membrane is gradually reduced in the filtration direction, such as two, three, four, five or six layers, preferably three, four or five layers.
  • Filter membrane according to the characteristics of the virus and cells of the cell culture mixture, the nominal pore size of the first filter membrane is about 2 ⁇ ⁇ , and the nominal pore size of the last membrane is about 0.1 ⁇ 0.2 ⁇ , if the first membrane is If the nominal pore size is less than 2 ⁇ , then more contaminants or impurities will quickly block the pores, so that the filtration can not proceed smoothly. If the nominal pore size of the first filtration membrane is larger than ⁇ , then some of the cells and other pollutants will pass through the first layer.
  • the membrane is filtered so as to accumulate on the second membrane, so that the filtration efficiency is lowered. If the nominal pore size of the last membrane is less than ⁇ . ⁇ , the filtration resistance is increased, which is not conducive to the filtration efficiency, if greater than 0.2 ⁇ , the final filtrate will have too much impurity and will not reach the required purity.
  • the first filter membrane can filter out large-diameter contaminants or impurities.
  • the last layer is to filter out small-diameter pollutants and impurities as much as possible. According to the characteristics of the cell culture fluid, it is filtered layer by layer to obtain higher purity. Virus filtrate.
  • the nominal pore size of the intermediate filtration membrane is between the nominal pore size of the first filtration membrane and the last filtration membrane.
  • the nominal pore size of the intermediate filtration membrane can be adjusted according to the characteristics of the cell culture mixture, for example, when using a three-layer filtration membrane.
  • the nominal pore size of the first filter membrane is about 2 ⁇ 10 ⁇
  • the nominal pore size of the second filter membrane is about 0.2 ⁇ 2 ⁇
  • the nominal pore size of the third filter membrane is about 0.1 ⁇ 0.2 ⁇ , using a multi-layer filter membrane. Structure, can achieve the purpose of deep filtration.
  • the first filter membrane with a nominal pore size of about 2 ⁇ 10 ⁇ can filter most of the coarse impurities, such as: cells, cell debris; filtered through layers, finally to achieve sterilization, removal of useful components of the mycoplasma level
  • the membrane is filtered to form a filtrate product of substantially the same quality, which product can be used for further post-treatment, such as: addition of adjuvant, freeze drying, sub-packaging, and the like.
  • the above filter membrane can be selected from all filter membranes of suitable pore size on the market today.
  • the material of the filter membrane is preferably a fiber bonded to an inorganic filter aid such as pearl carbon and diatomaceous earth and a binder, and the fiber may be selected from the group consisting of polypropylene fiber, glass fiber, cellulose acetate, nitrocellulose, and the like.
  • the mixed fibers of the fibers and the like may be the same or different materials of the respective filtration membranes, and the fibers preferably used are polypropylene fibers.
  • "nominal pore size" refers to the nominal pore size marked on the membrane product.
  • the multi-layer filter membrane may be a multi-layer filter membrane that is compounded together, or may be a single layer of appropriate pore size.
  • the multilayer filtration membrane formed by stacking the filtration membranes together may also be a filter membrane having a plurality of different pore size filtration layers integrally formed. As long as the relevant aperture requirements are met.
  • the amount of treatment can be determined according to the selection of the filtration membrane and the characteristics of the cell culture mixture.
  • the treatment amount is preferably about 100 to 200 L/(m 2 .h.l00 mbar), that is, 100 mbar (O.OlMpa).
  • the flow rate per square meter of filtration membrane under pressure differential is preferably about 120-180 L/(m 2 .h.100 mbar), more preferably about 140-170 L/(m 2 .h.100 mbar). In the range of 100 to 200 L/(m 2 .h.l00 mbar), a product of stable quality can usually be obtained. If it is too high, some undesired impurities may enter the filtrate, which may reduce the filtration effect; if it is too low, it is unfavorable for improving the filtration efficiency.
  • an animal virus culture mixture such as a foot-and-mouth disease virus culture mixture, a porcine reproductive and respiratory syndrome virus culture mixture, and a classical swine fever virus culture mixture is preferable.
  • filtration membranes may be appropriately selected. Specifically, for the culture mixture of the foot-and-mouth disease virus, since the virus has a diameter of about 20-25 nm and a small diameter, the filtration efficiency is improved. From the viewpoint of the above, it is preferred to use a double-layered filtration membrane having a nominal pore size of about 2 to ⁇ , and a second membrane membrane having a nominal pore diameter of about 0.1 to 0 ⁇ 2 ⁇ m.
  • the virus is about 50-60 nm in diameter, preferably using three layers of filter membrane.
  • the nominal pore size of the first membrane is about 2 ⁇ 10 ⁇ ⁇
  • the nominal pore size of the second membrane is about
  • the nominal pore size of the last filter membrane is about 0.1 ⁇ 0.2 ⁇ m.
  • the above filtration is preferably carried out by pressurizing before filtration and/or decompressing after filtration to increase the filtration rate.
  • a pressurized pump such as a peristaltic pump
  • a compressed gas is used to pressurize the culture mixture to pass through the filter membrane.
  • a vacuum pump such as a vacuum pump on the side of the filter to promote the passage of the liquid through the filter membrane.
  • the speed, without affecting the quality of the filtrate is preferably about 0.05-0.1 MPa, more preferably about 0.06-0.08 MPa, across the entire filter membrane (i.e., before and after filtration).
  • the pressure difference it can be appropriately adjusted according to the pore size of the filtration membrane and the number of filtration membranes, so as to maximize the filtration efficiency under the premise of ensuring the quality.
  • the filter membrane For the specific form of the filter membrane, it can be set as needed.
  • a filter in a folded form can be used, the filter includes a casing, and the folding type
  • the filter membrane divides the interior of the housing into a central zone and an edge zone.
  • the fluid to be filtered flows into the edge region of the casing, flows into the intermediate zone through the folded filter membrane, and the target product is obtained.
  • the filter area of such a folded form filter can be about 0.6 square meters - 1.8 square meters / 10 inches (0.6 m 2 ⁇ 1.8 m 2 /10"), such a filter area can achieve a large single processing capacity , up to hundreds of liters, which makes it easy to expand production scale.
  • Example 1 Separation and purification of BHK21 cells and foot-and-mouth disease virus culture mixture by deep filtration method BHK21 cells were recovered from the cell working seed liquid nitrogen pool, firstly passaged and expanded in a square bottle, followed by a spinner flask, a 5L bioreactor, and a 120L biological reaction.
  • the sequence of the apparatus was sequentially expanded and cultured, and then cell suspension was prepared by trypsin digestion, and inoculated into a CLAVORUSTM 650L reactor (Beijing Tianhe Rui Biotechnology Co., Ltd.) for large-scale expansion culture, and the working volume of the bioreactor was 400L, after 5 days of culture, the foot-and-mouth disease virus was inoculated on the obtained BHK21 cells, and the cell culture solution in the bioreactor was replaced with a virus culture solution, and the inoculated BHK21 cells were cultured, and the bioreactor was cultured for 12-24 hours, and then harvested once.
  • the harvested 400 L culture mixture was stored at a low temperature of 4 ° C in a liquid storage tank.
  • the obtained culture mixture has a virus concentration of about 4.5 g/mL, which contains BHK21 cells, BHK21 cell debris, proteins, nucleic acids, polysaccharides, bovine serum, etc., and the above culture mixture is divided into two equal parts, one for the following Experiment, another one for Comparative Example 1.
  • the nominal pore diameters of the layers of the filtration membrane were: 3 ⁇ , 0.2 ⁇ , and the total area of the filtration membrane was lm 2 .
  • the 200 L culture supernatant in the liquid storage tank is driven into the space between the filter housing and the filter element through the inlet pipe through the inlet pipe, and the supernatant liquid is filtered through the filter membrane step by step. All of the above 200 L culture mixture was processed in minutes to obtain a filtrate, during which the pressure difference across the filtration membrane was increased from about 0.05 MPa to about 0.06 MPa.
  • the liquid in the collection container is collected for further use in downstream reactions.
  • the virus concentration in the obtained filtrate is about 3.7 g/mL, which has an 80% recovery rate compared with the culture medium, wherein no cell is identified, no bacteria, mold, or mycoplasma contamination, and the removal rate of the impurity protein reaches 99%.
  • DNA content ⁇ 100 pg/mL, endotoxin content ⁇ 10 EU/ml.
  • Example 2 Separation and purification of Marcl45 cells and porcine reproductive and respiratory syndrome virus (Blue ear virus) culture mixture by depth filtration
  • the Marcl45 cells were recovered from the cell working seed liquid nitrogen pool, firstly passaged and expanded in a square bottle, and then sequentially expanded and cultured in the order of a spinner flask, a 5 L bioreactor, and a 120 L bioreactor, followed by trypsin digestion to prepare cells.
  • the suspension was inoculated in a CLAVORUSTM 650L reactor (Beijing Tianhe Rui Biotechnology Co., Ltd.) for large-scale expansion culture.
  • the working volume of the bioreactor was 300 L. After 4 days of culture, the resulting Marl45 cells were inoculated with pigs.
  • Respiratory Syndrome Virus replace the cell culture medium in the bioreactor with the virus culture solution, culture the inoculated Marcl45 cells, start perfusion culture after 6 hours of virus culture, and start harvesting the virus solution from the first day, and harvest the harvested virus.
  • the 800 L culture mixture was stored at 4 ° C in a liquid storage tank at a low temperature.
  • the obtained culture mixture contains Marc 145 cells, Marc 145 cell debris, protein, nucleic acid, polysaccharide, bovine serum and the like in addition to virus, and the above culture mixture is divided into two equal portions, one for the following experiment, and the other Used for Comparative Example 2.
  • the nominal pore diameter of each layer of the filter membrane is: 8 ⁇ , 0.45 ⁇ , 0.1 ⁇ .
  • the operating pressure difference is 0.05-0.08 MPa. All cultures were processed in 30 minutes. It can achieve a recovery rate of about 83%, no cell, no bacteria, fungi, mycoplasma contamination, 99% protein removal rate, residual DNA content ⁇ 100 8/11 ⁇ , endotoxin content ⁇ 10EU/ml.
  • the above indicators are tested for bacteria and mycoplasma according to the sterility test method and mycoplasma test method in the third appendix of the 2005 edition of the Veterinary Pharmacopoeia of the People's Republic of China.
  • the content of the protein is determined by enzyme-linked immunosorbent assay and the nucleic acid content is determined by spectrometry. , gel method to determine endotoxin content. From the above test results, it can be seen that the obtained filtrate only needs to be subjected to the steps of virus inactivation, vaccine emulsification and vaccine packaging, thereby obtaining a safe vaccine product which can be directly used for injection.
  • the other 200 L culture mixture obtained in Example 1 was directly filtered through a single-layer filtration membrane having a nominal pore diameter of 0.45 ⁇ m. During the period, the pressure difference between the two sides of the filtration membrane was increased from 0.05 MPa to 0.5 MPa, and only 100 L of the culture mixture was processed. Complete purification separation.
  • the properties of the liquid collected in the collection container are: viral particles, residual DNA, protein, endotoxin, wherein the residual DNA content is 500. Pg/mL, the impurity protein removal rate was only 50%, and the endotoxin content was 30 EU/ml. The virus recovery rate is only 25%.
  • the above indicators are also tested for bacteria and mycoplasma according to the sterility test method and mycoplasma test method in the third appendix of the 2005 edition of the Veterinary Pharmacopoeia of the People's Republic of China.
  • the determination of the content of heteroproteins by enzyme-linked immunosorbent assay and the determination of nucleic acids by spectrometry The content of the endotoxin was determined by gel method.
  • Example 2 The other 400 L Marc 145 cells obtained in Example 2 and the porcine reproductive and respiratory syndrome virus culture mixture were directly filtered through a single-layer filtration membrane having a nominal pore size of 0.1 ⁇ , and the membrane was clogged only for 20 minutes, and the entire purification separation could not be completed.

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Description

细胞培养混合物的分离纯化方法 技术领域
本发明涉及一种生物制品的分离纯化的方法， 特别涉及用以获得目的生 物制品的细胞培养混合物的分离纯化方法。
背景技术
目的生物制品的培养混合物的分离纯化是生物制品生产中最关键步骤之 一， 它是收获目的培养混合物与进一步下游加工之间的枢纽步骤， 对生物制 品的产量、 质量、 可重复性有着直接的影响， 也决定了生物制品整体生产工 艺的效率和成本耗费。
现有生物制品生产工艺中常用的目的培养混合物分离纯化方法多为如离 心、 凝胶层析、 过滤等几种方法的联合使用， 多存在着许多问题， 首先培养 混合物在设备之间的转移容易造成产品的污染， 其次， 所使用的设备单批次 处理量较少， 不易进行生产规模扩大， 再次， 所用设备重复使用需进行反复 清洗， 增加了劳动力与生产成本的耗费。 另外， 现有工艺常常不能完全分离 纯化目的培养混合物， 造成最终的生物制品异源物质比较多。
目前， 人类、 家禽、 家畜的常规和非常规免疫是保障人类生命、 健康的 重要手段， 所以各种病毒疫苗的需求量也在不断地增加。 而大多数病毒疫苗 都是通过细胞培养后分离纯化获得的， 所以对于高效率、 高品质地分离纯化 细胞培养混合物的需求越来越受到人们的关注。
发明内容
使用现有的技术手段来分离纯化细胞培养混合物， 由于存在上述问题， 分离纯化过程复杂， 不能大量、 快速且高纯度地分离纯化细胞培养混合物， 造成分离纯化的成本较高， 生产时间长等缺陷， 另外， 最终的生物制品异源 物质比较多， 使用时副反应比较大。 可见， 现有的分离纯化方法已无法满足 现实生产、 生活的需要。
因此， 本发明特别针对细胞培养混合物的分离提纯进行了潜心研究， 发 现细胞培养混合物具有组分包含复杂、 目的产物的含量普遍较低、 起始用量 较大、 不同病毒颗粒大小具有一定差异等特点。 对于细胞培养混合物这类生物制品， 本发明人开创性地提出了对其进行 分离纯化的新方法， 克服了上述缺陷。
本发明提供了一种细胞培养混合物的分离纯化方法， 1. 一种细胞培养 混合物的分离纯化方法， 包括使细胞培养混合物经过滤器进行过滤， 该过滤 器具有至少两层孔径不同的过滤膜， 过滤膜的各层膜的公称孔径在滤液流动 方向上逐级变小， 而且第一层过滤膜的公称孔径约为 2 ~ ΙΟμηι, 最后一层过 滤膜的公称孔径约为 0.1 ~ 0.2μηι。
2. 根据技术方案 1 所述的细胞培养混合物的分离纯化方法， 其中， 细 胞培养混合物选自动物用病毒细胞培养混合物、 人用病毒细胞培养混合物或 者分泌抗体的杂交瘤细胞培养混合物。
3. 根据技术方案 1 所述的细胞培养混合物的分离纯化方法， 其中， 所 述过滤器具有三层、 四层或五层过滤膜。
4. 根据技术方案 1所述的细胞培养混合物的分离纯化方法， 其中， 在 过滤过程中， 过滤膜前后的压差约为 0.05-0.1MPa。
5. 根据技术方案 1至 4任一项所述的细胞培养混合物的分离纯化方法， 其中， 所述过滤器是过滤膜为折叠形式的过滤器。
6. 根据技术方案 5 所述的细胞培养混合物的分离纯化方法， 其中， 所 述过滤器设置有
壳体；
折叠式的过滤膜将壳体内部分为中央区和边缘区。
7. 根据技术方案 1至 4任一项所述的细胞培养混合物的分离纯化方法， 其中， 所以细胞培养混合物选自： 口蹄疫病毒培养混合物， 猪繁殖与呼吸综 合征病毒培养混合物， 猪瘟病毒培养混合物， 狂犬病毒培养混合物， 乙型肝 炎病毒培养混合物， 流行性乙型脑炎病毒培养混合物， 曱型肝炎病毒培养混 合物或者脊髓灰质炎病毒培养混合物。
8. 根据技术方案 1至 4任一项所述的细胞培养混合物的分离纯化方法， 其中， 待过滤液的处理量为： 每 O.OlMpa压差下每平方米过滤膜的流量范围 约为 100~200L。
9. 根据技术方案 1所述的细胞培养混合物的分离纯化方法， 其中， 所 述细胞培养混合物是口蹄疫病毒培养混合物， 所述过滤膜是两层过滤膜， 第 一层过滤膜的公称孔径约为 2 ~ 10μηι, 第二层过滤膜的公称孔径约为 0.1 ~ 0·2μηι。
10. 根据技术方案 1所述的细胞培养混合物的分离纯化方法， 其中， 所 述细胞培养混合物是猪繁殖与呼吸综合征病毒培养混合物， 所述过滤膜是三 层过滤膜， 第一层过滤膜的公称孔径约为 2 ~ 10μηι, 第二层过滤膜的公称孔 径约为 0.2 ~ 2μηι, 第三层过滤膜的公称孔径约为 0.1 ~ 0.2μηι。
本发明的一种细胞培养混合物的分离纯化方法， 具有针对性强， 处理量 大、 分离纯化步骤筒单、 产品质量稳定、 成本低等优点， 特别适合用于分离 纯化口蹄疫病毒培养混合物或猪繁殖与呼吸综合征病毒培养混合物等， 得到 纯度较高的病毒疫苗混合物。
另外， 本发明细胞培养混合物的分离纯化方法中， 可达到较大的单次处 理量， 高至几百升， 从而易于扩大生产规模； 并且， 本发明细胞培养混合物 的分离纯化方法对目的物分离纯化较为彻底， 产物中的细胞碎片及其他较大 物质均被截留， 从而减少了生物制品的免疫接种副反应， 减少了目的培养物 的转移暴露， 从而减少了目的物被污染的几率。
本发明细胞培养混合物的分离纯化方法过程控制筒易， 使用过滤元件可 以为一次性使用产品， 无清洗、 清洗验证等过程， 节省劳动力和生产成本， 提高生产效率；
本发明细胞培养混合物的分离纯化方法过程控制筒易， 可以根据不同分 离纯化目的物选择不同大小过滤元件及其组合， 使用灵活， 应用范围广泛。 具体实施方式
本发明中的术语 "动物"， 指的是非人类的各种动物， 特别优选的是畜类 或禽类动物， 比如： 猪、 狗、 牛、 羊、 鸡、 鸭和 /或鹅等。
本发明中的术语 "细胞培养混合物"， 是指在现有的条件下培养病毒、抗 体等所获得的细胞培养液混合物。 具体地讲， 所述细胞培养混合物， 包括动 物用病毒细胞培养混合物、 人用病毒细胞培养混合物及分泌抗体的杂交瘤细 胞培养混合物。 其中所述细胞培养混合物中主要含有病毒颗粒（或抗体）， 以 及培养细胞、 培养细胞碎片、 蛋白质、 核酸、 多糖等杂质， 如果所用的细胞 培养基为含血清细胞培养基， 则所得混合物中还含有残余血清。 所得滤出液 中要求具有较高含量的目的病毒， 而所含杂质例如宿主蛋白、 宿主核酸等尽 量控制在较低水平， 无菌类污染。
病毒直径通常在 20nm-200nm之间， 而针对不同的病毒， 采用不同的细 胞进行培养， 发明人仔细地研究了不同病毒及抗体的细胞培养混合物， 所述 动物用病毒细胞培养混合物具体如下： 口蹄疫病毒培养混合物， 病毒直径约 20-25nm, 常用培养细胞为幼仓鼠肾传代细胞（ BHK21细胞）； 猪繁殖与呼吸 综合征病毒培养混合物， 病毒直径约 50-60nm, 常用培养细胞为 Marcl45细 胞； 猪瘟病毒培养混合物， 病毒直径约 70nm, 常用培养细胞为牛睾丸原代细 胞（ BT细胞）、 猪肾细胞（ PK细胞）及猪睾丸细胞（ ST细胞）； 狂犬病毒培 养混合物， 病毒长约 170-180nm, 宽约 75-80nm, 常用的培养细胞为 BHK21。
所述人用病毒细胞培养混合物如下： 狂犬病毒培养混合物， 病毒长约 170-180nm, 宽约 75-80nm, 常用的培养细胞为非洲绿猴肾细胞（ Vera细胞）； 乙型肝炎病毒培养混合物， 病毒直径约 22nm, 常用的培养细胞为中国仓鼠卵 巢细胞（CHO细胞）； 流行性乙型脑炎病毒培养混合物， 病毒直径约 40nm, 常用的培养细胞为 Vera 细胞； 曱型肝炎病毒培养混合物， 病毒直径约 27-32nm, 常用的培养细胞为人二倍体细胞 KMB17或 2BS; 脊髓灰质炎病毒 培养混合物， 病毒直径约 27-30nm, 常用的培养细胞为人二倍体细胞 2BS。
由上述可知， 大多数病毒的直径约为 20-100nm, 个别病毒的直径约在 100-200nm之间。
对细胞培养混合物进行分离得到的目标混合物要求无污染物， 并且杂质 的含量应尽量控制到最低水平， 其中所述污染物如细胞、 细菌、 霉菌和 /或支 原体等， 所述杂质如杂蛋白、 核酸和 /或牛血清等， 不同生物制品对此有不同 的要求和限制。
发明人发现各种污染物的直径与病毒的直径相比， 均存在相当的差异， 而在细胞培养混合物中， 动物细胞的直径通常约为 10-100μηι, 细菌细胞直径 通常约为 0.5-2.0μηι, 真菌细胞直径通常约为 10-40μηι, 支原体直径通常约为 0.2-0.3μηι。 因此， 通过选择恰当过滤条件， 可以大大地提高过滤效率， 从而 减少得到最终产物的步骤， 提高生产效率， 降低成本。 本发明提供了一种细胞培养混合物的分离纯化方法， 该方法包括将细胞 培养混合物经双层或多层过滤膜进行过滤， 得到分离纯化好的滤出液。 该双 层或多层过滤膜的各层膜的公称孔径在过滤方向上逐级变小， 比如二层、 三 层、 四层、 五层或六层， 优选为三层、 四层或五层过滤膜， 根据细胞培养混 合物的病毒与细胞的特点， 第一层过滤膜的公称孔径约为 2 ~ ΙΟμηι, 最后一 层过滤膜的公称孔径约为 0.1~0.2μηι, 如果第一层过滤膜的公称孔径小于 2μηι, 那么较多污染物或杂质会很快堵塞各孔道， 从而使过滤无法顺利进行， 如果第一层过滤膜的公称孔径大于 ΙΟμηι,那么有部分细胞等污染物会通过第 一层过滤膜， 从而积存在第二层过滤膜上， 从而使过滤效率变低； 该如果最 后一层过滤膜的公称孔径小于 Ο.ΐμηι, 会增加过滤阻力， 不利于提高过滤效 率， 如果大于 0.2μηι, 会使最终滤出液的杂质含量太多， 无法达到所要求的 纯度。 第一层过滤膜可以将较大直径的污染物或杂质过滤掉， 最后一层是尽 量将小直径的污染物与杂质过滤掉， 根据细胞培养液的特点， 逐层过滤， 从 而得到纯度较高的病毒滤出液。
中间的过滤膜的公称孔径介于第一层过滤膜与最后一层过滤膜的公称孔 径之间， 中间过滤膜的公称孔径可以根据细胞培养混合物的特点进行调整， 比如在使用三层过滤膜时， 第一层过滤膜的公称孔径约为 2 ~ 10μηι, 第二层 过滤膜的公称孔径约为 0.2 ~ 2μηι,第三层过滤膜的公称孔径约为 0.1 ~ 0.2μηι, 使用多层过滤膜的结构， 可以达到深层过滤的目的。 公称孔径约为 2 ~ 10μηι 的第一层过滤膜可以过滤大部分较粗大的杂质， 比如： 细胞、 细胞碎片； 经 过层层逐级过滤， 最终达到除菌、 去除支原体级别的有用组分滤过过滤膜， 形成质量基本达标的过滤液产物， 该产物可以用于进一步后处理， 比如： 加 入佐剂、 冷冻干燥、 分包装等。
上述过滤膜可以选用现在市场上的所有孔径合适的过滤膜。 过滤膜的材 料优选为纤维与无机助滤剂如珍珠碳和硅藻土以及粘接剂粘合而成， 所述纤 维可以选自聚丙烯纤维、 玻璃纤维、 醋酸纤维素、 硝酸纤维素以及以上纤维 的混合纤维等， 各层过滤膜的材料可以相同或不同， 优选使用的纤维为聚丙 烯纤维。 在本文中 "公称孔径" 指的过滤膜产品上标注的公称孔径。
多层过滤膜可以是复合在一起的多层过滤膜， 也可以是适当孔径的单层 过滤膜叠置在一起而形成的多层过滤膜， 也可以是一体制成的具有多个不同 孔径过滤层的过滤膜。 只要符合相关的孔径要求即可。
处理量可以根据过滤膜的选择与细胞培养混合物的特点来确定， 对于以 上例举的细胞培养混合物， 优选处理量为约 100~200L/(m2.h.l00mbar) , 即 lOOmbar ( O.OlMpa ) 压差下每平方米过滤膜的流量范围， 优选约 120-180L/(m2.h.l00mbar) , 进一步优选约 140~170L/(m2.h.l00mbar)。 在该 100~200L/(m2.h.l00mbar)范围内， 通常可以得到质量稳定的产品。 如果太高， 部分不希望的杂质可能会进入过滤液， 使过滤效果降低； 如果太低， 对于提 高过滤效率不利。
对于上述细胞培养混合物， 优选为动物用病毒培养混合物， 例如： 口蹄 疫病毒培养混合物、 猪繁殖与呼吸综合征病毒培养混合物、 猪瘟病毒培养混 合物。
根据动物用病毒培养混合物中所培养的病毒的不同， 可以适当选择不同 的过滤膜， 具体地， 对于为口蹄疫病毒培养混合物， 由于其病毒直径约 20-25nm,直径较小，所以从提高过滤效率的角度考虑，优选使用双层过滤膜， 第一层过滤膜的公称孔径约为 2 ~ ΙΟμηι,第 2层过滤膜的公称孔径约为 0.1 ~ 0·2μηι。
对于猪繁殖与呼吸综合征病毒培养混合物， 病毒直径约 50-60nm, 优选 使用 3层过滤膜， 第一层过滤膜的公称孔径约为 2 ~ 10 μ ηι, 第二层过滤膜的 公称孔径约为 0.2 ~ 2μηι, 最后一层过滤膜的公称孔径约为 0.1 ~ 0.2 μ m。
上述过滤优选在过滤前加压和 /或在过滤后减压的方式来提高过滤速度。 比如使用加压泵（如蠕动泵）或压缩气对培养混合物进行加压， 使其通过过 滤膜， 也可以使用在过滤后一侧设置真空泵等减压设备， 促使液体通过过滤 膜， 为了提高过滤速度， 又不影响滤出液的质量， 整个过滤膜两侧 （即过滤 前后）的压差优选约为 0.05-0.1MPa, 更优选约 0.06-0.08MPa。 当然， 对于该 压差， 可以根据过滤膜的孔径以及过滤膜的数量进行适当调整， 以在保证质 量的前提下， 最大可能地提高过滤效率为准。
对于过滤膜的具体形式， 可以根据需要进行设置， 为了增加使单位体积 的过滤膜的面积， 可以使用折叠形式的过滤器， 过滤器包括壳体， 折叠式的 过滤膜将壳体内部分为中央区和边缘区。 待过滤的流体流入壳体的边缘区， 经过折叠式的过滤膜流入中间区， 从而得到目标产物。 这样的折叠形式的过 滤器的过滤面积可以约为 0.6平方米 -1.8平方米 /10英寸（0.6m2 ~ 1.8m2/10" ), 这样的过滤面积， 可达到较大的单次处理量， 高至上百升， 从而易于扩大生 产规模。
为了进一步清晰地说明本发明的以上实施方式， 提供了以下实施例。 该 实施例仅为了进一步说明本发明的方法，对本发明的保护范围不起限定作用。
实施例 1 用深层过滤法分离纯化 BHK21细胞、 口蹄疫病毒培养混合物 从细胞工作种子液氮库中复苏 BHK21 细胞， 先在方瓶中传代扩增， 接 着通过转瓶、 5L 生物反应器、 120L生物反应器的顺序依次扩增培养， 之后 用胰蛋白酶消化制成细胞悬液， 接种于 CLAVORUS™ 650L反应器（北京天 和瑞生物科技公司）中进行大规模扩增培养，生物反应器的工作体积为 400L, 培养 5天后，在所得的 BHK21细胞上接种口蹄疫病毒，将生物反应器中的细 胞培养液更换为病毒培养液， 培养接种后的 BHK21 细胞， 生物反应器培养 12 - 24hr左右后一次性收获病毒液，将所收获的 400L培养混合物 4°C低温储 存于储液罐中。 所得培养混合物中病毒浓度约为 4.5 g/mL, 其中含有 BHK21 细胞、 BHK21细胞碎片、 蛋白、 核酸、 多糖以及牛血清等， 将上述培养混合 物分为等量的两份， 一份用于下述实验， 另一份用于对比例 1。
将三层不同孔径过滤膜组装至过滤器中，过滤膜各层的公称孔径分别为： 3μηι, 0.2μηι, 过滤膜总面积为 lm2。 利用无菌压缩空气将储液罐中的 200L 培养上清液经连接管道通过进液口打入过滤器壳体与过滤元件之间的空间 中， 上清液通过滤膜逐级过滤， 经 20分钟处理完所有上述 200L培养混合物， 得到滤出液， 期间过滤膜两侧的压差从约 0.05 MPa增加到约 0.06MPa。 收集 收集容器中的液体， 可进一步用于下游反应。 所得滤出液中病毒浓度约为 3.7 g/mL, 与培养液相比具有 80%的回收率， 其中经鉴定无细胞， 无细菌、 霉菌、 支原体的污染， 杂蛋白去除率达到 99%, 残余 DNA含量 <100 pg/mL, 内毒素含量 <10EU/ml。
以上各项指标， 按照中华人民共和国兽药典二零零五年版第三部附录中 无菌检验法、 支原体检验法进行细菌、 支原体检验， 利用酶联免疫法测定杂 蛋白含量， 光谱法测定核酸含量， 凝胶法测定内毒素含量。 从以上检测结果 可以看出， 所得滤出液仅需要再经病毒灭活、 疫苗乳化和疫苗包装的步骤， 就能够获得可直接用于注射的安全的疫苗产品。
实施例 2 用深层过滤法分离纯化 Marcl45细胞与猪繁殖与呼吸综合征病 毒（蓝耳病病毒）培养混合物
从细胞工作种子液氮库中复苏 Marcl45细胞， 先在方瓶中传代扩增， 接 着通过转瓶、 5L 生物反应器、 120L生物反应器的顺序依次扩增培养， 之后 用胰蛋白酶消化制成细胞悬液， 接种于 CLAVORUS™ 650L反应器（北京天 和瑞生物科技公司）中进行大规模扩增培养，生物反应器的工作体积为 300L, 培养 4天后， 在所得的 Marcl45细胞上接种猪繁殖与呼吸综合征病毒， 将生 物反应器中的细胞培养液更换为病毒培养液， 培养接种后的 Marcl45细胞， 病毒培养 6hr后开始灌注培养， 并从第一天起开始收获病毒液， 将所收获的 800L培养混合物 4°C低温储存于储液罐中。 所得培养混合物中除病毒外含有 Marc 145 细胞、 Marc 145 细胞碎片、 蛋白、 核酸、 多糖以及牛血清等， 将上 述培养混合物分为等量的两份， 一份用于下述实验， 另一份用于对比例 2。
过滤膜各层的公称孔径分别为： 8μηι, 0.45μηι, 0.1μηι。 操作压力差为 0.05-0.08MPa。 30分钟即处理完所有培养液。 可达到约 83%的回收率， 无细 胞、 无细菌、 真菌、 支原体的污染， 杂蛋白去除率达到 99%, 残余 DNA含 量<100 8/11^ , 内毒素含量 <10EU/ml。
以上各项指标， 按照中华人民共和国兽药典二零零五年版第三部附录中 无菌检验法、 支原体检验法进行细菌、 支原体检验， 利用酶联免疫法测定杂 蛋白含量， 光谱法测定核酸含量， 凝胶法测定内毒素含量。 从以上检测结果 可以看出， 所得滤出液仅需要再经病毒灭活、 疫苗乳化和疫苗包装的步骤， 就能够获得可直接用于注射的安全的疫苗产品。
对比例 1
将实施例 1中得到的另外 200L培养混合物通过公称孔径为 0.45μηι的单 层过滤膜直接过滤， 经期间过滤膜两侧的压差从 0.05MPa增加到 0.5MPa, 仅 处理 100L的培养混合物，无法完成全部纯化分离。收集收集容器中的液体的 性质为： 病毒颗粒、 残留 DNA、 蛋白质， 内毒素， 其中残留 DNA含量 500 pg/mL,杂蛋白去除率仅 50%,内毒素含量为 30EU/ml。病毒回收率仅为 25%。 以上各项指标， 同样按照中华人民共和国兽药典二零零五年版第三部附录中 无菌检验法、 支原体检验法进行细菌、 支原体检验， 利用酶联免疫法测定杂 蛋白含量， 光谱法测定核酸含量， 凝胶法测定内毒素含量。
对比例 2
将实施例 2中得到的另外 400L Marc 145细胞与猪繁殖与呼吸综合征病毒 培养混合物， 通过公称孔径为 0.1 μηι的单层过滤膜直接过滤， 仅 20min过滤 膜即堵塞， 无法完成全部纯化分离。

Claims

权利 要求 书
1. 一种细胞培养混合物的分离纯化方法， 包括使细胞培养混合物经过滤 器进行过滤， 该过滤器具有至少两层孔径不同的过滤膜， 过滤膜的各层膜的 公称孔径在滤液流动方向上逐级变小， 而且第一层过滤膜的公称孔径为 2 ~ ΙΟμηι, 最后一层过滤膜的公称孔径为 0.1 ~ 0.2μηι。
2. 根据权利要求 1所述的细胞培养混合物的分离纯化方法， 其中， 细胞 培养混合物选自动物用病毒细胞培养混合物、 人用病毒细胞培养混合物或者 分泌抗体的杂交瘤细胞培养混合物。
3. 根据权利要求 1所述的细胞培养混合物的分离纯化方法， 其中， 所述 过滤器具有三层、 四层或五层过滤膜。
4. 根据权利要求 1所述的细胞培养混合物的分离纯化方法， 其中， 在过 滤过程中， 过滤膜前后的压差为 0.05-0.1MPa。
5. 根据权利要求 1至 4任一项所述的细胞培养混合物的分离纯化方法， 其中， 所述过滤器是过滤膜为折叠形式的过滤器。
6. 根据权利要求 5所述的细胞培养混合物的分离纯化方法， 其中， 所述 过滤器设置有
壳体；
折叠式的过滤膜将壳体内部分为中央区和边缘区。
7. 根据权利要求 1至 4任一项所述的细胞培养混合物的分离纯化方法， 其中， 所述细胞培养混合物选自： 口蹄疫病毒培养混合物， 猪繁殖与呼吸综 合征病毒培养混合物， 猪瘟病毒培养混合物， 狂犬病毒培养混合物， 乙型肝 炎病毒培养混合物， 流行性乙型脑炎病毒培养混合物， 曱型肝炎病毒培养混 合物或者脊髓灰质炎病毒培养混合物。
8. 根据权利要求 1至 4任一项所述的细胞培养混合物的分离纯化方法， 其中， 待过滤液的处理量为： 每 O.OlMpa压差下每平方米过滤膜的流量范围 为 100~200L。
9. 根据权利要求 1所述的细胞培养混合物的分离纯化方法， 其中， 所述 细胞培养混合物是口蹄疫病毒培养混合物， 所述过滤膜是两层过滤膜， 第一 层过滤膜的公称孔径为 2 ~ ΙΟμηι, 第二层过滤膜的公称孔径为 0.1 ~ 0.2μηι。
10. 根据权利要求 1所述的细胞培养混合物的分离纯化方法， 其中， 所述 细胞培养混合物是猪繁殖与呼吸综合征病毒培养混合物， 所述过滤膜是三层 过滤膜， 第一层过滤膜的公称孔径为 2 ~ 10μηι, 第二层过滤膜的公称孔径为 0.2 ~ 2μηι, 第三层过滤膜的公称孔径为 0.1 ~ 0.2μηι。
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