WO2020259532A1 - 一种生产病毒的方法及收获液组合物 - Google Patents

一种生产病毒的方法及收获液组合物 Download PDF

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WO2020259532A1
WO2020259532A1 PCT/CN2020/097901 CN2020097901W WO2020259532A1 WO 2020259532 A1 WO2020259532 A1 WO 2020259532A1 CN 2020097901 W CN2020097901 W CN 2020097901W WO 2020259532 A1 WO2020259532 A1 WO 2020259532A1
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virus
cells
harvest
cell
liquid composition
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PCT/CN2020/097901
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French (fr)
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柳云帆
王文波
张立明
陈茜
康三毛
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杭州康万达医药科技有限公司
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Priority to US17/596,732 priority Critical patent/US20220267713A1/en
Priority to EP20832620.7A priority patent/EP3992282A4/en
Priority to JP2021576078A priority patent/JP2022539511A/ja
Priority to CN202080036017.6A priority patent/CN113874495A/zh
Publication of WO2020259532A1 publication Critical patent/WO2020259532A1/zh

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
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    • C12N2710/16011—Herpesviridae
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    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
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    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • C12N2710/24011—Poxviridae
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    • C12N2710/24151—Methods of production or purification of viral material
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    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16051—Methods of production or purification of viral material

Definitions

  • the present invention relates to the field of biotechnology, in particular, to a method for producing viruses and a harvest liquid composition, and more specifically, to a method for producing viruses through cell culture, and for harvesting cells cultured Virus harvest liquid composition.
  • Viral biological products including oncolytic virus drugs, viral vaccines or recombinant viral vectors, generally include: first inoculating the virus seed on uninfected cells of the appropriate generation, and harvesting when a certain degree of cytopathic disease is reached After infecting the cells, lysing the cells in different ways, harvesting the virus, and then purifying by density gradient centrifugation or chromatography to obtain the original solution, adding a certain buffer solution to become a semi-finished product, and the semi-finished product is lyophilized or aliquoted into a finished product.
  • vaccinia virus, herpes simplex virus, varicella-zoster virus, adenovirus, etc. belong to intracellular viruses and have strong cell binding activity, the cells must be collected and broken during virus harvesting to obtain free virus particles.
  • Recombinant viral vectors based on lentivirus, retrovirus, and adeno-associated virus At present, in the actual packaging and production process, only free virus outside the cell is harvested, and a large amount of intracellular virus is not fully utilized.
  • freeze-thaw methods are used to lyse cells to harvest intracellular viruses.
  • the freeze-thaw method has the disadvantages of high energy consumption, long cycle, multiple steps, difficult to scale up, and unstable yield, which limits the production scale of viral biological products.
  • hypotonic or chemical lysis to harvest intracellular viruses, but a large number of viruses bound to the cell membrane are not fully released, and many chemical lysis reagents can also destroy enveloped viruses (such as vaccinia virus, herpes simplex virus, etc.) The envelope makes the virus lose its biological activity.
  • the present invention provides a method for producing viruses and a harvest liquid composition.
  • the present invention provides:
  • a method of producing a virus comprising:
  • Culturing cells wherein the cells are cells that have been inoculated with viruses or have been transfected with viral packaging elements;
  • the cultured cells are contacted with the harvesting fluid composition to harvest the virus in one step, wherein the harvesting fluid composition comprises: pancreatin, a pH buffer and an optional nuclease, and the pH of the harvesting fluid composition is greater than 7.5 And does not exceed 10.5.
  • the concentration of the nuclease is 1-100 IU/ml, preferably 1-50 IU/ml, It is also preferably 1-5 IU/ml.
  • the osmotic pressure of the harvest liquid composition ranges from 0-50 mOsmol/kg or 800-2500 mOsmol/kg, preferably 0-20 mOsmol/kg or 1785-2000 mOsmol/kg, more preferably 1-20 mOsmol/kg.
  • the virus includes: vaccinia virus, varicella-zoster virus, rotavirus, EV71 virus, hepatitis A virus, simple Herpes virus, lentivirus, retrovirus, adenovirus, adeno-associated virus, measles virus, Semliki forest virus, vesicular stomatitis virus, polio virus, reovirus, vesicular stomatitis virus, spinal cord
  • the poliovirus, Seneca Valley virus, Echo-type enterovirus, Coxsackie virus, Newcastle disease virus and Malaba virus are preferably enveloped viruses.
  • the harvest liquid composition comprises Tris salt buffer, pancreatin and nuclease
  • concentration of the Tris salt buffer is 1-50 mM, preferably 1-10 mM.
  • a harvesting fluid composition for harvesting cell-cultured viruses characterized in that the harvesting fluid composition comprises: pancreatin, a pH buffer and an optional nuclease, and the harvesting fluid composition
  • the pH of the substance is greater than 7.5 and not more than 10.5.
  • the virus comprises: vaccinia virus, varicella-zoster virus, rotavirus, EV71 virus, hepatitis A Virus, herpes simplex virus, lentivirus, retrovirus, adenovirus, adeno-associated virus, measles virus, Semliki forest virus,
  • the present invention has the following advantages and positive effects:
  • the present invention proposes for the first time a one-step harvesting method for virus production, so that in the virus production method of the present invention, by contacting the cultured cells with the harvesting liquid composition of the present invention, the virus can be harvested in one step.
  • the inventors of the present invention have obtained the harvesting fluid composition of the present invention through a lot of research.
  • the harvesting fluid composition can completely lyse the cells and harvest the virus, so there is no need to implement other cell lysis methods such as freezing and thawing, ultrasound, and mechanical disruption. step.
  • the use of the method and the harvest liquid composition of the present invention can greatly simplify the production process, reduce the production cost, and can further obtain high-concentration, high-purity, and high-activity virus products.
  • Figure 1 shows the results of harvesting viruses with different treatment solutions in an embodiment of the present application.
  • the abscissa represents different groups, and the ordinate represents the average yield per unit area (pfu/cm 2 ) of the virus.
  • the virus production method of the present invention includes: culturing cells, wherein the cells are cells that have been inoculated with viruses or transfected with virus packaging elements; and contacting the cultured cells with the harvesting liquid composition of the present invention to harvest the virus in one step.
  • the harvesting fluid composition of the present invention includes pancreatin, a pH buffer and optional nuclease, and the pH of the harvesting fluid composition is greater than 7.5 and not greater than 10.5.
  • the “element for virus packaging” mentioned herein refers to the cis-acting element and trans-acting element required for the packaging of lentivirus, retrovirus, adeno-associated virus, etc. by recombinant viral vectors. ).
  • the "one-step virus harvesting" as used herein means that when cells obtained by culture are in contact with the harvesting fluid composition of the present invention, the harvesting fluid composition can completely lyse the cells and harvest the virus, so there is no need to perform freezing and thawing, Ultrasound, mechanical disruption and other cell lysis methods or steps.
  • the inventor of the present invention found that if only the hypotonic buffer in the prior art is used, the purpose of harvesting the virus in one step cannot be achieved, and the virus yield is still low; but the inventor of the present invention unexpectedly found that the use of the present invention When the inventive harvesting liquid composition is used, the virus can be harvested in one step, and the virus yield is significantly improved. Compared with the prior art method, the virus yield obtained by the present invention can be increased by more than 5 times.
  • the concentration of pancreatin may be 0.01-0.12% (the% is the mass volume ratio (w/v), that is, the number of grams of pancreatin contained in 100 ml of the harvest liquid composition 0.01-0.12g), such as 0.03, 0.05, 0.07, 0.09, 0.11% (w/v), preferably 0.03-0.06% (w/v).
  • the pancreatin may be commercially available, and may be traditional pancreatin derived from animals, or recombinant pancreatin.
  • the concentration of the nuclease may be 1-100 IU/ml, for example 1, 3, 5, 10, 20, 40, 60, 80 IU/ml, preferably 1 -50IU/ml, preferably 1-5IU/ml.
  • a certain concentration of Mg 2+ (such as 1-10 mM Mg 2+ , preferably 1-2 mM Mg 2+ ; for example, 1 mM MgCl 2 ) can be added to make the nuclease work.
  • the osmotic pressure of the harvest liquid composition can range from 0-50 mOsmol/kg (for example, 1, 5, 10, 15, 30, 45 mOsmol/kg) or 800-2500 mOsmol/kg (for example, 1000, 1250, 1500, 1700, 1900, 2100, 2300mOsmol/kg).
  • the osmotic pressure of the harvest liquid composition is 0-20 mOsmol/kg or 1785-2000 mOsmol/kg, more preferably 1-20 mOsmol/kg, further preferably 1-10 mOsmol/kg.
  • the pH of the harvest liquid composition is alkaline, more specifically greater than 7.5 and not more than 10.5, such as 7.6, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, preferably 8.5-9.5.
  • pH of the harvest liquid composition is less than or equal to 7.5, cell lysis is incomplete and the virus yield is low; when the pH of the harvest liquid composition is greater than 10.5, the virus activity is affected.
  • the pH buffer in the harvest liquid composition can be selected from Tris salt buffer, sodium bicarbonate buffer; preferably Tris salt buffer.
  • the harvest fluid composition of the present invention includes Tris salt buffer, pancreatin, and nuclease.
  • concentration of the Tris salt buffer may be 1-50 mM, for example 1, 3, 5, 8, 10, 20, 30, 40 mM, preferably 1-10 mM.
  • the amount of the harvest liquid composition can be 35 ⁇ l or more of the harvest liquid composition/cm 2 cell, preferably 70 ⁇ l or more of the harvest liquid composition/cm 2
  • the cells are, for example, 75, 100, 125, 150, 200 ⁇ l of harvest solution composition/cm 2 cells.
  • square centimeter cells refers to the sum of all cells grown on an area of 1 square centimeter from adherent cultured cells.
  • the amount of the harvest liquid composition may be 35 ⁇ l or more of the harvest liquid composition/10 5 cells, preferably 70 ⁇ l or more of the harvest liquid composition/10 5 cells, for example, 75 ⁇ l or more. 100, 125, 150, 200 ⁇ l harvesting fluid composition/10 5 cells.
  • the contact time between the cultured cells and the harvest liquid composition generally does not exceed 60 minutes, usually within a contact time of 5-60 minutes (for example, 10, 20, 30, 40, 50 minutes). It can be completely cracked.
  • the lysis of the cells can be observed through a microscope. When the intact cells cannot be observed under the microscope, the cells are deemed to have been completely lysed.
  • virus inoculation or virus packaging, and cell culture can be carried out in the usual manner in the art.
  • the amount of virus inoculation can be 0.001-0.2 MOI, for example, 0.005, 0.01, 0.02, 0.03, 0.05, 0.08, 0.1, 0.15 MOI.
  • cell culture can be carried out, for example, through cell flasks (scale: 25-225cm 2 cells/flask), cell factories (scale: 500-800cm 2 cells/layer, for example, 632cm 2 cells/layer) or fermentation Tank (scale: 0.5-500m 2 cells/tank).
  • the culture method can include adherent culture and suspension culture.
  • the medium can be those commonly used in the art, such as DMEM and MEM medium.
  • the method of the present invention may further include the step of separating the cultured cells from the culture medium before contacting the cultured cells with the harvest liquid composition.
  • the harvested virus can be further purified by means of tangential flow centrifugation and/or column chromatography.
  • Cells that can be used in the present invention include (but are not limited to): Vero cells, 293 cells, CEF cells (ie, chicken embryo fibroblasts), HeLa cells.
  • the Vero cell line was established by Y. Yasumura and Y. Kawakita of Chiba University in Japan from the kidneys of normal adult African green monkeys.
  • the cell is an anchorage-dependent fibroblast and can support the proliferation of a variety of viruses, including herpes simplex virus, vaccinia virus, Japanese encephalitis, polio, rabies and other viruses. WHO believes that Vero cells are safe to use within 150 generations, are non-tumorogenic, and have been approved for the production of human virus vaccines.
  • 293 cells are transformed with type 5 adenovirus 75 strain, a human embryonic kidney hypotriploid cell line containing the E1 region of type 5 adenovirus, which is a complementary cell line deficient in E1 region. It was constructed by F.L. Graham and J.S. Miley of McMaster University in Canada in 1976 using DNA transfection technology. There are many derivative strains of 293 cells, such as HEK293, Ad293, 293T/17, AAV-293 and so on. The 293 cell line is widely used in adenovirus, retrovirus, lentivirus, vaccinia virus production, gene expression and protein expression.
  • Chicken embryo cells are the earliest objects to be used in tissue culture. Tissue culture workers have done a lot of research work with chicken embryos.
  • Chicken embryo fibroblasts (CEF) have convenient sources, simple preparation, good tolerance, and are suitable for the growth and reproduction of many viruses, so they are widely used in vaccine production, virus cultivation, and some cell and molecular biology research (For example, refer to the literature: Meiser A., et al., Comparison of virus production in chicken embryo fibroblasts infected with the WR, IHD-J and MVA strains of vaccine: IHD-J is the most effective in trans-Golgi and network extracellular enveloped virus release. J Gen Virol, 2003, 84(Pt 6): p.1383-92).
  • HeLa cells are the first aneuploid epithelioid cell line obtained from human tissues through continuous culture. It was established by G.O.Gey et al. in 1951 from a 31-year-old female black cervical cancer tissue. Compared with other cancer cell lines, this cell line proliferates abnormally quickly, is sensitive to polio virus, adenovirus, vaccinia virus, etc., and shows obvious cell degeneration, which has great value in virus research and production. It is also widely used in tumor research, biological experiments, cell culture, etc.
  • Viruses that can be produced by the present invention include (but are not limited to): vaccinia virus, varicella-zoster virus, rotavirus, EV71 virus, hepatitis A virus, herpes simplex virus (herpes simplex virus) ), lentivirus, retrovirus, adenovirus, adenovirus-associated virus, measles virus, Semliki Forest virus, Vesicular stomatitis virus (vesicular stomatitis virus), poliovirus (poliovirus), reovirus (reovirus), vesicular stomatitis virus (vesicular stomatitis virus), polio virus, Seneca valley virus (Seneca) Valley Virus, Echo enterovirus, Coxsackie virus, Newcastle disease virus and Maraba virus, preferably enveloped viruses.
  • herpes simplex virus herpes simplex virus
  • lentivirus retrovirus
  • adenovirus adenovirus-
  • enveloped viruses include: vaccinia virus, varicella-zoster virus, rotavirus, EV71 virus, Coxsackie virus (CA16 virus), hepatitis A virus, herpes simplex virus, lentivirus, retrovirus.
  • Viruses that can be produced in the present invention include oncolytic viruses, which can selectively replicate in tumor cells.
  • the oncolytic viruses of the present invention include genetically mutated viruses with oncolytic effects and wild-type viruses with oncolytic effects.
  • the genetically mutated viruses with oncolytic effect include (but are not limited to): adenovirus (adenovirus), pox virus (also known as vaccinia virus), herpes simplex virus (HSV), Measles virus, Semliki Forest virus, vesicular stomatitis virus, poliovirus and retrovirus; said to have oncolysis
  • the wild-type viruses that act include (but are not limited to): reovirus, vesicular stomatitis virus, polio virus, Seneca Valley virus (Seneca Valley Virus), Echo type Enterovirus (echo enterovirus), Coxsackie virus (Coxsackie virus), Newcastle disease virus (Newcastle disease virus) and Maraba virus (
  • Vaccinia virus (or vaccinia virus, abbreviated as VV) belongs to the family of poxviruses and plays an important role in the history of humans fighting infections and diseases. Before 1980, vaccinia virus was widely used as a smallpox vaccine, and finally variola virus was completely eliminated all over the world. With the successful expression of foreign genes in vaccinia virus, combined with its good safety in population vaccination, vaccinia virus has gradually been developed into gene expression vectors, preventive and therapeutic vaccine vectors, immunotherapy vectors, etc.
  • IMV intracellular mature virus
  • IEV intracellular enveloped virus particles
  • CEV cell-bound enveloped virus
  • EEV extracellular enveloped virus
  • Oncolytic poxviruses include (but are not limited to): Pexa-vac (available from Jennerex Biotherapeutics), JX-963 (available from Jennerex Biotherapeutics), JX-929 (available from Jennerex Biotherapeutics), VSC20 (preparation The method can be found in the scientific literature: "McCart, JA, et al. Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kind and vaccinia growth factor genes. Cancer Res (2001) 61:8751–8757.”), GL- ONC1 (available from Genelux), TG6002 (available from Transgene), DDvv-IL21 (see PCT Patent Application Publication No. WO2019/062234A1) and the like.
  • VZV Varicella-Zoster virus
  • VZV varicella-Zoster virus
  • VZV belongs to the alpha herpesvirus subfamily and is a double-stranded DNA virus. Its primary infection is varicella. When the latent infection is reactivated, it can cause herpes zoster. Vaccination is the most effective way to prevent varicella and shingles caused by VZV. To date, the live attenuated varicella vaccine (Oka strain) is the only vaccine approved for the prevention of diseases caused by VZV. Vaccination is recognized as the most effective measure to prevent varicella caused by VZV.
  • Herpes Simplex Virus belongs to the alphavirus subfamily of the Herpesvirus family. According to the difference in antigenicity, the virus is currently divided into type 1 and type 2.
  • Herpes simplex virus can use human cells as host cells, and has advantages such as a wide range of human cell types that can infect, accidental proliferation can be prevented with anti-herpes drugs, neither the lysis period nor the incubation period integrates the host cell genome, and the risk of insertion mutations is small. It is widely used in gene therapy of tumors or degenerative diseases of the nervous system.
  • HSV is a lysis pathway and mainly exists in cells.
  • Oncolytic herpes simplex viruses include (but are not limited to): HSV-1 and HSV-2 herpes simplex viruses; specifically including (for example): (Available from Amgen), G207 (available from Medigene), HF10 (available from Takara Bio), Seprehvir (available from Virttu Biologics), OrienX010 (available from Beijing Aoyuan Heli Biologics) , NV1020 (available from Catherax), etc.
  • Lentivirus (Lentivirus, abbreviated as LV) belongs to the retroviral family and is an RNA virus.
  • the overall size of lentivirus is about 100 nanometers. It has advantages such as large capacity of carrying gene fragments, high transfection efficiency, wide host range, and long-term stable expression. It has become an ideal vector for the transfer of target genes and is used in clinical treatment. It is a CAR-T One of the common viruses in cells.
  • Two CAR-T drugs targeting CD19 that have been marketed in the United States, namely Novartis’ Kymriah and Kate’s Yescarta, both use lentivirus as their carrier.
  • lentivirus Since a total amount of virus of 1.0E+12TU is required for one gene therapy, improving the packaging and production level of lentivirus can effectively promote the development of lentivirus-mediated gene therapy.
  • the production of lentivirus is to produce a limited number of packaged viruses by transiently transducing a plasmid into a cell matrix.
  • the general construction process is to package the three necessary proteins Gag/Pol, Rev, VSV-G (instead of HIV-1 Env) They were placed on the three plasmids independently, the vector of the target gene was constructed on the plasmid pLenti-gene, and then the four plasmids were co-transfected into the host cells in proportion, and the supernatant was collected after culturing to purify the virus.
  • Retroviruses are single-stranded RNA viruses.
  • the retroviral genome is about 10Kb and contains three important genes, from 5'to 3'respectively Gag (encoding the core protein of the virus), Pol (reverse transcriptase), and Env (the envelope glycoprotein on the surface of the virus particle).
  • LTR long terminal repeats
  • plasmids are often used to transfect cell substrates, to construct and screen stable toxin-producing cell lines.
  • the integration sites of retroviral vectors are usually located in the open region and transcriptionally active region of the chromosome, which greatly improves the probability of obtaining a cell line that expresses the foreign target gene efficiently.
  • retrovirus is also one of the common viral vectors for constructing CAR-T cells.
  • Adenovirus (Adenovirus, abbreviated as AdV) is a spherical particle without envelope, and its DNA exists in the form of linear double strands. Adenovirus has no envelope, and the nucleocapsid has a diameter of 70-80 nanometers and is icosahedral stereosymmetric. Adenovirus is a relatively safe vector.
  • More than 50% of the population has antibodies to type 5 adenovirus, and because of its high transgene efficiency, a wide range of transduced cell types, easy preparation and purification, only after entering the host cell Transient expression without integration into the host cell genome and other advantages, so it has become more and more prominent in gene therapy, gene immunization, and vaccine preparation, and is widely used in vaccine development, immunotherapy, gene therapy and other fields.
  • Oncolytic adenoviruses include (but are not limited to): human type 5 adenovirus or human chimeric adenovirus; specifically including (for example): Onyx-015 (available from Onyx Pharmaceuticals), H101 (available from Shanghai Sanwei Bio Technology Co., Ltd.), Ad5-yCD/mutTKSR39rep-hIL12 (available from Henry Ford Health System), CG0070 (available from Cold Genesys), DNX-2401 (available from DNAtrix), OBP-301 (available from since Oncolys BioPharma Corporation), ONCOS-102 (available from Targovax Oy company / Oncos Therapeutics Corporation), ColoAd1 (available from PsiOxus Therapeutics Corporation), VCN-01 (available from VCN Biosciences Corporation), ProstAtak TM (available from Advantagene) and so on.
  • Onyx-015 available from Onyx Pharmaceuticals
  • H101 available from Shanghai Sanwei Bio Technology Co., Ltd.
  • Adenovirus-associated virus belongs to the Parvoviridae family. It is a non-enveloped single-stranded linear DNA virus with genomic DNA less than 5Kb. Adeno-associated virus has advantages such as high safety, low immunogenicity, wide host range, stable expression, and can infect dividing and non-dividing cells at the same time. It has a very attractive prospect in the biomedical industry, especially in the field of gene therapy. Most of the recombinant AAV remains in the producer cell.
  • the present invention uses 293 cells to produce vaccinia virus (including oncolytic poxvirus).
  • the production method includes:
  • the inoculation amount can be 0.001-0.2 MOI, for example, 0.005, 0.01, 0.02, 0.03, 0.05, 0.08, 0.1, 0.15 MOI;
  • Culturing the inoculated cells to make the cell pathological preferably until the cells are completely pathologically diseased (usually 48-96h), for example, cell flasks can be used for culture, and the culture method is, for example, adherent culture; and
  • the harvest fluid composition comprises: pancreatin, pH buffer and optional nuclease, and the pH of the harvest fluid composition is greater than 7.5 and not more than 10.5, preferably 8.5-9.5; preferably, the The harvesting fluid composition comprises Tris salt buffer, pancreatin and nuclease, wherein the concentration of Tris salt buffer can be 1-50 mM, for example 1, 3, 5, 8, 10, 20, 30, 40 mM, preferably 1 -10mM; the concentration of pancreatin can be 0.01-0.12% (w/v), such as 0.03, 0.05, 0.07, 0.09, 0.11% (w/v), preferably 0.03-0.06% (w/v); nucleic acid
  • the concentration of the enzyme can be 1-100 IU/ml, for example 1, 3, 5, 10, 20, 40, 60, 80 IU/ml, preferably 1-50 IU/ml, and more preferably 1-5 IU/ml;
  • the osmotic pressure of the harvest liquid composition is preferably hypotonic, and the pH is preferably
  • the amount of the harvesting fluid composition can be greater than or equal to 35 ⁇ l harvesting fluid composition/cm 2 cells, preferably greater than or equal to 70 ⁇ l harvesting fluid composition/cm 2 cells, for example 75, 100, 125, 150, 200 ⁇ l harvesting fluid composition /cm 2 cells;
  • the time for the cultured cells to contact the harvesting fluid composition generally does not exceed 60 minutes, usually within a contact time of 5-60 minutes (for example, 10, 20, 30, 40, 50 minutes). Was completely cracked.
  • the virus production method of the present invention adopts gentle means to obtain the virus in cultured cells, does not involve cell lysis methods or steps such as freezing and thawing, ultrasound, mechanical disruption, etc., has simple process, convenient operation, easy amplification, and is very suitable for large-scale Cell culture is used to produce viruses, and compared with the traditional and commonly used prior art methods (such as freeze-thaw methods), the virus yield is significantly increased, which is 5-10 times that of the prior art methods, while ensuring the integrity of virus particles. It will not damage the biological activity of the virus, thus making large-scale virus production possible.
  • the technical means used in the following examples are conventional means well known to those skilled in the art; the reagents used are commercially available products. Unless otherwise specified below, the percentage concentration (%) of each reagent refers to the volume percentage concentration (%(v/v)) of the reagent.
  • the DMEM, MEM medium, serum, and reagents used are all common commercially available products, and the preparation of the harvest solution composition (also referred to as the treatment solution or the harvest solution) is carried out according to the instructions of Preparation Example 1.
  • Tris base obtained from Biosharp, article number 77-86-1)
  • Tris base mother liquor preparation Weigh 1.938g Tris base, add a small amount of water for injection to dissolve, and then dilute to 40ml with water for injection. 1mM Tris is prepared by diluting 400mmol/L Tris base mother liquor with water for injection.
  • the NaHCO 3 was prepared into a 7.5% (w/v) NaHCO 3 solution with ultrapure water.
  • Methyl cellulose (source: Shenggong Biology; product number: A600615-0250)
  • 4% methylcellulose Weigh 8.0g methylcellulose, add 160ml ultrapure water and shake, then dilute the volume to 200ml with a graduated cylinder, sterilize at 121°C for 20min, cool to room temperature, store at 4°C, shake it every day until methyl The cellulose is completely dissolved.
  • Semi-solid medium Take 360ml DMEM medium, 15ml FBS, 125ml 4% methylcellulose, shake and mix, and store at 4°C.
  • the composition of the buffer PBS is: 137mM NaCl, 2.7mM KCl, 10mM Na 2 HPO 4 , 2mM KH 2 PO 4 , pH 7.2.
  • composition of the buffer "potassium dihydrogen phosphate and disodium hydrogen phosphate” is: 0.29% (g/ml) potassium dihydrogen phosphate, 0.026% (g/ml) disodium hydrogen phosphate, pH 7.2-7.4.
  • the potassium dihydrogen phosphate used was obtained from Shanghai Sinopharm, CAS: 7778-77-0; the disodium hydrogen phosphate was obtained from Shanghai test, catalog number: 10020318; all were of analytical grade.
  • vaccinia virus is the recombinant oncolytic poxvirus DDvv-hIL21 described in PCT Patent Application Publication No. WO2019/062234A1, and its preparation method is described in preparation examples 1 and 2 in the patent document.
  • HEK-293 cells are from ATCC, catalog number CRL-1573. Vero cells are from ATCC, catalog number CCL-81. HeLa cells are from ATCC, catalog number CCL-2).
  • the SLF-1 cell line is from the China Common Microbial Species Collection and Management Center, and the number is CGMCCNo. 4875.
  • each dilution Set up 5 parallel wells each plate set up 1 cell maintenance liquid hole as a negative control, gently shake, 37°C for 2h, gently shake once every 30-60min; aspirate the liquid in the well, add 2ml DMEM medium to rinse Once to remove unadsorbed free virus, add 3-4ml of semi-solid medium and incubate in a 5% CO 2 incubator at 37°C for 2-3 days .
  • virus titer is calculated by the following formula.
  • the 293 cells were made into a suspension with a concentration of about 1 ⁇ 10 5 /ml with DMEM culture solution, and seeded in a 96-well plate at 100 ⁇ l/well (at the same time, a 10-fold dilution was prepared to infect the above cells respectively).
  • the first 10 wells of each row were added with 100 ⁇ l of the same concentration of virus dilution, and the 11th and 12th wells were added with an equal volume of 2% BCS DMEM as a negative control. Place them in a CO 2 incubator at 37°C for 10 days, then observe under a fluorescent inverted microscope to judge and record the cytopathic effect (CPE) of each row.
  • CPE cytopathic effect
  • the processed CEF cells were cultured overnight at 37°C, their morphology and quality were examined under a microscope, the cells were counted by trypan blue staining, confirmed by a live cell counter, and the viability of the prepared CEF cells was calculated. The microscopic examination of the cell morphology is complete and in good condition.
  • T75 cell culture flasks (cell culture area of each flask 75cm 2 ) were used to culture 293, Vero, and HeLa cells. After the cells grew to a monolayer, they were used for the cell lysis efficiency test of the harvest solution.
  • the 293, Vero, and HeLa cells that grow to a single layer are discarded from the original growth medium, and the adherent cells are respectively taken from 2-5ml of the AE group treatment solution in Preparation Example 1 to wash the cell surface 1-2 times, and then add 8ml of the same fresh treatment solution. Let it stand at room temperature and observe the changes in cell morphology under a microscope. When intact cells are not observed under the microscope (considered as completely lysed), centrifuge the suspension at 300g for 10 minutes to see if there is any precipitate after centrifugation. Each experiment was repeated more than three times.
  • test results show:
  • Treatment solution D and E groups can basically lyse cells as the lysis time is prolonged. Observation under the microscope shows that the cell fragments are large. After the cell suspension is centrifuged, there is no obvious precipitation and the supernatant is not sticky. Cell nucleic acid The removal effect is obvious. The cell lysis ratio is less than 100%; there is no significant difference between 293, Vero and HeLa cells;
  • Cultivation and harvest of vaccinia virus inoculate 293, HeLa, and CEF cells into T75 cell culture flasks, and when the cells grow to adherent confluence greater than 80%, inoculate working seeds of vaccinia virus to each cell at MOI of 0.02.
  • the cell bottle inoculated with virus is kept at 37.0 ⁇ 1.0°C for 2 hours; after the adsorption is completed, the virus maintenance solution containing 2% FBS is added and placed in a 37.0 ⁇ 1.0°C incubator to continue culturing until the cells are completely diseased (usually 48-96h) , Discard the culture solution, add the AE group treatment solution described in Preparation Example 1, and treat for 10-60 min. Observe the cell status under a microscope.
  • freeze-thaw control group (referred to as the freeze-thaw group) is set as follows: the cells after the complete disease are pipetted down, and the cells and infection medium are transferred to a 15 ml sterile centrifuge tube.
  • the plaque method (that is, the plaque forming unit method) was used to determine the virus titer, and according to the test results, the harvest effect of each treatment solution and the influence on the virus titer were evaluated. The results are shown in Table 4 below.
  • Both treatment solution A and B groups can completely lyse the cells within 10 minutes; the cell fragments are small, after the cell suspension is centrifuged, there is no obvious precipitation and the supernatant is not sticky, and the nucleic acid removal effect is obvious.
  • the cell lysis ratio is 100%; there is no significant difference between 293 and HeLa cells;
  • Treatment solution C group (control group) has a poor cell lysis effect. After 60 minutes of lysis, many cell morphologies can still be seen under the microscope. After the cell suspension is centrifuged, obvious precipitation can be seen; the supernatant is sticky, indicating that some cells have been lysed and released Intracellular substances such as nucleic acids;
  • Treatment solution D and E groups can basically lyse cells as the lysis time increases. Observation under the microscope shows that the cell fragments are large. After the cell suspension is centrifuged, there is no obvious precipitation and the supernatant is not sticky. Nucleic acid is removed The effect is obvious. The cell lysis ratio is less than 100%; there is no significant difference between 293 and HeLa cells;
  • group F freeze-thaw control group
  • group F freeze-thaw control group
  • the cell morphology and cell fragments were still visible under the microscope, but after the cell suspension was centrifuged, there was no obvious precipitation, and the supernatant was sticky and had flocculent floats.
  • the cells are lysed, releasing nucleic acid and other intracellular substances.
  • the cell lysis ratio is less than 100%; there is no significant difference between 293 and HeLa cells.
  • Example 4 The effect of different treatment methods on harvesting viruses
  • the virus solution was collected. Centrifuge the virus solution at 300 g for 10 min and then take the supernatant to detect the virus titer.
  • the freeze-thaw control group was set up in the following way: the completely diseased cells were pipetted down, and the cells and infection medium were transferred to a 15 ml sterile centrifuge tube. Place it in a refrigerator at -80°C and quickly freeze it for 120 minutes, then quickly put it in a 37°C water bath to thaw for 10 minutes, repeat 3 times, centrifuge the virus solution at 750g for 10 minutes and take the supernatant to detect the virus titer. The titer of virus harvested after treatment with the treatment solution was evaluated. Each experiment was repeated more than three times, and the average value was taken for statistical analysis.
  • FIG. 1 The results of this experiment are shown in Figure 1.
  • the figure shows that when the cells are completely lysed to release the virus, the results obtained vary greatly due to the different ways of processing the intracellular virus. Among them: the yield of the freeze-thaw treatment is low; the treatment solution G group (1mM Tris+ The yield after treatment with 0.06% (w/v) pancreatin + 5IU/ml nuclease) was about 6 times that of the freeze-thaw group; treatment solution H group (7.5% NaHCO 3 + 0.06% (w/v) pancreatin + 5IU/ ml nuclease) is about 5 times that of the freeze-thaw group.
  • Inoculate 293 into a T75 cell culture flask When the cells grow to adherent and confluent> 80%, inoculate the cells with working seeds of vaccinia virus at MOI of 0.02, and place the virus-inoculated cell flask at 37.0 ⁇ 1.0°C for adsorption 0-2 Hours: After the adsorption is over, add a virus maintenance solution containing 2% FBS, and place it in a 37.0 ⁇ 1.0°C incubator to continue culturing. After the cells are completely diseased, the virus is harvested.
  • the medium was discarded, in group A were added to the treatment liquid according to Example 1 was prepared, according to the area of cultured cells, according to 35 ⁇ l / cm 2, 70 ⁇ l / cm 2, 150 ⁇ l / cm 2, respectively, to the processing 10,30min, cells were observed under a microscope Status, collect virus fluid and record cell disruption. Centrifuge the virus solution at 750g for 10 minutes, and then take the supernatant to detect the virus titer, and record whether there is precipitation after centrifugation and whether the supernatant is viscous. At the same time, the freeze-thaw control group was set up in the following way: the completely diseased cells were pipetted down, and the cells and infection medium were transferred to a 15 ml sterile centrifuge tube.
  • the treatment dosage and treatment time have a significant impact on the cell lysis effect and virus yield. Treating the harvested intracellular virus with the amount of 35, 70, and 150 ⁇ l/cm 2 treatment solution of group A can effectively lyse the cells within 30 minutes, the cell lysis ratio is 100%, and the supernatant is not sticky.
  • the cell lysis efficiency was positively correlated with the dosage of the treatment solution and the treatment time. When the dosage of the treatment solution was 150 ⁇ l/cm 2 and the treatment time was 10 minutes, the virus titer was the highest, which was more than 6 times that of the freeze-thaw group.
  • Example 6 Cultivation and harvest of various exemplary viruses
  • Cultivation and harvest of varicella virus Inoculate SLF-1 into T25 cell culture flasks, and when the cells grow to a single layer, use the Oka strain of varicella-zoster virus (from ATCC, numbered as VR-795) to work seeds according to MOI of 0.001- 0.1 Inoculate SLF-1 cells, place the virus-inoculated cell bottle at 35.0 ⁇ 1.0°C for 0.5-2 hours; add virus MEM maintenance solution after the adsorption is over, place it in a 35.0 ⁇ 1.0°C incubator and continue culturing for 48-96h, discard Remove the culture solution, add the harvest solution of the AE group as described in Preparation Example 1, after standing at room temperature for 10 minutes, observe the cell status under a microscope, and shake the cells to detach when the cells are significantly expanded, and collect the virus solution.
  • HSV Place Vero cells in a 37°C, 5% CO 2 incubator for expansion and cultivation. After the cells grow to a single layer, they are infected with MOI values of 0.001-0.1 respectively. Harvest the infected cells and the supernatant when the cells become rounded and not yet fall off, add the AE group treatment solution described in Preparation Example 1, respectively, treat for 10-60 minutes, observe the cell status under a microscope, and wait until the cells are completely lysed (up to 60 minutes of lysis) , Collect the virus liquid, record the cell lysis time and cell disruption.
  • the plaque method was used to determine the virus titer, and according to the test results, the harvesting effect of each treatment solution at different time points and the impact on the virus titer were evaluated.
  • Cultivation and harvest of adenovirus Place 293 cells in a 37°C, 5% CO 2 incubator for amplification and culture. After the cells grow to a single layer, they are infected with MOI values of 1-100. Harvest the infected cells and the supernatant when the cells become rounded and not yet fall off, add the AE group treatment solution described in Preparation Example 1, respectively, treat for 10-60 minutes, observe the cell status under a microscope, and wait until the cells are completely lysed (up to 60 minutes of lysis) , Collect the virus liquid, record the cell lysis time and cell disruption.
  • the virus titer was determined according to the TCID50 method, and the harvest effect of each treatment solution at different time points and the impact on the virus titer were evaluated at the same time.
  • Cultivation and harvest of lentivirus Place 293 cells in a 37°C, 5% CO 2 incubator for amplification and culture. Add 4 kinds of plasmids (that is, the 3 proteins Gag/Pol, Rev, VSV-G (instead of HIV-1 Env) necessary for virus packaging) and place them on the 3 plasmids independently.
  • the vector of the target gene is constructed on the plasmid pLenti- Gene, get 4 kinds of plasmids), infect the cells, harvest the infected cells and the supernatant when the cells become round and not fall off, add the AE group treatment solution described in Preparation Example 1, respectively, treat for 10-60min, and observe under a microscope Cell status, after the cells are completely lysed (up to 60min), collect the virus solution, and record the cell lysis time and cell disruption. Centrifuge the virus solution at 300g for 10 minutes, and then take the supernatant to detect the virus titer, and record whether there is precipitation after centrifugation and whether the supernatant is viscous. At the same time, a freeze-thaw harvest control group and a supernatant control group are set. According to the test results, evaluate the harvest effect of each treatment solution at different time points and the influence on the virus titer.
  • the 293 cells are expanded and cultured with DMEM medium containing 10% calf serum.
  • the cell factory is inoculated, and the cells are digested into cells after culturing in a CO 2 incubator for about 48 hours.
  • Suspension, used to inoculate cell tank obtained from NBS company, model CelliGen Plus/310, surface area is about 22 square meters; cells are cultured by perfusion, after 6-8 days, vaccinia virus is infected with MOI 0.02, and the infection is about 48-72 Harvesting started after 1 hour, wherein the G group treatment solution described in Preparation Example 1 can be used to harvest 1.3 ⁇ 10 13 PFU/tank of virus.
  • the cell pellet was equilibrated to room temperature with 3L treatment solution (Approximately 157 ⁇ l/cm 2 cells) were lysed and harvested.
  • the treatment solution consisted of 1 mM Tris, 0.03% recombinant pancreatin, 5 U/ml nuclease, pH 9.0, and 1 mM MgCl 2 was added at the same time.
  • the virus with 7.80 ⁇ 10 10 PFU can be harvested (yield per unit area: 4.09 ⁇ 10 6 PFU/cm 2 ).
  • Resuscitate 293 cells to 3 T225 cell culture flasks brand: Corning, item number: 431082
  • expand and culture with DMEM medium containing 10% calf serum and inoculate 1 when the cell confluence is 80%-90% 10-layer cell factory (brand: Corning, model: "CellSTACK Chamber, 10 STACK, POLYSTYRENE, STERILE, 1/6", total area: 6360cm 2 ), digested cells to make a cell suspension after culturing in a CO 2 incubator for about 72 hours
  • the solution is used to inoculate the cell tank (purchased from Eppendorf, the model is BioFlo 320, the surface area of the 5L cell tank is about 150,000 cm 2 ); the inoculation volume is 470 ml, and the total number of cells: 1.78 ⁇ 10 9 cells.
  • the composition of the treatment solution is 1 mM Tris, 0.03% recombinant trypsin, 5 U/ml nuclease, pH 9.3, and 1 mM MgCl 2 is added at the same time; the common treatment solution is about 13.5 L (about 90 ⁇ l/cm 2 cells).
  • the virus can be harvested at 4.41 ⁇ 10 11 PFU/pot (yield per unit area: 2.94 ⁇ 10 6 PFU/cm 2 ).
  • Resuscitate 293 cells to 3 T225 cell culture flasks brand: Corning, item number: 431082
  • expand and culture with DMEM medium containing 10% calf serum and inoculate 1 when the cell confluence is 80%-90%
  • Cell factory brand: Corning, model: "CellSTACK Chamber, 10 STACK, POLYSTYRENE, STERILE, 1/6", total area: 6360cm 2
  • digested cells in a CO 2 incubator for about 72 hours to make a cell suspension Used for inoculating cell tank (purchased from Eppendorf company, model BioFlo 320, 5L cell tank surface area is about 150,000 cm 2 ); inoculation volume 430 ml, total cell volume: 1.97 ⁇ 10 9 cells.
  • the composition of the treatment solution is 1mM Tris, 0.03% recombinant pancreatin, 5U/ml nuclease, pH 9.3, and 1mM MgCl 2 is added at the same time; the common treatment solution is about 15L (about 100 ⁇ l/cm 2 cells).
  • the virus can be harvested at 2.41 ⁇ 10 11 PFU/pot (the yield per unit area is 1.61 ⁇ 10 6 PFU/cm 2 ).
  • the composition of the treatment solution is 1mM Tris, 0.03% recombinant pancreatin, 5U/ml nuclease, pH 9.3, and 1mM MgCl 2 is added at the same time; the common treatment solution is about 50L (about 110 ⁇ l/cm 2 cells).
  • the virus can be harvested at 1.24 ⁇ 10 12 PFU/pot (yield per unit area: 2.76 ⁇ 10 6 PFU/cm 2 ).
  • the production process can be greatly simplified, which is particularly suitable for large-scale production of viruses.
  • the yield of the virus obtained by harvesting is high and the impurities are low, which further makes the subsequent purification process easier to implement, and the obtained virus purity and biological activity are also higher.

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Abstract

提供一种生产病毒的方法及收获液组合物。所述方法包括：培养已接种病毒或已转染病毒包装用元件的细胞，使培养得到的细胞与收获液组合物接触，一步收获病毒；所述收获液组合物包含：胰酶、pH缓冲液和任选的核酸酶，pH大于7.5且不超过10.5。

Description

一种生产病毒的方法及收获液组合物 技术领域
本发明涉及生物技术领域，具体而言，涉及一种生产病毒的方法、以及收获液组合物，更具体而言，涉及一种通过细胞培养来生产病毒的方法、以及用于收获经细胞培养的病毒的收获液组合物。
背景技术
病毒类生物制品，包括溶瘤病毒药物、病毒类疫苗或重组病毒载体等，其制备过程一般包括：先将毒种接种于未感染的适宜代次的细胞上，当达到一定程度细胞病变时收获感染细胞，通过不同的方式裂解细胞后，收获病毒，然后通过密度梯度离心或层析方式纯化后得到原液，加入一定的缓冲液即成半成品，半成品经冻干或分装后即成为成品。
目前病毒类生物制品生产用细胞的大规模培养，主要采用细胞工厂或发酵罐的方式，大部分扩增的病毒在胞内，因此在病毒的收获工艺中需要有效地破碎裂解细胞使病毒释放，并保证病毒颗粒的完整性。而不同的收获工艺对细胞的裂解程度和病毒颗粒的完整性的影响不同。收获方式直接影响病毒滴度，而收获的病毒原液的有效高滴度值是影响成品品质的重要因素。
由于痘苗病毒、单纯疱症病毒、水痘带状疱疹病毒、腺病毒等属于细胞内病毒，具有很强的细胞结合活性，所以病毒收获时必须收集并破碎细胞才能得到游离的病毒颗粒。基于慢病毒、逆转录病毒、腺相关病毒的重组病毒载体，目前在实际包装生产过程中，只收获了细胞外的游离病毒，而大量的胞内病毒并没有得到充分的利用。
目前在针对这些病毒的大规模生产过程中，大多采用了冻融法来裂解细胞收获胞内病毒。但冻融法存在能耗高、周期长、步骤多、不易放大、产量不稳定的缺点，限制了病毒类生物制品的生产规模。 也有通过低渗或化学裂解的方法来收获细胞内病毒，但大量结合在细胞膜上的病毒并未充分释放，而且很多化学裂解试剂同样会破坏有包膜病毒(如痘苗病毒、单纯疱疹病毒等)的包膜，使得病毒失去生物活性。
因此，仍然需要能够通过细胞培养来有效地生产病毒的方法、以及能够有效地收获经细胞培养的病毒的收获液，这对于获得高品质的病毒类疫苗或重组病毒载体等产品来说是至关重要的。
发明内容
为了解决上述现有技术中所存在的一个或多个问题，本发明提供了一种生产病毒的方法、以及收获液组合物。
具体而言，本发明提供了：
(1)一种生产病毒的方法，所述方法包括：
培养细胞，其中所述细胞为已接种病毒或已转染病毒包装用元件的细胞；以及
使培养得到的细胞与收获液组合物接触，一步收获病毒，其中所述收获液组合物包含：胰酶、pH缓冲液和任选的核酸酶，并且所述收获液组合物的pH为大于7.5且不超过10.5。
(2)根据(1)所述的方法，其特征在于，在所述收获液组合物中，所述胰酶的浓度为0.01-0.12％(w/v)，优选为0.03-0.06％(w/v)。
(3)根据(1)或(2)所述的方法，其特征在于，在所述收获液组合物中，所述核酸酶的浓度为1-100IU/ml，优选为1-50IU/ml，还优选为1-5IU/ml。
(4)根据(1)-(3)中任一项所述的方法，其特征在于，所述收获液组合物的渗透压的范围为0-50mOsmol/kg或800-2500mOsmol/kg，优选为0-20mOsmol/kg或1785-2000mOsmol/kg，更优选为1-20mOsmol/kg。
(5)根据(1)-(4)中任一项所述的方法，其特征在于，所述收获液组合物的pH为8.5-9.5。
(6)根据(1)-(5)中任一项所述的方法，其特征在于，所 述pH缓冲液选自Tris盐缓冲液、碳酸氢钠缓冲液。
(7)根据(1)-(6)中任一项所述的方法，其特征在于，所述培养是通过细胞瓶、细胞工厂或发酵罐进行的，培养方式包括贴壁培养和悬浮培养。
(8)根据(1)-(7)中任一项所述的方法，其特征在于，当所述培养的方式为贴壁培养时，所述收获液组合物的用量为大于等于35μl收获液组合物/cm 2细胞，优选为大于等于70μl收获液组合物/cm 2细胞；当所述培养的方式为悬浮培养时，所述收获液组合物的用量为大于等于35μl收获液组合物/10 5个细胞，优选为大于等于70μl收获液组合物/10 5个细胞。
(9)根据(1)-(8)中任一项所述的方法，其特征在于，所述培养得到的细胞与所述收获液组合物接触的时间为5-60分钟。
(10)根据(1)-(9)中任一项所述的方法，其特征在于，所述细胞选自Vero细胞、293细胞、CEF细胞、HeLa细胞。
(11)根据(1)-(10)中任一项所述的方法，其特征在于，所述病毒包括：痘苗病毒、水痘-带状疱疹病毒、轮状病毒、EV71病毒、甲肝病毒、单纯疱疹病毒、慢病毒、逆转录病毒、腺病毒、腺相关病毒、麻疹病毒、塞姆利基森林病毒、水疱性口炎病毒、脊髓灰质炎病毒、呼肠孤病毒、水疱性口炎病毒、脊髓灰质炎病毒、塞内卡谷病毒、埃可型肠道病毒、柯萨奇病毒、新城疫病毒和马拉巴病毒，优选为有包膜的病毒。
(12)根据(1)-(11)中任一项所述的方法，其特征在于，所述细胞为293细胞，所述病毒为痘苗病毒。
(13)根据(1)-(5)、(7)-(12)中任一项所述的方法，其特征在于，所述收获液组合物包含Tris盐缓冲液、胰酶和核酸酶，所述Tris盐缓冲液的浓度为1-50mM，优选为1-10mM。
(14)一种用于收获经细胞培养的病毒的收获液组合物，其特征在于，所述收获液组合物包含：胰酶、pH缓冲液和任选的核酸酶，并且所述收获液组合物的pH为大于7.5且不超过10.5。
(15)根据(14)所述的收获液组合物，其特征在于，在所述 收获液组合物中，所述胰酶的浓度为0.01-0.12％(w/v)，优选为0.03-0.06％(w/v)。
(16)根据(14)或(15)所述的收获液组合物，其特征在于，在所述收获液组合物中，所述核酸酶的浓度为1-100IU/ml，优选为1-50IU/ml，还优选为1-5IU/ml。
(17)根据(14)-(16)中任一项所述的收获液组合物，其特征在于，所述收获液组合物的渗透压的范围为0-50mOsmol/kg或800-2500mOsmol/kg，优选为0-20mOsmol/kg或1785-2000mOsmol/kg，更优选为1-20mOsmol/kg。
(18)根据(14)-(17)中任一项所述的收获液组合物，其特征在于，所述收获液组合物的pH为8.5-9.5。
(19)根据(14)-(18)中任一项所述的收获液组合物，其特征在于，所述pH缓冲液选自Tris盐缓冲液、碳酸氢钠缓冲液。
(20)根据(14)-(19)中任一项所述的收获液组合物，其特征在于，所述培养是通过细胞瓶、细胞工厂或发酵罐进行的，培养方式包括贴壁培养和悬浮培养。
(21)根据(14)-(20)中任一项所述的收获液组合物，其特征在于，所述细胞选自Vero细胞、293细胞、CEF细胞、HeLa细胞。
(22)根据(14)-(21)中任一项所述的收获液组合物，其特征在于，所述病毒包括：痘苗病毒、水痘-带状疱疹病毒、轮状病毒、EV71病毒、甲肝病毒、单纯疱疹病毒、慢病毒、逆转录病毒、腺病毒、腺相关病毒、麻疹病毒、塞姆利基森林病毒、水疱性口炎病毒、脊髓灰质炎病毒、呼肠孤病毒、水疱性口炎病毒、脊髓灰质炎病毒、塞内卡谷病毒、埃可型肠道病毒、柯萨奇病毒、新城疫病毒和马拉巴病毒，优选为有包膜的病毒。
(23)根据(14)-(22)中任一项所述的收获液组合物，其特征在于，所述细胞为293细胞，所述病毒为痘苗病毒。
(24)根据(14)-(18)、(20)-(23)中任一项所述的收获液组合物，其特征在于，所述收获液组合物包含Tris盐缓冲液、胰酶和核酸酶，所述Tris盐缓冲液的浓度为1-50mM，优选为1-10mM。
本发明与现有技术相比具有以下优点和积极效果：
本发明首次提出用于病毒生产的一步收获法，使得在本发明的病毒生产方法中，通过使培养得到的细胞与本发明的收获液组合物接触，就能够实现一步收获病毒。本发明的发明人经大量研究得到本发明的收获液组合物，所述收获液组合物能使所述细胞完全裂解而收获病毒，因此无需实施冻融、超声、机械破碎等其它细胞裂解方法或步骤。本发明的病毒生产方法具有操作简便、容易放大、产量稳定等优点，而且与现有技术方法相比产量也出乎意料地显著提高，同时能保证病毒颗粒的完整性，而不会损害病毒的生物活性，因此非常适合大规模生产病毒。
在大规模生产过程中，采用本发明方法及收获液组合物能极大地简化生产工艺，降低生产成本，并能进一步得到高浓度、高纯度、高活性的病毒产品。
附图说明
图1示出本申请一个实施方案中不同处理液处理收获病毒的结果。其中横坐标表示不同组别，纵坐标表示病毒的平均单位面积产量(pfu/cm 2)。
具体实施方式
以下通过具体实施方式的描述并参照附图对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的范围之内。
本发明的病毒生产方法包括：培养细胞，其中所述细胞为已接种病毒或已转染病毒包装用元件的细胞；以及使培养得到的细胞与本发明的收获液组合物接触，一步收获病毒。本发明的收获液组合物包含：胰酶、pH缓冲液和任选的核酸酶，并且所述收获液组合物的pH为大于7.5且不超过10.5。
本文中所述的“病毒包装用元件”是指重组病毒载体包装慢病 毒、逆转录病毒、腺相关病毒等需要的顺式作用元件(cis-acting element)和反式作用元件(trans-acting element)。
本文中所述的“一步收获病毒”是指培养得到的细胞与本发明的收获液组合物接触时，所述收获液组合物能使所述细胞完全裂解而收获病毒，因此无需实施冻融、超声、机械破碎等其它细胞裂解方法或步骤。
本发明的发明人发现，如果仅采用现有技术中的低渗缓冲液，并不能达到一步收获病毒的目的，病毒产量仍然较低；而本发明的发明人出乎意料地发现，在使用本发明的收获液组合物时，能够实现一步收获病毒，且病毒产量得到显著提高。与现有技术方法相比，通过本发明得到的病毒产量可以提高5倍以上。
在所述收获液组合物中，胰酶的浓度可为0.01-0.12％(所述％为质量体积比(w/v)，即，每100ml收获液组合物中所含的胰酶的克数为0.01-0.12g)，例如为0.03、0.05、0.07、0.09、0.11％(w/v)，优选为0.03-0.06％(w/v)。所述胰酶可以是商购得到的，并且可以是动物来源的传统胰酶，也可以是重组胰酶。
在所述收获液组合物中，如果包含核酸酶，则核酸酶的浓度可为1-100IU/ml，例如为1、3、5、10、20、40、60、80IU/ml，优选为1-50IU/ml，还优选为1-5IU/ml。可添加一定浓度的Mg 2+(如1-10mM Mg 2+，优选1-2mM Mg 2+；例如1mM MgCl 2)，可使核酸酶发挥作用。
所述收获液组合物的渗透压的范围可为0-50mOsmol/kg(例如为1、5、10、15、30、45mOsmol/kg)或800-2500mOsmol/kg(例如为1000、1250、1500、1700、1900、2100、2300mOsmol/kg)。优选地，所述收获液组合物的渗透压为0-20mOsmol/kg或1785-2000mOsmol/kg，更优选为1-20mOsmol/kg，进一步优选为1-10mOsmol/kg。
所述收获液组合物的pH为碱性的，更具体为大于7.5且不超过10.5，例如为7.6、8.0、8.5、9.0、9.5、10.0、10.5，优选为8.5-9.5。当所述收获液组合物的pH小于等于7.5时，细胞裂解不完全，病毒 产量低；当所述收获液组合物的pH大于10.5时，病毒的活性受影响。
所述收获液组合物中的pH缓冲液可选自Tris盐缓冲液、碳酸氢钠缓冲液；优选为Tris盐缓冲液。
在一个实施方案中，本发明的收获液组合物包含Tris盐缓冲液、胰酶和核酸酶。所述Tris盐缓冲液的浓度可为1-50mM，例如为1、3、5、8、10、20、30、40mM，优选为1-10mM。
在本发明的方法中，当培养细胞采用贴壁培养的方式时，收获液组合物的用量可为大于等于35μl收获液组合物/cm 2细胞，优选为大于等于70μl收获液组合物/cm 2细胞，例如为75、100、125、150、200μl收获液组合物/cm 2细胞。在本文中，平方厘米细胞(cm 2细胞)是指贴壁培养的细胞在1平方厘米的面积上生长的所有细胞之和。当培养细胞采用悬浮培养的方式时，收获液组合物的用量可为大于等于35μl收获液组合物/10 5个细胞，优选为大于等于70μl收获液组合物/10 5个细胞，例如为75、100、125、150、200μl收获液组合物/10 5个细胞。
在本发明中，培养得到的细胞与收获液组合物接触的时间一般不超过60分钟，通常在5-60分钟的接触时间内(例如为10、20、30、40、50分钟)所述细胞就可被完全裂解。可通过显微镜来观察细胞的裂解情况，当在显微镜下观察不到完整细胞时就视为细胞已完全裂解。
在本发明中，病毒接种或病毒包装、以及细胞培养可按照本领域通常的方式进行。病毒接种量可为0.001-0.2MOI，例如为0.005、0.01、0.02、0.03、0.05、0.08、0.1、0.15MOI。本文所用的术语“MOI”或“感染复数”(Multiplicity of infection)也即，病毒与细胞个数比，是指用以起始病毒感染的每个细胞感染病毒数。MOI＝pfu/细胞，即细胞个数×MOI＝总PFU。
在本发明中，细胞培养例如可通过细胞瓶(规模可为：25-225cm 2细胞/瓶)、细胞工厂(规模可为：500-800cm 2细胞/层，例如632cm 2细胞/层)或发酵罐(规模可为：0.5-500m 2细胞/罐)进行，培养方式可包括贴壁培养和悬浮培养。培养基可采用本领域常用的那些，例如 为DMEM、MEM培养基。本发明的方法还可包括：在使培养得到的细胞与收获液组合物接触前，将培养得到的细胞与培养基分离的步骤。另外，对于收获的病毒，可以通过切向流离心和/或柱层析等方式将其进一步纯化。
可用于本发明的细胞包括(但不限于)：Vero细胞、293细胞、CEF细胞(即鸡胚成纤维细胞)、HeLa细胞。
Vero细胞株是日本千叶大学的Y.Yasumura和Y.Kawakita从正常成年非洲绿猴的肾脏建株的。该细胞是贴壁依赖性的成纤维细胞，能支持多种病毒的增殖，包括单纯疱疹病毒、痘苗病毒、乙型脑炎、脊髓灰质炎、狂犬病等病毒。WHO认为Vero细胞在150代以内使用是安全的，无致肿瘤性，已被准许用于生产人用病毒疫苗。
293细胞是用5型腺病毒75株系转化，含有5型腺病毒E1区的人胚肾亚三倍体细胞系，是一种E1区缺陷互补细胞系。它是加拿大麦克马斯特大学(McMaster University)的F.L.Graham与J.S.Miley于1976年用DNA转染技术构建而成的。293细胞有多种衍生株，如HEK293、Ad293、293T/17、AAV-293等。293细胞系被广泛用于腺病毒、逆转录病毒、慢病毒、痘苗病毒生产、基因表达和蛋白表达。
鸡胚细胞是组织培养最早被利用的对象，组织培养工作者曾用鸡胚做过大量研究工作。鸡胚成纤维细胞(CEF)的来源方便，制备简单，耐受性好，且适合于许多病毒的生长繁殖，因此被广泛用于疫苗的生产、病毒的培养以及一些细胞和分子生物学的研究(例如，参见文献：Meiser A.,et al.,Comparison of virus production in chicken embryo fibroblasts infected with the WR,IHD-J and MVA strains of vaccinia virus:IHD-J is most efficient in trans-Golgi network wrapping and extracellular enveloped virus release.J Gen Virol,2003,84(Pt 6):p.1383-92)。
HeLa细胞是第一个来自人体组织经连续培养获得的非整倍体上皮样细胞系，它由G.O.Gey等在1951年从31岁女性黑人的宫颈癌组织建立。此细胞系跟其它癌细胞系相比，增殖异常迅速，对脊髓灰质炎病毒、腺病毒、痘苗病毒等有敏感性，并显示有明显的细胞变性， 在病毒研究、生产方面具有较大利用价值，同时还被广泛应用于肿瘤研究、生物实验、细胞培养等。
本发明可生产的病毒包括(但不限于)：痘苗病毒(vaccinia virus)、水痘-带状疱疹病毒(varicella-zoster virus)、轮状病毒、EV71病毒、甲肝病毒、单纯疱疹病毒(herpes simplex virus)、慢病毒(lentivirus)、逆转录病毒(retrovirus)、腺病毒(adenovirus)、腺相关病毒(adenovirus-associated virus)、麻疹病毒(measles virus)、塞姆利基森林病毒(Semliki Forest virus)、水疱性口炎病毒(vesicular stomatitis virus)、脊髓灰质炎病毒(poliovirus)、呼肠孤病毒(reovirus)、水疱性口炎病毒(vesicular stomatitis virus)、脊髓灰质炎病毒、塞内卡谷病毒(Seneca Valley Virus)、埃可型肠道病毒(echo enterovirus)、柯萨奇病毒(Coxsackie virus)、新城疫病毒(Newcastle disease virus)和马拉巴病毒(maraba virus)，优选为有包膜的病毒。例如，有包膜的病毒包括：痘苗病毒、水痘-带状疱疹病毒、轮状病毒、EV71病毒、柯萨奇病毒(CA16病毒)、甲肝病毒、单纯疱疹病毒、慢病毒、逆转录病毒。
本发明可生产的病毒包括溶瘤病毒，所述溶瘤病毒能够选择性地在肿瘤细胞中复制。本发明所述的溶瘤病毒包括具有溶瘤作用的经基因突变的病毒和具有溶瘤作用的野生型病毒。所述具有溶瘤作用的经基因突变的病毒包括(但不限于)：腺病毒(adenovirus)、痘病毒(也称痘苗病毒(vaccinia virus))、单纯疱疹病毒(herpes simplex virus(HSV))、麻疹病毒(measles virus)、塞姆利基森林病毒(Semliki Forest virus)、水疱性口炎病毒(vesicular stomatitis virus)、脊髓灰质炎病毒(poliovirus)和逆转录病毒(retrovirus)；所述具有溶瘤作用的野生型病毒包括(但不限于)：呼肠孤病毒(reovirus)、水疱性口炎病毒(vesicular stomatitis virus)、脊髓灰质炎病毒、塞内卡谷病毒(Seneca Valley Virus)、埃可型肠道病毒(echo enterovirus)、柯萨奇病毒(Coxsackie virus)、新城疫病毒(Newcastle disease virus)和马拉巴病毒(maraba virus)。所述溶瘤病毒的基因组中可以整合有外源基因，所述外源基因包括外源免疫调节基因、外源筛选基因、外 源报告基因等。所述溶瘤病毒的基因组中也可以不整合任何外源基因。
痘苗病毒(或称痘病毒)(vaccinia virus，缩写为VV)属痘病毒科，在人类对抗感染和疾病的历史中扮演着重要的角色。1980年以前，痘苗病毒主要作为天花疫苗而广泛使用，并最终在全世界范围内彻底清除了天花病毒。随着外源基因在痘苗病毒内的成功表达，结合其在人群接种中表现出的良好的安全性，痘苗病毒逐渐被开发为基因表达载体、预防和治疗性疫苗载体、免疫治疗载体等。根据其感染细胞的不同阶段，在痘苗病毒的整个生命周期中，可以产生出四种不同的感染形式：细胞内成熟病毒颗粒(intracellular mature virus，缩写为IMV)、细胞内包膜病毒颗粒(intracellular enveloped virus，缩写为IEV)、细胞结合包膜病毒颗粒(cellular enveloped virus，缩写为CEV)和细胞外包膜病毒颗粒(extracellular enveloped virus，缩写为EEV)。在绝大多数痘苗病毒株中(包括WR株、哥本哈根株、安卡拉株、天坛株等)，IMV占总子代病毒量的90％以上，这部分病毒在病毒工厂中组装完成后即滞留在细胞中，直到细胞裂解时才释放出胞外。
溶瘤痘病毒包括(但不限于)：Pexa-vac(可得自Jennerex Biotherapeutics公司)、JX-963(可得自Jennerex Biotherapeutics公司)、JX-929(可得自Jennerex Biotherapeutics公司)、VSC20(制备方法可参见科技文献：“McCart,JA,et al.Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes.Cancer Res(2001)61:8751–8757.”)、GL-ONC1(可得自Genelux公司)、TG6002(可得自Transgene公司)、DDvv-IL21(参见PCT专利申请公开No.WO2019/062234A1)等。
水痘-带状疱疹病毒(Varicella-Zoster virus，缩写为VZV)属α疱疹病毒亚科，为双链DNA病毒，其原发感染为水痘，潜伏感染再激活时可引发带状疱疹。接种疫苗是预防由VZV引起的水痘与带状疱疹的最有效措施。迄今为止，水痘减毒活疫苗(Oka株)是唯一获准用于预防由VZV引起的疾病的疫苗。接种该疫苗是公认的预防由 VZV引起的水痘的最有效措施。
单纯疱疹病毒(Herpes Simplex Virus，缩写为HSV)属于疱疹病毒科α病毒亚科。根据抗原性的差别，目前把该病毒分为1型和2型。单纯疱疹病毒能以人类细胞作为宿主细胞，并且具有诸如能够感染的人类细胞类型广、发生意外增殖可以用抗疱疹药物阻止、裂解期和潜伏期都不整合宿主细胞基因组、插入突变危险小等优点，在肿瘤或神经系统退行性疾病的基因治疗中广泛应用。HSV为溶胞途径，主要存在于细胞中。
溶瘤单纯疱疹病毒包括(但不限于)：HSV-1、HSV-2型单纯疱疹病毒；具体包括(例如)：
Figure PCTCN2020097901-appb-000001
(可得自Amgen公司)、G207(可得自Medigene公司)、HF10(可得自Takara Bio公司)、Seprehvir(可得自Virttu Biologics公司)、OrienX010(可得自北京奥源和力生物公司)、NV1020(可得自Catherax公司)等。
慢病毒(Lentivirus，缩写为LV)属于逆转录病毒科，为RNA病毒。慢病毒整体大小约100纳米，具有诸如携带基因片段容量大、转染效率高、宿主范围广、长期稳定表达等优点，现已经成为转移目的基因的理想载体并用于临床治疗，是构建CAR-T细胞的常见病毒之一。在美国已经上市的两个靶向CD19的CAR-T药品，即，诺华的Kymriah和凯特的Yescarta，均采用慢病毒作为其载体。由于1次基因治疗需要1.0E+12TU的病毒总量，因此提高慢病毒的包装与生产水平可有效地促进慢病毒介导的基因治疗的发展。目前，慢病毒生产是通过质粒瞬转细胞基质产生有限次数的包装病毒，其一般构建流程是将病毒包装所必需的3个蛋白Gag/Pol、Rev、VSV-G(代替HIV-1的Env)分别独立地放置在3个质粒上，目的基因的载体构建在质粒pLenti-gene上，然后将四个质粒按比例共转染宿主细胞，进行培养后收集上清，纯化病毒。
逆转录病毒为单链RNA病毒。逆转录病毒基因组大约10Kb，含有三个重要的基因，从5’到3’分别为Gag(编码病毒的核心蛋白)、Pol(逆转录酶)、Env(病毒颗粒表面的被膜糖蛋白)。同时有长末端重复序列(LTR)和包装信号ψ顺式作用元件。逆转录病毒的生产 常选用质粒转染细胞基质、构建和筛选稳定产毒细胞株的方式。逆转录病毒载体的整合位点通常位于染色体开放区和转录活性区，大大提高了获得高效表达外源目的基因细胞系的机率。同时，逆转录病毒也是构建CAR-T细胞的常见病毒载体之一。
腺病毒(Adenovirus，缩写为AdV)是一种没有包膜的球状颗粒，其DNA以线性双链形式存在。腺病毒没有囊膜，核衣壳的直径为70-80纳米，呈二十面体立体对称。腺病毒是一种比较安全的载体，超过50％的人群体内均有5型腺病毒的抗体，并且由于其具有诸如转基因效率高、转导细胞类型广泛、易于制备和纯化、进入宿主细胞后仅瞬时表达而不整合到宿主细胞基因组等优点，因此在基因治疗、基因免疫以及疫苗制备方面的地位越来越突出，被广泛应用于疫苗开发、免疫治疗、基因治疗等各个领域。
溶瘤腺病毒包括(但不限于)：人5型腺病毒或人嵌合型腺病毒；具体包括(例如)：Onyx-015(可得自Onyx Pharmaceuticals公司)、H101(可得自上海三维生物技术有限公司)、Ad5-yCD/mutTKSR39rep-hIL12(可得自Henry Ford Health System公司)、CG0070(可得自Cold Genesys公司)、DNX-2401(可得自DNAtrix公司)、OBP-301(可得自Oncolys BioPharma公司)、ONCOS-102(可得自Targovax Oy公司/Oncos Therapeutics公司)、ColoAd1(可得自PsiOxus Therapeutics公司)、VCN-01(可得自VCN Biosciences公司)、ProstAtak TM(可得自Advantagene公司)等。
腺相关病毒(Adenovirus-associated virus，缩写为AAV)属细小病毒科，为无包膜的单链线状DNA病毒，基因组DNA小于5Kb。腺相关病毒具有诸如安全性高、免疫原性低、宿主范围广、表达稳定、同时能感染分裂细胞和非分裂细胞等优点，在生物医药行业尤其是基因治疗领域具有非常诱人的前景。重组AAV大多留在生产细胞内。
在一个实施方案中，本发明通过采用293细胞来生产痘苗病毒(包括溶瘤痘病毒)。该生产方法包括：
将痘苗病毒工作种子接种至293细胞，接种量可为0.001-0.2MOI，例如为0.005、0.01、0.02、0.03、0.05、0.08、0.1、0.15MOI；
对接种细胞进行培养使细胞病变，优选培养至细胞完全病变(一般为48-96h)，例如可采用细胞瓶进行培养，培养方式例如为贴壁培养；以及
使培养得到的细胞与本发明的收获液组合物接触，一步收获病毒，
其中所述收获液组合物包含：胰酶、pH缓冲液和任选的核酸酶，并且所述收获液组合物的pH为大于7.5且不超过10.5，优选为8.5-9.5；优选地，所述收获液组合物包含Tris盐缓冲液、胰酶和核酸酶，其中Tris盐缓冲液的浓度可为1-50mM，例如为1、3、5、8、10、20、30、40mM，优选为1-10mM；胰酶的浓度可为0.01-0.12％(w/v)，例如为0.03、0.05、0.07、0.09、0.11％(w/v)，优选为0.03-0.06％(w/v)；核酸酶的浓度可为1-100IU/ml，例如为1、3、5、10、20、40、60、80IU/ml，优选为1-50IU/ml，还优选为1-5IU/ml；所述收获液组合物的渗透压优选为低渗的，pH优选为8.5-9.5；
其中收获液组合物的用量可为大于等于35μl收获液组合物/cm 2细胞，优选为大于等于70μl收获液组合物/cm 2细胞，例如为75、100、125、150、200μl收获液组合物/cm 2细胞；
其中所述培养得到的细胞与收获液组合物接触的时间一般不超过60分钟，通常在5-60分钟的接触时间内(例如为10、20、30、40、50分钟)所述细胞就可被完全裂解。
本发明的病毒生产方法采用温和的手段，获得经培养的细胞内的病毒，不涉及冻融、超声、机械破碎等细胞裂解方法或步骤，工艺简单、便于操作、容易放大、非常适合通过大规模细胞培养来生产病毒，而且，与传统常用的现有技术方法(如冻融方法)相比，病毒产量显著提高，是现有技术方法的5-10倍，同时能保证病毒颗粒的完整性，而不会损害病毒的生物活性，因此使得大规模病毒生产成为可能。
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
本文中的所有数值都应理解为用词语“约”修饰。由端值表示的数值范围包括该范围内的所有数值和子集(例如，1到100包括1、10、30、50、100等)，虽然受篇幅所限只能具体列出几个例子，但之间的数值的所有可能组合(包括所给的最低值和最高值)被认为是在本申请中明确述及的。
例子
若未特别指明，以下例子中所用的技术手段为本领域技术人员所熟知的常规手段；所用的试剂为市售商品。以下除非特别说明，否则各试剂的百分浓度(％)均指该试剂的体积百分浓度(％(v/v))。所用的DMEM、MEM培养基、血清、试剂均为常用市售产品，并按照制备例1的说明进行收获液组合物(也称为处理液或收获液)的配制。
以下例子中所用的材料说明如下：
1.Tris base(得自Biosharp，货号77-86-1)
400mmol/L Tris base母液配制：称取1.938g Tris base，加入少量注射用水溶解，再用注射用水定容至40ml。1mM Tris是采用注射用水将400mmol/L Tris base母液稀释而成的。
2.NaHCO 3(得自Gibco，货号25080-094)
用超纯水将NaHCO 3配制成7.5％(w/v)的NaHCO 3溶液。
3.DMEM(来源：Gibco；货号：C11965500CP)
甲基纤维素(来源：生工生物；产品编号：A600615-0250)
4％甲基纤维素：称取甲基纤维素8.0g加入160ml超纯水摇动，再用量筒定容至200ml，121℃灭菌20min，冷却至室温，4℃保存，每天摇动，直至甲基纤维素完全溶解。
半固体培养基：取DMEM培养基360ml、FBS 15ml、4％甲基纤维素125ml摇动混匀，4℃保存。
4.胰酶(得自Gibco，货号A12177-01)
使用时用处理液稀释至终浓度0.01-0.12％(w/v)。
5.核酸酶(得自Merck，货号70746-10KUN)
使用时用处理液稀释至终浓度1-100IU/ml。可添加(例如)1mM MgCl 2，使核酸酶发挥作用。
6.缓冲液PBS的组成为：137mM NaCl、2.7mM KCl、10mM Na 2HPO 4、2mM KH 2PO 4，pH 7.2。
缓冲液“磷酸二氢钾和磷酸氢二钠”的组成为：0.29％(g/ml)磷酸二氢钾、0.026％(g/ml)磷酸氢二钠，pH 7.2-7.4。
所用的磷酸二氢钾得自上海国药，CAS:7778-77-0；磷酸氢二钠得自沪试，货号：10020318；均为分析纯。
7.痘苗病毒工作种子：痘苗病毒为PCT专利申请公开No.WO2019/062234A1中所述的重组溶瘤痘病毒DDvv-hIL21，其制备方法参见该专利文献中的制备例1和2。
9. 293[HEK-293]细胞来源于ATCC，货号
Figure PCTCN2020097901-appb-000002
CRL-1573。Vero细胞来源于ATCC，货号
Figure PCTCN2020097901-appb-000003
CCL-81。HeLa细胞来源于ATCC，货号
Figure PCTCN2020097901-appb-000004
CCL-2)。SLF-1细胞株来源于中国普通微生物菌种保藏管理中心，编号CGMCCNo.4875。
以下例子中所用的测试方法说明如下：
1、台盼蓝染色法计数
将细胞用PBS洗后，用胰蛋白酶消化，细胞悬浮在PBS中，加入终浓度为0.04％(w/v)的台盼蓝染液，显微镜下计数，死细胞会染成蓝色，活细胞为透明无色。取活细胞数为最终数据。
2、病毒滴度测定
(1)噬斑形成单位(plaque forming unit，缩写为pfu)法测定病毒滴度
将Vero细胞接种于6孔板中，待细胞铺满6孔板的80％以上时，轻轻吸出细胞培养液，加入经细胞维持液稀释好的待测滴度病毒样品500μl，每个稀释度设5个平行孔，每板设1个细胞维持液孔作为阴性对照，轻轻摇匀，37℃作用2h，每隔30-60min轻摇1次；吸弃孔内液体，加入DMEM培养基2ml漂洗1次，以除去未吸附的游离病毒，加入半固体培养基3-4ml，在37℃下5％CO 2孵箱静止培养2-3d， 期间注意观察病毒蚀斑的形成；(以下步骤不要求无菌)取出6孔板，吸弃半固体培养基，加入1ml 1％结晶紫染色液，室温静止30min-3h；流水轻柔洗弃染色液，计数(或干燥后计数)每孔蚀斑个数。按下式计算病毒滴度。
病毒滴度(pfu/ml)＝病毒蚀斑平均数×稀释倍数/病毒加入体积(ml)
(2)组织培养半数感染剂量(TCID50)法测定滴度
293细胞用DMEM培养液制成浓度约为1×10 5/ml的悬液，以100μl/孔接种于96孔板(同时按10倍稀释制备病毒稀释液分别感染上述细胞)。每排前10个孔分别加入100μl同一浓度的病毒稀释液，第11、12孔加入等体积2％BCS的DMEM做阴性对照。置于CO 2孵箱内37℃培养10d，然后在荧光倒置显微镜下观察，判断并记录每排细胞病变效应(CPE)情况。判断的标准是只要有少量细胞发生CPE即为阳性，若不能判断是CPE还是细胞死亡时，则与后面的阴性对照比较。
按照下述公式计算滴度：T＝10 1+d(S-0.5)IU/ml
其中d＝Log10稀释倍数，S＝从第一次稀释起的阳性比率之和，2次平行实验得到的滴度值应相差≤10 0.7。
3、病毒颗粒数测定
用病毒裂解液充分裂解，以病毒保存液为空白，测定本品OD260。
病毒样品经SDS(十二烷基硫酸钠)处理后通过测定A260nm值(UV-SDS法)来确定病毒颗粒数：取病毒250μl，加等体积0.2％SDS溶液裂解，振荡混匀，56℃水浴中放置10min。等温度降至室温时，瞬时离心。以病毒稳定液与0.2％SDS溶液等体积混匀后的溶液作空白对照，测定波长260nm与280nm处的吸光值，平行实验2次。
计算公式：病毒颗粒数＝A260nm×稀释倍数×1.1×10 12
制备例1
1、用于收获的处理液(也称收获液)的配制
于无菌环境中配制收获液，步骤如下：
(1)向注射用水中根据表格1分别加入相应量的试剂，充分溶解；
(2)将步骤(1)已充分溶解的溶液采用注射用水定容至目标体积；
(3)调至相应的pH值；
(4)过滤除菌，室温保存，备用；
(5)采用全自动冰点渗透压计法测定各溶液渗透压，结果如下：
1mM Tris渗透压：1mOsmol/kg
7.5％NaHCO 3渗透压：1785mOsmol/kg
表1处理液主要成分组成
Figure PCTCN2020097901-appb-000005
2、细胞培养
鸡胚成纤维细胞(CEF)的制备：
取7-9日龄SPF鸡胚，使用碘酒和75％的酒精棉球擦拭鸡蛋表面，取鸡胚后，去除鸡胚的头部、四肢和内脏，剪碎剩余部分组织块，使用胰酶在室温静置消化组织块15-20min，加入DMEM完全培养基，自动移液枪反复吹洗，放置5min沉降，DMEM稀释组织后，用四层无菌纱布过滤处理后的组织3次，最后进行单个细胞的计数，将处理好的CEF细胞进行37℃过夜培养，显微镜下镜检其形态和质量，细 胞台盼蓝染色计数，活细胞计数仪确认，计算制备的CEF细胞的活性。镜检细胞形态完整、状态良好。
采用常规技术培养293、Vero、HeLa等细胞，过程如下：将汇合率80％-90％的贴壁细胞于37℃、5％CO 2培养箱中取出，先用75％的酒精喷洒于培养瓶表面，将细胞培养瓶旋转放于生物安全柜中，用无菌移液管吸净其中的培养基，加一定量预热的PBS洗涤一次，吸净PBS。向细胞瓶中加入0.25％(w/v)胰酶，摇动使其充分平铺于细胞表面。盖好瓶盖，置于37℃、5％CO 2培养箱中消化，显微镜下观察细胞是否变圆，若未完全变圆可继续消化，直至细胞完全变圆；加一定体积含10％FBS的培养基终止反应，用移液管轻轻将瓶上的细胞吹下，并反复吹打将细胞重悬。取重悬后细胞至新细胞培养瓶中，加入10％FBS的培养基，于37℃、5％CO 2培养箱中培养，每天观察，至汇合率80％-90％时再次进行传代。
实施例2处理液裂解细胞效力试验
采用T75细胞培养瓶(每瓶细胞培养面积75cm 2)培养293、Vero、HeLa细胞，待细胞长至单层后，用于收获液裂解细胞效力试验。
(1)处理液的配制：按照制备例1所述的处理液配方进行配制。
(2)破碎细胞效力试验(胰酶消化后细胞裂解情况)
将长至单层的293、Vero、HeLa细胞弃原生长液，用1ml 0.25％(w/v)胰酶，37℃消化2min使细胞消化完全，加入5-10ml 10％FBS+DMEM培养基终止消化，混合均匀后，300g离心10min，弃上清，细胞沉淀分别取制备例1中A-E组处理液8ml，混合均匀后，室温静置(最长120min)，计时并显微镜下观察细胞形态改变情况，取少量细胞悬液于显微镜下观察细胞破碎情况，等显微镜下观察不到完整细胞时(视为完全裂解)，将悬液置300g离心10min，看离心后是否有沉淀。每组实验重复三次以上。
(3)破碎细胞效力试验(原位细胞裂解)
将长至单层的293、Vero、HeLa细胞弃原生长液，贴壁细胞分 别取制备例1中A-E组处理液2-5ml清洗细胞表面1-2次，再加入8ml相同的新鲜处理液，室温静置，计时并显微镜下观察细胞形态改变情况，待显微镜下观察不到完整细胞时(视为完全裂解)，将悬液置300g离心10min，看离心后是否有沉淀。每组实验重复三次以上。
试验结果分别见表2、表3。
表2胰酶消化后细胞裂解情况
Figure PCTCN2020097901-appb-000006
表3原位细胞裂解情况
Figure PCTCN2020097901-appb-000007
Figure PCTCN2020097901-appb-000008
本实施例中，无论是胰酶消化后细胞裂解试验，还是原位细胞裂解试验，试验结果均显示：
处理液A、B组均可以在15分钟内使细胞完全裂解，且B组细胞完全裂解时间较A组短；细胞碎片小，细胞悬液离心后，无明显沉淀且上清不粘，细胞核酸去除效果明显。细胞裂解比例为100％；293、Vero、HeLa细胞无明显差异；
处理液C组(对照组)细胞裂解效果差，裂解120min时，显微镜下仍可见很多细胞形态，细胞悬液离心后，可见明显沉淀；上清中较粘，说明有部分细胞已经裂解，释放出核酸等胞内物质；
处理液D、E组(对照组)随着裂解时间的延长，基本也能使细胞裂解，显微镜下观察可见细胞碎片较大，细胞悬液离心后，无明显沉淀且上清不粘，细胞核酸去除效果明显。细胞裂解比例达不到100％；293、Vero、HeLa细胞无明显差异；
比较表2和表3的胰酶消化后细胞裂解情况与细胞原位裂解情况，可发现各组收获液裂解效果基本一致，细胞先用胰酶消化比原位裂解细胞时间要短，胰酶消化后用收获液处理效果要稍好于原位裂解。处理液A、B组不管是用胰酶消化还是原位裂解293、Vero、HeLa细胞，均能在30min内有效裂解，说明本发明所述收获液在293、Vero、 HeLa细胞中均适用。
实施例3病毒的培养和处理液对病毒滴度的影响
痘苗病毒的培养和收获：将293、HeLa、CEF细胞分别接种至T75细胞培养瓶，待细胞长至贴壁汇合度大于80％时，将痘苗病毒工作种子按照MOI为0.02接种至各细胞，将接种病毒的细胞瓶置37.0±1.0℃吸附2小时；吸附结束后补加含有2％FBS的病毒维持液，置37.0±1.0℃培养箱中继续培养，待细胞完全病变后(一般48-96h)，弃去培养液，分别加入制备例1所述的A-E组处理液，处理10-60min，显微镜下观察细胞状态，待细胞完全裂解后(最多裂解60min)，收集病毒液，记录细胞裂解时间、细胞破碎情况。将病毒液300g离心10min后取上清检测病毒滴度，并记录离心后是否有沉淀和上清是否粘稠等情况。同时，以如下方式设置冻融对照组(简称冻融组)：将完全病变后的细胞用移液器吹打下来，将细胞和感染培养基转移至15ml无菌离心管中。置于-80℃冰箱中快速冷冻120min，再迅速放入37℃水浴中解冻10min，反复3次，将病毒液750g离心10min后取上清检测病毒滴度。每组实验重复三次以上，取平均值做统计学分析。
采用空斑法(即噬斑形成单位法)测定病毒滴度，根据各项检测结果，评价各组处理液的收获效果以及对病毒滴度的影响。结果见下表4。
表4处理液收获痘苗病毒试验结果
Figure PCTCN2020097901-appb-000009
本实验结果显示：
处理液A、B组均可以在10分钟内使细胞完全裂解；细胞碎片小，细胞悬液离心后，无明显沉淀且上清不粘，核酸去除效果明显。细胞裂解比例为100％；293、HeLa细胞无明显差异；
处理液C组(对照组)细胞裂解效果差，裂解60min时，显微镜下仍可见很多细胞形态，细胞悬液离心后，可见明显沉淀；上清中较粘，说明有部分细胞已经裂解，释放出核酸等胞内物质；
处理液D、E组(对照组)随着裂解时间的延长，基本也能使细胞裂解，显微镜下观察可见细胞碎片较大，细胞悬液离心后，无明显沉淀且上清不粘，核酸去除效果明显。细胞裂解比例达不到100％；293、HeLa细胞无明显差异；
F组(冻融对照组)经冻融3次后，显微镜下仍可见细胞形态、细胞碎片较大，但细胞悬液离心后，无明显沉淀，上清较粘且有絮状 飘浮物，说明经冻融3次后，细胞裂解，释放出核酸等胞内物质。细胞裂解比例达不到100％；293、HeLa细胞无明显差异。
处理液A、B组通过原位裂解293、HeLa细胞，均能在10min内有效裂解，说明本发明所述收获液在293、HeLa细胞中均适用。(病毒滴度值偏差≤30％pfu/ml视为可接受的检测误差范围，下同。)经过体外体内抗肿瘤活性检测，所制备的溶溜病毒生物活性均未受损害。
实施例4不同处理方式对收获病毒的影响
培养293细胞，待细胞长至贴壁汇合度大于80％时，将痘苗病毒工作种子按照MOI为0.02接种至细胞，将接种病毒的T75细胞瓶置37.0±1.0℃吸附2小时；吸附结束后补加含有2％FBS的病毒维持液，置37.0±1.0℃培养箱中继续培养，培养48小时。待细胞完全病变后收获胞内病毒和上清(胞外病毒)。胞内病毒分别加入8ml收获用处理液G和收获用处理液H，处理10min，显微镜下观察细胞状态，待细胞完全裂解后，收集病毒液。将病毒液300g离心10min后取上清检测病毒滴度。同时，以如下方式设置冻融对照组：将完全病变后的细胞用移液器吹打下来，将细胞和感染培养基转移至15ml无菌离心管中。置于-80℃冰箱中快速冷冻120min，再迅速放入37℃水浴中解冻10min，反复3次，将病毒液750g离心10min后取上清检测病毒滴度。评价处理液处理后收获的病毒滴度。每组实验重复三次以上，取平均值做统计学分析。
本实验结果如图1所示。该图示出，在细胞完全裂解释放出病毒时收获，所得到的结果因处理胞内病毒的方式不同而相差较大，其中：冻融处理方式的产量低；处理液G组(1mM Tris+0.06％(w/v)胰酶+5IU/ml核酸酶)处理后产量是冻融组的约6倍；处理液H组(7.5％NaHCO 3+0.06％(w/v)胰酶+5IU/ml核酸酶)是冻融组的约5倍。
实施例5不同用量处理液处理对收获病毒的影响
将293接种至T75细胞培养瓶，待细胞长至贴壁汇合度＞80％ 时，将痘苗病毒工作种子按照MOI为0.02接种至细胞，将接种病毒的细胞瓶置37.0±1.0℃吸附0-2小时；吸附结束后补加含有2％FBS的病毒维持液，置37.0±1.0℃培养箱中继续培养，待细胞完全病变后收获病毒。弃去培养液，分别加入制备例1所述的A组处理液，根据培养细胞面积，按35μl/cm 2、70μl/cm 2、150μl/cm 2、分别于处理10、30min，显微镜下观察细胞状态，收集病毒液，记录细胞破碎情况。将病毒液750g离心10min后取上清检测病毒滴度，并记录离心后是否有沉淀和上清是否粘稠等情况。同时，以如下方式设置冻融对照组：将完全病变后的细胞用移液器吹打下来，将细胞和感染培养基转移至15ml无菌离心管中。置于-80℃冰箱中快速冷冻120min，再迅速放入37℃水浴中解冻10min，反复3次，将病毒液750g离心10min后取上清检测病毒滴度。采用空斑法测定病毒滴度。根据各项检测结果，评价以不同用量和不同时间的处理液处理收获病毒对收获效果和滴度的影响。结果见下表5。
表5处理液用量和处理时间的不同与冻融组相对照的试验结果
Figure PCTCN2020097901-appb-000010
本实验结果显示：
处理用量与处理时间对细胞的裂解效果和病毒的产量影响显著。用用量为35、70、150μl/cm 2的A组处理液处理收获的胞内病毒，均能在30min内有效裂解细胞，细胞裂解比例为100％，且上清不粘。细胞裂解效率与处理液用量和处理时间呈正相关，处理液用量为 150μl/cm 2、处理时间10min时，病毒滴度最高，为冻融组的6倍以上。
实施例6多种示例性病毒的培养和收获
水痘病毒的培养和收获：将SLF-1接种至T25细胞培养瓶，待细胞长至单层，将Oka株水痘带状疱疹病毒(来自ATCC，编号为VR-795)工作种子按照MOI为0.001-0.1接种至SLF-1细胞，将接种病毒的细胞瓶置35.0±1.0℃吸附0.5-2小时；吸附结束后补加病毒MEM维持液，置35.0±1.0℃培养箱中继续培养48-96h，弃去培养液，分别加入制备例1所述的A-E组收获液，室温下静置10min后，显微镜下观察细胞状态，待细胞发生明显膨胀时震摇使细胞脱落，收集病毒液。进行细胞膨胀时间、细胞脱落比例、细胞破碎情况和病毒滴度等各项检测。同时，设置冻融收获对照组。根据各项检测结果，评价各组收获液的收获效果以及对病毒滴度的影响。
HSV的培养与收获：将Vero细胞置37℃，5％CO 2培养箱中扩增培养。待细胞长至单层后，以MOI值分别为0.001-0.1感染。待细胞出现变圆而尚未脱落时收获感染细胞和上清，分别加入制备例1所述的A-E组处理液，处理10-60min，显微镜下观察细胞状态，待细胞完全裂解后(最多裂解60min)，收集病毒液，记录细胞裂解时间、细胞破碎情况。将病毒液300g离心10min后取上清检测病毒滴度，并记录离心后是否有沉淀和上清是否粘稠等情况。同时，设置冻融收获对照组和上清对照组。采用空斑法测定病毒滴度，根据各项检测结果，评价各组处理液处理不同时间点收获效果以及对病毒滴度的影响。
腺病毒的培养与收获：将293细胞置37℃，5％CO 2培养箱中扩增培养。待细胞长至单层后，以MOI值分别为1-100感染。待细胞出现变圆而尚未脱落时收获感染细胞和上清，分别加入制备例1所述的A-E组处理液，处理10-60min，显微镜下观察细胞状态，待细胞完全裂解后(最多裂解60min)，收集病毒液，记录细胞裂解时间、细胞破碎情况。将病毒液300g离心10min后取上清检测病毒滴度， 并记录离心后是否有沉淀和上清是否粘稠等情况。同时，设置冻融收获对照组和上清对照组。根据TCID50法测定病毒滴度，同时评价各组处理液处理不同时间点收获效果以及对病毒滴度的影响。
慢病毒的培养与收获：将293细胞置37℃，5％CO 2培养箱中扩增培养。加入4种质粒(即将病毒包装所必需的3个蛋白Gag/Pol、Rev、VSV-G(代替HIV-1的Env)分别独立地放置在3个质粒上，目的基因的载体构建在质粒pLenti-gene上，得到4种质粒)后，感染细胞，待细胞出现变圆而尚未脱落时收获感染细胞和上清，分别加入制备例1所述的A-E组处理液，处理10-60min，显微镜下观察细胞状态，待细胞完全裂解后(最多裂解60min)，收集病毒液，记录细胞裂解时间、细胞破碎情况。将病毒液300g离心10min后取上清检测病毒滴度，并记录离心后是否有沉淀和上清是否粘稠等情况。同时，设置冻融收获对照组和上清对照组。根据各项检测结果，评价各组处理液处理不同时间点收获效果以及对病毒滴度的影响。
实施例7病毒生产工艺的放大试验
293细胞复苏后用含10％小牛血清的DMEM培养液进行扩增培养，当细胞汇合度为80％-90％时接种细胞工厂，于CO 2培养箱培养约48小时后消化细胞制成细胞悬液，用于接种细胞罐(得自NBS公司，型号为CelliGen Plus/310，表面积约22平方米)；经灌流培养细胞，6-8天后以MOI 0.02感染痘苗病毒，感染病毒约48-72小时后开始收获，其中用制备例1所述的G组处理液可收获得到1.3×10 13PFU/罐的病毒。
实施例8细胞工厂工艺放大实验
293细胞复苏后用含10％小牛血清的DMEM培养液进行扩增至3个10层细胞工厂(品牌：Corning，型号：“CellSTACK Chamber,10 STACK,POLYSTYRENE,STERILE,1/6”，3个10层细胞工厂总面积：6360*3＝19080cm 2)。当细胞汇合度为80％-90％时，以MOI＝0.02感染痘苗病毒，感染培养基：2％血清DMEM培养液。感染病毒约72小时后，将细胞拍打下来，和培养基一起转移至无菌离心瓶中， 配平，于4℃、2000rpm离心30分钟，弃上清，细胞沉淀用已经平衡至室温的3L处理液(约157μl/cm 2细胞)进行裂解收获，处理液组成为1mM Tris，0.03％重组胰酶，5U/ml核酸酶，pH9.0，同时添加1mM MgCl 2。可收获得到7.80×10 10PFU的病毒(单位面积产量为：4.09×10 6PFU/cm 2)。
实施例9发酵罐(5L)工艺放大实验
293细胞复苏至3个T225细胞培养瓶(品牌：Corning，货号：431082)，用含10％小牛血清的DMEM培养液进行扩增培养，当细胞汇合度为80％-90％时接种1个10层细胞工厂(品牌：Corning，型号：“CellSTACK Chamber,10 STACK,POLYSTYRENE,STERILE,1/6”，总面积：6360cm 2)，于CO 2培养箱培养约72小时后消化细胞制成细胞悬液，用于接种细胞罐(购自Eppendorf公司，型号为BioFlo 320，5L细胞罐表面积约150000cm 2)；接种体积470ml，细胞总量：1.78×10 9个细胞。经灌流培养细胞，6-8天后以MOI＝0.025感染痘苗病毒，感染病毒约72小时后开始用处理液原位裂解收获。处理液组成为1mMTris，0.03％重组胰酶，5U/ml核酸酶，pH9.3，同时添加1mM MgCl 2，；共用处理液约13.5L(约90μl/cm 2细胞)。可收获得到4.41×10 11PFU/罐的病毒(单位面积产量为：2.94×10 6PFU/cm 2)。
实施例10发酵罐(5L)工艺放大实验
293细胞复苏至3个T225细胞培养瓶(品牌：Corning，货号：431082)，用含10％小牛血清的DMEM培养液进行扩增培养，当细胞汇合度为80％-90％时接种1个细胞工厂(品牌：Corning，型号：“CellSTACK Chamber,10 STACK,POLYSTYRENE,STERILE,1/6”，总面积：6360cm 2)，于CO 2培养箱培养约72小时后消化细胞制成细胞悬液，用于接种细胞罐(购自Eppendorf公司，型号为BioFlo 320，5L细胞罐表面积约150000cm 2)；接种体积430ml，细胞总量：1.97×10 9个细胞。经灌流培养细胞，6-8天后以MOI＝0.06感染痘苗病毒，感染病毒约5天后用处理液进行原位裂解收获。处理液组成为1mM Tris， 0.03％重组胰酶，5U/ml核酸酶，pH9.3，同时添加1mM MgCl 2；共用处理液约15L(约100μl/cm 2细胞)。可收获得到2.41×10 11PFU/罐的病毒(单位面积产量为：1.61×10 6PFU/cm 2)。
实施例11发酵罐(14L)工艺放大实验
293细胞复苏至3个T225细胞培养瓶(品牌：Corning，货号：431082)，用含10％小牛血清的DMEM培养液进行扩增培养，当细胞汇合度为80％-90％时接种3个10层细胞工厂(品牌：Corning，型号：“CellSTACK Chamber,10 STACK,POLYSTYRENE,STERILE,1/6”，3个10层细胞工厂总面积：6360*3＝19080cm 2)，于CO 2培养箱培养约72小时后消化细胞制成细胞悬液，用于接种细胞罐(购自Eppendorf公司，型号为BioFlo 320，14L罐表面积约450000cm 2)；接种体积1400ml，细胞总量：3.19×10 9个细胞。经灌流培养细胞，6-8天后以MOI＝0.05感染痘苗病毒，感染病毒约72小时后用处理液开始原位裂解收获。处理液组成为1mM Tris，0.03％重组胰酶，5U/ml核酸酶，pH9.3，同时添加1mM MgCl 2；共用处理液约50L(约110μl/cm 2细胞)。可收获得到1.24×10 12PFU/罐的病毒(单位面积产量为：2.76×10 6PFU/cm 2)。
由上述实施例可见，通过采用本发明方法以及用于收获的处理液，能够大大简化生产工艺，从而特别适合对病毒进行大规模生产。采用本发明方法以及用于收获的处理液收获得到的病毒产量高，杂质低，进一步使得后续纯化工艺更加易于实施，获得的病毒纯度和生物活性也更高。
可以理解的是，以上实施方式仅仅是为了说明本发明的原理而采用的示例性实施方式，然而本发明并不局限于此。对于本领域内的普通技术人员来说，在不脱离本发明的精神和实质的情况下，可以做出各种变型和修改，这些变型和修改也在本发明的保护范围内。

Claims (24)

  1. 一种生产病毒的方法，所述方法包括：
    培养细胞，其中所述细胞为已接种病毒或已转染病毒包装用元件的细胞；以及
    使培养得到的细胞与收获液组合物接触，一步收获病毒，其中所述收获液组合物包含：胰酶、pH缓冲液和任选的核酸酶，并且所述收获液组合物的pH为大于7.5且不超过10.5。
  2. 根据权利要求1所述的方法，其特征在于，在所述收获液组合物中，所述胰酶的浓度为0.01-0.12％(w/v)，优选为0.03-0.06％(w/v)。
  3. 根据权利要求1所述的方法，其特征在于，在所述收获液组合物中，所述核酸酶的浓度为1-100IU/ml，优选为1-50IU/ml，还优选为1-5IU/ml。
  4. 根据权利要求1所述的方法，其特征在于，所述收获液组合物的渗透压的范围为0-50mOsmol/kg或800-2500mOsmol/kg，优选为0-20mOsmol/kg或1785-2000mOsmol/kg，更优选为1-20mOsmol/kg。
  5. 根据权利要求1所述的方法，其特征在于，所述收获液组合物的pH为8.5-9.5。
  6. 根据权利要求1所述的方法，其特征在于，所述pH缓冲液选自Tris盐缓冲液、碳酸氢钠缓冲液。
  7. 根据权利要求1所述的方法，其特征在于，所述培养是通过细胞瓶、细胞工厂或发酵罐进行的，培养方式包括贴壁培养和悬浮培养。
  8. 根据权利要求1所述的方法，其特征在于，当所述培养的方式为贴壁培养时，所述收获液组合物的用量为大于等于35μl收获液组合物/cm 2细胞，优选为大于等于70μl收获液组合物/cm 2细胞；当所述培养的方式为悬浮培养时，所述收获液组合物的用量为大于等于35μl收获液组合物/10 5个细胞，优选为大于等于70μl收获液组合物/10 5个细胞。
  9. 根据权利要求1所述的方法，其特征在于，所述培养得到的细胞与所述收获液组合物接触的时间为5-60分钟。
  10. 根据权利要求1所述的方法，其特征在于，所述细胞选自Vero细胞、293细胞、CEF细胞、HeLa细胞。
  11. 根据权利要求1所述的方法，其特征在于，所述病毒包括：痘苗病毒、水痘-带状疱疹病毒、轮状病毒、EV71病毒、甲肝病毒、单纯疱疹病毒、慢病毒、逆转录病毒、腺病毒、腺相关病毒、麻疹病毒、塞姆利基森林病毒、水疱性口炎病毒、脊髓灰质炎病毒、呼肠孤病毒、水疱性口炎病毒、脊髓灰质炎病毒、塞内卡谷病毒、埃可型肠道病毒、柯萨奇病毒、新城疫病毒和马拉巴病毒，优选为有包膜的病毒。
  12. 根据权利要求1所述的方法，其特征在于，所述细胞为293细胞，所述病毒为痘苗病毒。
  13. 根据权利要求1-5、7-12中任一项所述的方法，其特征在于，所述收获液组合物包含Tris盐缓冲液、胰酶和核酸酶，所述Tris盐缓冲液的浓度为1-50mM，优选为1-10mM。
  14. 一种用于收获经细胞培养的病毒的收获液组合物，其特征在 于，所述收获液组合物包含：胰酶、pH缓冲液和任选的核酸酶，并且所述收获液组合物的pH为大于7.5且不超过10.5。
  15. 根据权利要求14所述的收获液组合物，其特征在于，在所述收获液组合物中，所述胰酶的浓度为0.01-0.12％(w/v)，优选为0.03-0.06％(w/v)。
  16. 根据权利要求14所述的收获液组合物，其特征在于，在所述收获液组合物中，所述核酸酶的浓度为1-100IU/ml，优选为1-50IU/ml，还优选为1-5IU/ml。
  17. 根据权利要求14所述的收获液组合物，其特征在于，所述收获液组合物的渗透压的范围为0-50mOsmol/kg或800-2500mOsmol/kg，优选为0-20mOsmol/kg或1785-2000mOsmol/kg，更优选为1-20mOsmol/kg。
  18. 根据权利要求14所述的收获液组合物，其特征在于，所述收获液组合物的pH为8.5-9.5。
  19. 根据权利要求14所述的收获液组合物，其特征在于，所述pH缓冲液选自Tris盐缓冲液、碳酸氢钠缓冲液。
  20. 根据权利要求14所述的收获液组合物，其特征在于，所述培养是通过细胞瓶、细胞工厂或发酵罐进行的，培养方式包括贴壁培养和悬浮培养。
  21. 根据权利要求14所述的收获液组合物，其特征在于，所述细胞选自Vero细胞、293细胞、CEF细胞、HeLa细胞。
  22. 根据权利要求14所述的收获液组合物，其特征在于，所述 病毒包括：痘苗病毒、水痘-带状疱疹病毒、轮状病毒、EV71病毒、甲肝病毒、单纯疱疹病毒、慢病毒、逆转录病毒、腺病毒、腺相关病毒、麻疹病毒、塞姆利基森林病毒、水疱性口炎病毒、脊髓灰质炎病毒、呼肠孤病毒、水疱性口炎病毒、脊髓灰质炎病毒、塞内卡谷病毒、埃可型肠道病毒、柯萨奇病毒、新城疫病毒和马拉巴病毒，优选为有包膜的病毒。
  23. 根据权利要求14所述的收获液组合物，其特征在于，所述细胞为293细胞，所述病毒为痘苗病毒。
  24. 根据权利要求14-18、20-23中任一项所述的收获液组合物，其特征在于，所述收获液组合物包含Tris盐缓冲液、胰酶和核酸酶，所述Tris盐缓冲液的浓度为1-50mM，优选为1-10mM。
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