A method for producing viruses and a harvesting fluid composition

Abstract

Provided are a method for producing viruses and a harvesting fluid composition. The method comprises: culturing cells inoculated with viruses or transfected with virus packaging elements, contacting the cultured cells with a harvesting fluid composition, and harvesting the viruses in one step; the harvesting fluid composition comprises: pancreatin, pH buffer, and optional nuclease, with a pH greater than 7.5 and not exceeding 10.5.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular, to a method for producing viruses and a harvesting fluid composition, and more particularly, to a method for producing viruses by cell culture and a harvesting fluid composition for harvesting virus cultured by cells. Background Art

[0002] The preparation process of viral biological products, including oncolytic virus drugs, viral vaccines or recombinant viral vectors, generally includes the following steps: first, the virus is inoculated into uninfected cells of appropriate generation, and the infected cells are harvested when a certain degree of cytopathic effect is reached. After lysing the cells by different methods, the virus is harvested, and then purified by density gradient centrifugation or chromatography to obtain the stock solution. A certain buffer solution is added to obtain the semi-finished product, and the semi-finished product becomes the finished product after freeze-drying or subpackaging.

[0003] At present, large-scale cultivation of cells for the production of viral biological products mainly adopts the method of cell factories or fermenters. Most of the amplified viruses are intracellular. Therefore, in the virus harvesting process, it is necessary to effectively break and lyse the cells to release the viruses and ensure the integrity of the virus particles. Different harvesting processes have different effects on the degree of cell lysis and the integrity of virus particles. The harvesting method directly affects the virus titer, and the effective high titer value of the harvested virus stock solution is an important factor affecting the quality of the finished product.

[0004] Since vaccinia virus, herpes simplex virus, varicella zoster virus, adenovirus, etc. are intracellular viruses with strong cell binding activity, the cells must be collected and broken to obtain free virus particles when the virus is harvested. In the actual packaging and production process of recombinant viral vectors based on lentivirus, retrovirus, and adeno-associated virus, only extracellular free viruses are harvested, while a large number of intracellular viruses are not fully utilized.

[0005] At present, in the large-scale production process of these viruses, the freeze-thaw method is mostly used to lyse cells and harvest intracellular viruses. However, the freeze-thaw method has the disadvantages of high energy consumption, long cycle, many steps, difficulty in amplification, and unstable yield, which limits the production scale of viral biological products. There are also methods of harvesting intracellular viruses by hypotonic or chemical lysis, but a large number of viruses bound to the cell membrane are not fully released, and many chemical lysis reagents will also destroy the envelope of enveloped viruses (such as vaccinia virus, herpes simplex virus, etc.), making the virus lose its biological activity.

[0006] Therefore, there is still a need for a method that can effectively produce viruses through cell culture, as well as a harvest fluid that can effectively harvest the virus cultured through cells, which is crucial for obtaining high-quality viral vaccines or recombinant viral vectors and other products. Summary of the invention

[0007] In order to solve one or more problems existing in the above-mentioned prior art, the present invention provides a method for producing viruses and a harvest fluid composition.

[0008] Specifically, the present invention provides:

[0009] (1) A method for producing a virus, the method comprising:

[0010] Cultivating cells, wherein the cells are cells that have been inoculated with a virus or transfected with a virus packaging element; and

[0011] The cultured cells are contacted with a harvesting fluid composition to harvest the virus in one step, wherein the harvesting fluid composition comprises: trypsin, pH buffer and optional nuclease, and the pH of the harvesting fluid composition is greater than 7.5 and not more than 10.5.

[0012] (2) The method according to (1), characterized in that the concentration of the pancreatic enzyme in the harvest fluid composition is 0.01-0.12% (w / v), preferably 0.03-0.06% (w / v).

[0013] (3) The method according to (1) or (2), characterized in that the concentration of the nuclease in the harvest fluid composition is 1-100 IU / ml, preferably 1-50 IU / ml, and more preferably 1-5 IU / ml.

[0014] (4) The method according to any one of (1) to (3), characterized in that the osmotic pressure of the harvest fluid composition is in the range of 0-50 mOsmol / kg or 800-2500 mOsmol / kg, preferably 0-20 mOsmol / kg or 1785-2000 mOsmol / kg, and more preferably 1-20 mOsmol / kg.

[0015] (5) The method according to any one of (1) to (4), characterized in that the pH of the harvest liquid composition is 8.5-9.5.

[0016] (6) The method according to any one of (1) to (5), characterized in that the pH buffer is selected from Tris salt buffer and sodium bicarbonate buffer.

[0017] (7) The method according to any one of (1) to (6), characterized in that the culture is carried out in a cell bottle, a cell factory or a fermenter, and the culture methods include adherent culture and suspension culture.

[0018] (8) The method according to any one of (1) to (7), characterized in that when the culture method is adherent culture, the amount of the harvesting liquid composition is greater than or equal to 35 μl of the harvesting liquid composition / cm 2 cells, preferably greater than or equal to 70 μl of harvest fluid composition / cm 2 cells; when the culture method is suspension culture, the amount of the harvesting liquid composition is greater than or equal to 35 μl of the harvesting liquid composition / 10 5 cells, preferably greater than or equal to 70 μl of the harvest composition / 10 5 cells.

[0019] (9) The method according to any one of (1) to (8), characterized in that the cultured cells are in contact with the harvesting fluid composition for 5 to 60 minutes.

[0020] (10) The method according to any one of (1) to (9), characterized in that the cells are selected from Vero cells, 293 cells, CEF cells, and HeLa cells.

[0021] (11) The method according to any one of (1) to (10), characterized in that the virus includes: 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, vesicular stomatitis virus, poliovirus, reovirus, vesicular stomatitis virus, poliovirus, Seneca Valley virus, echovirus, coxsackievirus, Newcastle disease virus and Maraba virus, preferably an enveloped virus.

[0022] (12) The method according to any one of (1) to (11), characterized in that the cells are 293 cells and the virus is vaccinia virus.

[0023] (13) The method according to any one of (1)-(5) and (7)-(12), characterized in that the harvesting fluid composition contains Tris saline buffer, pancreatic enzyme and nuclease, and the concentration of the Tris saline buffer is 1-50mM, preferably 1-10mM.

[0024] (14) A harvesting fluid composition for harvesting virus cultured by cells, characterized in that the harvesting fluid composition comprises: pancreatic enzyme, pH buffer and optional nuclease, and the pH of the harvesting fluid composition is greater than 7.5 and not more than 10.5.

[0025] (15) The harvesting fluid composition according to (14), characterized in that the concentration of the pancreatic enzyme in the harvesting fluid composition is 0.01-0.12% (w / v), preferably 0.03-0.06% (w / v).

[0026] (16) The harvesting fluid composition according to (14) or (15), characterized in that the concentration of the nuclease in the harvesting fluid composition is 1-100 IU / ml, preferably 1-50 IU / ml, and more preferably 1-5 IU / ml.

[0027] (17) The harvesting fluid composition according to any one of (14) to (16), characterized in that the osmotic pressure of the harvesting fluid composition is in the range of 0-50mOsmol / kg or 800-2500mOsmol / kg, preferably 0-20mOsmol / kg or 1785-2000mOsmol / kg, and more preferably 1-20mOsmol / kg.

[0028] (18) The harvesting liquid composition according to any one of (14) to (17), characterized in that the pH of the harvesting liquid composition is 8.5-9.5.

[0029] (19) The harvest fluid composition according to any one of (14) to (18), characterized in that the pH buffer is selected from Tris salt buffer and sodium bicarbonate buffer.

[0030] (20) The harvest fluid composition according to any one of (14) to (19), characterized in that the culture is carried out by a cell bottle, a cell factory or a fermenter, and the culture method includes adherent culture and suspension culture.

[0031] (21) The harvest fluid composition according to any one of (14) to (20), characterized in that the cells are selected from Vero cells, 293 cells, CEF cells, and HeLa cells.

[0032] (22) The harvest fluid composition according to any one of (14) to (21), characterized in that the viruses include: 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, vesicular stomatitis virus, poliovirus, reovirus, vesicular stomatitis virus, poliovirus, Seneca Valley virus, echovirus, coxsackievirus, Newcastle disease virus and Maraba virus, preferably an enveloped virus.

[0033] (23) The harvest fluid composition according to any one of (14) to (22), characterized in that the cells are 293 cells and the virus is vaccinia virus.

[0034] (24) The harvesting fluid composition according to any one of (14)-(18) and (20)-(23), characterized in that the harvesting fluid composition contains Tris saline buffer, pancreatic enzyme and nuclease, and the concentration of the Tris saline buffer is 1-50mM, preferably 1-10mM.

[0035] Compared with the prior art, the present invention has the following advantages and positive effects:

[0036] 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 liquid composition of the present invention after extensive research, and the harvesting liquid composition can completely lyse the cells to harvest the virus, so there is no need to implement other cell lysis methods or steps such as freeze-thaw, ultrasound, and mechanical disruption. The virus production method of the present invention has the advantages of simple operation, easy amplification, stable yield, etc., and the yield is unexpectedly significantly improved compared with the prior art method, while ensuring the integrity of the virus particles without damaging the biological activity of the virus, so it is very suitable for large-scale production of viruses.

[0037] In the large-scale production process, the use of the method and the harvest fluid composition of the present invention can greatly simplify the production process, reduce production costs, and further obtain high-concentration, high-purity, and high-activity virus products. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The results of virus harvesting by different treatment solutions in one embodiment of the present application are shown. The horizontal axis represents different groups, and the vertical axis represents the average unit area yield of the virus (pfu / cm 2 ). DETAILED DESCRIPTION

[0039] The present invention will be further illustrated below through the description of specific implementation methods and with reference to the accompanying drawings, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0040] The virus production method of the present invention comprises: 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 fluid composition of the present invention to harvest the viruses in one step. The harvesting fluid composition of the present invention comprises: pancreatin, pH buffer and optional nuclease, and the pH of the harvesting fluid composition is greater than 7.5 and not more than 10.5.

[0041] The "viral packaging elements" mentioned herein refer to the cis-acting elements and trans-acting elements required for the packaging of lentivirus, retrovirus, adeno-associated virus, etc. by recombinant viral vectors.

[0042] The "one-step virus harvesting" described herein means that when the cultured cells are contacted with the harvesting fluid composition of the present invention, the harvesting fluid composition can completely lyse the cells and harvest the viruses, so there is no need to implement other cell lysis methods or steps such as freeze-thaw, ultrasound, and mechanical disruption.

[0043] The inventors of the present invention have found that if only the hypotonic buffer in the prior art is used, the purpose of one-step virus harvesting cannot be achieved, and the virus yield is still low; however, the inventors of the present invention have unexpectedly found that when the harvesting liquid composition of the present invention 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.

[0044] In the harvesting fluid composition, the concentration of pancreatin can be 0.01-0.12% (the % is the mass volume ratio (w / v), that is, the number of grams of pancreatin contained in each 100 ml of the harvesting fluid composition is 0.01-0.12g), for example, 0.03, 0.05, 0.07, 0.09, 0.11% (w / v), preferably 0.03-0.06% (w / v). The pancreatin can be commercially available, and can be a traditional pancreatin of animal origin or a recombinant pancreatin.

[0045] In the harvesting fluid composition, if a nuclease is included, the concentration of the nuclease may be 1-100 IU / ml, for example, 1, 3, 5, 10, 20, 40, 60, 80 IU / ml, preferably 1-50 IU / ml, and more preferably 1-5 IU / ml. A certain concentration of Mg may be added 2+ (e.g. 1-10 mM Mg 2+ , preferably 1-2 mM Mg 2+ ; for example 1 mM MgCl2), allows the nuclease to function.

[0046] The osmotic pressure of the harvest liquid composition may range from 0-50 mOsmol / kg (e.g., 1, 5, 10, 15, 30, 45 mOsmol / kg) or 800-2500 mOsmol / kg (e.g., 1000, 1250, 1500, 1700, 1900, 2100, 2300 mOsmol / kg). Preferably, the osmotic pressure of the harvest liquid composition is 0-20 mOsmol / kg or 1785-2000 mOsmol / kg, more preferably 1-20 mOsmol / kg, and further preferably 1-10 mOsmol / kg.

[0047] The pH of the harvest liquid composition is alkaline, more specifically greater than 7.5 and not more than 10.5, for example, 7.6, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, preferably 8.5-9.5. When the 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 activity of the virus is affected.

[0048] The pH buffer in the harvest fluid composition can be selected from Tris saline buffer and sodium bicarbonate buffer; preferably Tris saline buffer.

[0049] In one embodiment, the harvesting fluid composition of the present invention comprises Tris salt buffer, trypsin and nuclease. The 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.

[0050] In the method of the present invention, when the cells are cultured in an adherent culture manner, the amount of the harvesting fluid composition may be greater than or equal to 35 μl of the harvesting fluid composition / cm 2 cells, preferably greater than or equal to 70 μl of harvest fluid composition / cm 2 cells, for example, 75, 100, 125, 150, 200 μl of harvest composition / cm 2 Cells. In this article, square centimeters of cells (cm 2 Cells) refers to the sum of all cells grown on an area of ​​1 square centimeter of adherent cultured cells. When the cells are cultured in suspension, the amount of the harvesting liquid composition may be greater than or equal to 35 μl of the harvesting liquid composition / 10 5 cells, preferably greater than or equal to 70 μl of the harvest composition / 10 5 cells, for example, 75, 100, 125, 150, 200 μl of the harvest composition / 10 5 cells.

[0051] In the present invention, the contact time between the cultured cells and the harvesting liquid composition is generally no more than 60 minutes, and the cells can be completely lysed within a contact time of 5-60 minutes (e.g., 10, 20, 30, 40, 50 minutes). The cell lysis can be observed under a microscope, and the cells are considered to be completely lysed when no intact cells are observed under the microscope.

[0052] In the present invention, virus inoculation or virus packaging, and cell culture can be carried out in a conventional manner in the art. The virus 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. The term "MOI" or "multiplicity of infection" as used herein, that is, the ratio of the number of viruses to the number of cells, refers to the number of viruses infected per cell used to initiate virus infection. MOI = pfu / cell, that is, the number of cells × MOI = total PFU.

[0053] In the present invention, cell culture can be carried out, for example, by cell flasks (the scale can be: 25-225cm 2 cells / bottle), cell factories (scale can be: 500-800cm 2 Cell / layer, e.g. 632cm 2 Cells / layer) or fermentation tank (scale can be: 0.5-500m 2 The culture medium may be any of those commonly used in the art, such as DMEM and MEM. The method of the present invention may further include: before contacting the cultured cells with the harvested liquid composition, separating the cultured cells from the culture medium. In addition, the harvested virus may be further purified by tangential flow centrifugation and / or column chromatography.

[0054] Cells that can be used in the present invention include (but are not limited to): Vero cells, 293 cells, CEF cells (chicken embryo fibroblasts), and HeLa cells.

[0055] 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 that can support the proliferation of a variety of viruses, including herpes simplex virus, vaccinia virus, Japanese encephalitis, poliomyelitis, rabies and other viruses. WHO believes that Vero cells are safe to use within 150 generations and are non-tumorigenic, and have been approved for the production of human viral vaccines.

[0056] 293 cells are a human embryonic kidney hypotriploid cell line transformed with adenovirus type 5 strain 75 and containing the E1 region of adenovirus type 5. It is an E1 region defect complementation cell line. It was constructed by FL Graham and JS 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, etc. 293 cell lines are widely used in adenovirus, retrovirus, lentivirus, vaccinia virus production, gene expression and protein expression.

[0057] Chicken embryo cells are the earliest objects utilized in tissue culture, and tissue culture workers have done a large amount of research work with chicken embryos. Chicken embryo fibroblasts (CEF) are convenient in source, simple to prepare, have good tolerance, and are suitable for the growth and reproduction of many viruses, and are therefore widely used in the production of vaccines, the cultivation of viruses, and the research of some cells and molecular biology (for example, referring to literature: Meiser A., ​​et al., Comparison of virus production in chicken embryofibroblasts infected with the WR, IHD-J and MVA strains of vaccinia virus: IHD-J is most efficient in trans-Golgi network wrapping and extracellularenveloped virus release. J Gen Virol, 2003, 84 (Pt 6): p.1383-92).

[0058] HeLa cells are the first aneuploid epithelial cell line obtained from human tissue through continuous culture. It was established by GOGey et al. in 1951 from cervical cancer tissue of a 31-year-old black woman. Compared with other cancer cell lines, this cell line proliferates abnormally rapidly, is sensitive to poliovirus, adenovirus, vaccinia virus, etc., and shows obvious cell degeneration. It has great utilization value in virus research and production, and is also widely used in tumor research, biological experiments, cell culture, etc.

[0059] The 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, lentivirus, retrovirus, adenovirus, adenovirus-associated virus, measles virus, Semliki Forest virus, vesicular stomatitis virus, poliovirus, reovirus, vesicular stomatitis virus, poliovirus, Seneca Valley Virus, echoenterovirus, Coxsackie virus, Newcastle disease virus and maraba virus, preferably an enveloped virus. For example, enveloped viruses include: vaccinia virus, varicella-zoster virus, rotavirus, EV71 virus, coxsackie virus (CA16 virus), hepatitis A virus, herpes simplex virus, lentivirus, and retrovirus.

[0060] The viruses that can be produced by the present invention include oncolytic viruses, which can selectively replicate in tumor cells. The oncolytic viruses described in the present invention include genetically mutated viruses with oncolytic effects and wild-type viruses with oncolytic effects. The genetically mutated viruses with oncolytic effects include, but are not limited to, adenovirus, poxvirus (also known as vaccinia virus), herpes simplex virus (HSV), measles virus, Semliki Forest virus, vesicular stomatitis virus, poliovirus and retrovirus; the wild-type viruses with oncolytic effects include, but are not limited to, reovirus, vesicular stomatitis virus, poliovirus, Seneca Valley virus, echo enterovirus, Coxsackie virus, Newcastle disease virus and maraba virus. Exogenous genes may be integrated into the genome of the oncolytic virus, and the exogenous genes include exogenous immune regulation genes, exogenous screening genes, exogenous reporter genes, etc. No exogenous genes may be integrated into the genome of the oncolytic virus.

[0061] Vaccinia virus (or poxvirus) (Vaccinia virus, abbreviated as VV) belongs to the Poxviridae family and plays an important role in the history of human resistance to infection and disease. Before 1980, vaccinia virus was widely used mainly as a smallpox vaccine, and eventually the smallpox virus was completely eliminated worldwide. 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. According to the different stages of its infected cells, four different infection forms can be produced in the entire life cycle of vaccinia virus: intracellular mature virus particles (IMV), intracellular enveloped virus particles (IEV), cell-bound enveloped virus particles (CEV) and extracellular enveloped virus particles (EEV). In the vast majority of vaccinia virus strains (including WR strain, Copenhagen strain, Ankara strain, Tiantan strain, etc.), IMV accounts for more than 90% of the total progeny virus. This part of the virus is retained in the cell after assembly in the virus factory and is not released outside the cell until the cell lyses.

[0062] 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 (the preparation method can be found in the scientific literature: "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 (available from Genelux), TG6002 (available from Transgene), DDvv-IL21 (see PCT patent application publication No. WO2019 / 062234A1), etc.

[0063] Varicella-Zoster virus (VZV) belongs to the alphaherpesvirus subfamily and is a double-stranded DNA virus. Its primary infection is chickenpox, and reactivation of latent infection can cause herpes zoster. Vaccination is the most effective measure to prevent chickenpox and herpes zoster 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 with this vaccine is recognized as the most effective measure to prevent chickenpox caused by VZV.

[0064] Herpes simplex virus (HSV) belongs to the alphavirus subfamily of the herpesviridae family. Based on 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 be infected, anti-herpes drugs can be used to prevent unexpected proliferation, no integration into the host cell genome during the lytic and latent phases, and low risk of insertion mutations. It is widely used in gene therapy for tumors or neurodegenerative diseases. HSV is a lytic pathway and is mainly present in cells.

[0065] Oncolytic herpes simplex viruses include (but are not limited to): HSV-1, HSV-2 herpes simplex viruses; specifically include (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 Biological Company), NV1020 (available from Catherax), etc.

[0066] Lentivirus (abbreviated as LV) belongs to the Retroviridae family and is an RNA virus. The overall size of lentivirus is about 100 nanometers. It has advantages such as large capacity for carrying gene fragments, high transfection efficiency, wide host range, and long-term stable expression. It has now become an ideal vector for transferring target genes and used in clinical treatment. It is one of the common viruses for constructing CAR-T cells. Two CAR-T drugs targeting CD19 that have been launched in the United States, namely, Novartis' Kymriah and Kite's Yescarta, both use lentivirus as their vector. Since one gene therapy requires a total amount of 1.0E+12TU of virus, improving the packaging and production level of lentivirus can effectively promote the development of lentivirus-mediated gene therapy. At present, lentivirus production is to produce a limited number of packaged viruses through transient transfection of plasmids into cell matrices. The general construction process is to place the three proteins Gag / Pol, Rev, and VSV-G (replacing HIV-1's Env) required for virus packaging independently on three plasmids, and the vector of the target gene is constructed on the plasmid pLenti-gene. The four plasmids are then co-transfected into host cells in proportion, and the supernatant is collected after culture to purify the virus.

[0067] Retrovirus is a single-stranded RNA virus. The retrovirus genome is about 10Kb and contains three important genes, from 5' to 3', Gag (encoding the core protein of the virus), Pol (reverse transcriptase), and Env (envelope glycoprotein on the surface of the virus particle). There are also long terminal repeats (LTR) and packaging signal ψ cis-acting elements. The production of retroviruses often uses plasmid transfection of cell matrices, construction and screening of stable virus-producing cell lines. The integration sites of retrovirus vectors are usually located in the open region and transcriptional active region of the chromosome, which greatly increases the probability of obtaining a cell line that efficiently expresses the exogenous target gene. At the same time, retrovirus is also one of the common viral vectors for constructing CAR-T cells.

[0068] Adenovirus (AdV) is a spherical particle without an envelope, and its DNA exists in a linear double-stranded form. Adenovirus has no envelope, and the diameter of the nucleocapsid is 70-80 nanometers, with icosahedral symmetry. Adenovirus is a relatively safe vector, and more than 50% of the population has antibodies to adenovirus type 5. In addition, due to its advantages such as high transgenic efficiency, wide range of transduced cell types, easy preparation and purification, and only transient expression after entering the host cell without integration into the host cell genome, it has become increasingly prominent in gene therapy, genetic immunity, and vaccine preparation, and is widely used in various fields such as vaccine development, immunotherapy, and gene therapy.

[0069] Oncolytic adenoviruses include, but are not limited to, human adenovirus type 5 or human chimeric adenovirus; specifically include, for example, Onyx-015 (available from Onyx Pharmaceuticals), H101 (available from Shanghai Sanwei Biotechnology 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 OncolysBioPharma), ONCOS-102 (available from Targovax Oy / Oncos Therapeutics), ColoAd1 (available from PsiOxus Therapeutics), VCN-01 (available from VCN Biosciences), ProstAtak TM (available from Advantagene Corporation) and the like.

[0070] Adenovirus-associated virus (AAV) belongs to the Parvoviridae family. It is a non-enveloped single-stranded linear DNA virus with a genomic DNA of less than 5Kb. Adenovirus has advantages such as high safety, low immunogenicity, wide host range, stable expression, and the ability to infect both dividing and non-dividing cells. It has very attractive prospects in the biopharmaceutical industry, especially in the field of gene therapy. Recombinant AAV mostly remains in the production cells.

[0071] In one embodiment, the present invention produces vaccinia virus (including oncolytic poxvirus) by using 293 cells. The production method comprises:

[0072] The vaccinia virus working seed is inoculated into 293 cells, and 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;

[0073] Cultivating the inoculated cells to make them cytopathic, preferably culturing them until they are completely cytopathic (generally 48-96 hours), for example, culturing them in a cell flask, and the culturing method is, for example, adherent culture; and

[0074] The cultured cells are contacted with the harvesting liquid composition of the present invention to harvest the virus in one step.

[0075] The harvesting fluid composition comprises: pancreatin, pH buffer and optional nuclease, and the pH of the harvesting fluid composition is greater than 7.5 and not more than 10.5, preferably 8.5-9.5; preferably, the harvesting fluid composition comprises Tris salt buffer, pancreatin and nuclease, wherein the concentration of Tris salt buffer may be 1-50 mM, for example, 1, 3, 5, 8, 10, 20, 30, 40 mM, preferably 1-10 mM; the concentration of pancreatin may be 0.01-0 .12% (w / v), for example, 0.03, 0.05, 0.07, 0.09, 0.11% (w / v), preferably 0.03-0.06% (w / v); the concentration of the nuclease may be 1-100 IU / ml, for example, 1, 3, 5, 10, 20, 40, 60, 80 IU / ml, preferably 1-50 IU / ml, and further preferably 1-5 IU / ml; the osmotic pressure of the harvest fluid composition is preferably hypotonic, and the pH is preferably 8.5-9.5;

[0076] The amount of the harvesting fluid composition can be greater than or equal to 35 μl of the harvesting fluid composition / cm 2 cells, preferably greater than or equal to 70 μl of harvest fluid composition / cm 2 cells, for example, 75, 100, 125, 150, 200 μl of harvest composition / cm 2 cell;

[0077] The contact time between the cultured cells and the harvested liquid composition is generally no more than 60 minutes, and the cells can usually be completely lysed within 5-60 minutes of contact time (e.g., 10, 20, 30, 40, 50 minutes).

[0078] The virus production method of the present invention adopts mild means to obtain the virus in the cultured cells, and does not involve cell lysis methods or steps such as freeze-thaw, ultrasound, and mechanical disruption. The process is simple, easy to operate, easy to scale up, and very suitable for producing viruses through large-scale cell culture. Moreover, compared with the traditional commonly used existing technology methods (such as freeze-thaw methods), the virus yield is significantly improved, which is 5-10 times that of the existing technology methods. At the same time, the integrity of the virus particles can be guaranteed without damaging the biological activity of the virus, thereby making large-scale virus production possible.

[0079] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0080] All numerical values ​​herein should be understood to be modified by the word "about". The numerical range represented by the end value includes all numerical values ​​and subsets within the range (for example, 1 to 100 includes 1, 10, 30, 50, 100, etc.), although only a few examples can be specifically listed due to space limitations, all possible combinations of numerical values ​​between (including the given minimum and maximum values) are considered to be explicitly stated in this application.

[0081] example

[0082] Unless otherwise specified, 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, the percentage concentration (%) of each reagent refers to the volume percentage concentration (% (v / v)) of the reagent. The DMEM, MEM culture medium, serum, and reagents used are all commonly available commercial products, and the harvesting fluid composition (also referred to as treatment fluid or harvesting fluid) is prepared according to the instructions of Preparation Example 1.

[0083] The materials used in the following examples are described below:

[0084] 1. Tris base (obtained from Biosharp, product number 77-86-1)

[0085] Preparation of 400mmol / L Tris base stock solution: weigh 1.938g Tris base, add a small amount of injection water to dissolve, and then dilute to 40ml with injection water. 1mM Tris is prepared by diluting 400mmol / L Tris base stock solution with injection water.

[0086] 2. NaHCO3 (from Gibco, product number 25080-094)

[0087] NaHCO3 was prepared into a 7.5% (w / v) NaHCO3 solution using ultrapure water.

[0088] 3.DMEM (Source: Gibco; Catalog Number: C11965500CP)

[0089] Methylcellulose (Source: Sangon Biotech; Product No.: A600615-0250)

[0090] 4% methylcellulose: Weigh 8.0 g of methylcellulose, add 160 ml of ultrapure water and shake, then use a measuring cylinder to make up to 200 ml, sterilize at 121°C for 20 min, cool to room temperature, store at 4°C, and shake every day until the methylcellulose is completely dissolved.

[0091] Semi-solid culture medium: Take 360 ​​ml of DMEM culture medium, 15 ml of FBS, and 125 ml of 4% methylcellulose, shake and mix well, and store at 4°C.

[0092] 4. Pancreatin (from Gibco, product number A12177-01)

[0093] When used, dilute with treatment solution to a final concentration of 0.01-0.12% (w / v).

[0094] 5. Nuclease (from Merck, Catalog No. 70746-10KUN)

[0095] When used, dilute with treatment solution to a final concentration of 1-100 IU / ml. For example, 1 mM MgCl2 may be added to allow nuclease to function.

[0096] 6. The composition of buffer PBS is: 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4, pH7.2.

[0097] The composition of the buffer "monopotassium phosphate and disodium hydrogen phosphate" is: 0.29% (g / ml) monopotassium phosphate, 0.026% (g / ml) disodium hydrogen phosphate, pH 7.2-7.4.

[0098] The potassium dihydrogen phosphate used was obtained from Shanghai Guoyao, CAS: 7778-77-0; the disodium hydrogen phosphate was obtained from Shanghai Test, product number: 10020318; both were analytical grade.

[0099] 7. Vaccinia virus working seed: The vaccinia virus is the recombinant oncolytic poxvirus DDvv-hIL21 described in PCT patent application publication No. WO2019 / 062234A1. The preparation method thereof refers to Preparation Examples 1 and 2 in the patent document.

[0100] 9. 293 [HEK-293] cells were obtained from ATCC, catalog number CRL-1573. Vero cells were obtained from ATCC, catalog number CCL-81. HeLa cells were obtained from ATCC, catalog number The SLF-1 cell line was obtained from the China General Microbiological Culture Collection Center, with the number CGMCC No.4875.

[0101] The test methods used in the following examples are described as follows:

[0102] 1. Trypan blue staining method

[0103] After washing the cells with PBS, digesting with trypsin, suspending the cells in PBS, adding trypan blue dye solution with a final concentration of 0.04% (w / v), counting under a microscope, dead cells will be stained blue, and live cells will be transparent and colorless. The number of live cells is taken as the final data.

[0104] 2. Virus titer determination

[0105] (1) Determination of virus titer by plaque forming unit (PFU) method

[0106] Vero cells were inoculated in a 6-well plate. When the cells covered more than 80% of the 6-well plate, the cell culture medium was gently aspirated, and 500 μl of the virus sample to be tested diluted with the cell maintenance solution was added. Five parallel wells were set for each dilution, and one cell maintenance solution well was set for each plate as a negative control. The wells were gently shaken and allowed to act at 37°C for 2 hours, and the wells were gently shaken once every 30-60 minutes. The liquid in the wells was aspirated and discarded, and 2 ml of DMEM medium was added to rinse once to remove the unabsorbed free virus. 3-4 ml of semi-solid culture medium was added, and the wells were incubated in a 5% CO2 incubator at 37°C for 2-3 days. During this period, the formation of virus plaques was observed. (The following steps do not require sterility) The 6-well plate was taken out, the semi-solid culture medium was aspirated and discarded, and 1 ml of 1% crystal violet staining solution was added, and the wells were left to stand at room temperature for 30 minutes to 3 hours. The staining solution was gently washed and discarded with running water, and the number of plaques in each well was counted (or counted after drying). The virus titer was calculated according to the following formula.

[0107] Virus titer (pfu / ml) = average number of virus plaques × dilution factor / virus added volume (ml)

[0108] (2) Titer determination by tissue culture median infectious dose (TCID50) method

[0109] 293 cells were cultured in DMEM at a concentration of about 1 × 10 5 / ml suspension, inoculated in 96-well plates at 100μl / well (simultaneously prepare virus dilutions at 10 times dilution to infect the above cells respectively). Add 100μl of virus dilutions of the same concentration to the first 10 wells of each row, and add an equal volume of 2% BCS DMEM to the 11th and 12th wells as negative controls. Culture in a CO2 incubator at 37°C for 10 days, then observe under a fluorescent inverted microscope to determine and record the cytopathic effect (CPE) of each row. The judgment standard is that as long as a small number of cells have CPE, it is positive. If it is not possible to determine whether it is CPE or cell death, compare it with the negative control at the back.

[0110] The titer was calculated according to the following formula: T = 10 1+d(S-0.5) IU / ml

[0111] Where d = Log10 dilution factor, S = the sum of the positive ratios from the first dilution, and the titer values ​​obtained from two parallel experiments should differ by ≤10 0.7 .

[0112] 3. Determination of viral particle number

[0113] The virus was fully lysed with virus lysis buffer, and the virus storage buffer was used as blank to measure the OD260 of this product.

[0114] The virus sample was treated with SDS (sodium dodecyl sulfate) and the number of virus particles was determined by measuring the A260nm value (UV-SDS method): 250 μl of virus was taken, and an equal volume of 0.2% SDS solution was added for lysis, and the mixture was shaken and placed in a 56°C water bath for 10 minutes. When the temperature dropped to room temperature, centrifuged instantly. The solution obtained by mixing an equal volume of virus stabilization solution and 0.2% SDS solution was used as a blank control, and the absorbance at wavelengths of 260nm and 280nm was measured. The experiment was repeated twice in parallel.

[0115] Calculation formula: Number of virus particles = A260nm × dilution factor × 1.1 × 10 12

[0116] Preparation Example 1

[0117] 1. Preparation of treatment solution for harvesting (also called harvesting solution)

[0118] Prepare the harvest solution in a sterile environment as follows:

[0119] (1) Add the corresponding amount of reagents according to Table 1 to water for injection and fully dissolve;

[0120] (2) diluting the fully dissolved solution in step (1) to a target volume using water for injection;

[0121] (3) Adjust to the corresponding pH value;

[0122] (4) Filter and sterilize, store at room temperature and set aside;

[0123] (5) The osmotic pressure of each solution was measured using a fully automatic freezing point osmometer method. The results are as follows:

[0124] 1mM Tris osmotic pressure: 1mOsmol / kg

[0125] 7.5% NaHCO3 osmotic pressure: 1785mOsmol / kg

[0126] Table 1 Main components of the treatment solution

[0127]

[0128] 2. Cell culture

[0129] Preparation of Chicken Embryo Fibroblasts (CEF):

[0130] Take 7-9 day old SPF chicken embryos, wipe the surface of the eggs with iodine and 75% alcohol cotton balls, remove the head, limbs and internal organs of the chicken embryos, cut up the remaining tissue blocks, use pancreatin to digest the tissue blocks at room temperature for 15-20 minutes, add DMEM complete medium, blow and wash repeatedly with an automatic pipette, let it settle for 5 minutes, dilute the tissue with DMEM, filter the treated tissue with four layers of sterile gauze for 3 times, and finally count the single cells, culture the treated CEF cells at 37°C overnight, examine their morphology and quality under a microscope, count the cells with trypan blue staining, confirm with a live cell counter, and calculate the activity of the prepared CEF cells. Microscopic examination shows that the cell morphology is intact and in good condition.

[0131] 293, Vero, HeLa and other cells were cultured using conventional techniques. The process was as follows: the adherent cells with a confluence rate of 80%-90% were taken out from a 37°C, 5% CO2 incubator, and 75% alcohol was first sprayed on the surface of the culture bottle. The cell culture bottle was rotated and placed in a biological safety cabinet. The culture medium was sucked out with a sterile pipette, and a certain amount of preheated PBS was added to wash once, and the PBS was sucked out. 0.25% (w / v) pancreatin was added to the cell bottle, and it was shaken to fully spread on the cell surface. The bottle cap was covered and placed in a 37°C, 5% CO2 incubator for digestion. The cells were observed under a microscope to see if they were rounded. If they were not completely rounded, digestion could be continued until the cells were completely rounded; a certain volume of culture medium containing 10% FBS was added to terminate the reaction, and the cells on the bottle were gently blown off with a pipette, and the cells were resuspended by repeated blowing. The resuspended cells were transferred to a new cell culture flask, and cultured with 10% FBS culture medium. The cells were cultured in a 37°C, 5% CO2 incubator and observed every day. The cells were passaged again when the confluence rate reached 80%-90%.

[0132] Example 2 Treatment solution cell lysis efficacy test

[0133] T75 cell culture flasks (75 cm per flask) were used. 2 ) Culture 293, Vero, and HeLa cells, and after the cells grow to a monolayer, use the harvesting fluid for cell lysis efficiency testing.

[0134] (1) Preparation of the treatment solution: The treatment solution was prepared according to the formulation of Preparation Example 1.

[0135] (2) Cell disruption efficiency test (cell lysis after trypsin digestion)

[0136] The original growth medium of 293, Vero, and HeLa cells grown to a monolayer was discarded, and 1 ml of 0.25% (w / v) pancreatin was used to digest the cells at 37°C for 2 min to completely digest the cells. 5-10 ml of 10% FBS+DMEM medium was added to terminate the digestion, and after mixing evenly, the cells were centrifuged at 300g for 10 min, and the supernatant was discarded. The cell precipitate was taken 8 ml of the AE group treatment solution in Preparation Example 1, mixed evenly, and allowed to stand at room temperature (up to 120 min), and the cell morphology was observed under a microscope for timing and microscopic observation. A small amount of cell suspension was taken to observe the cell fragmentation under a microscope. When no intact cells were observed under the microscope (considered to be completely lysed), the suspension was centrifuged at 300g for 10 min to see if there was precipitation after centrifugation. Each group of experiments was repeated three times or more.

[0137] (3) Cell disruption efficacy test (in situ cell lysis)

[0138] The original growth medium of 293, Vero, and HeLa cells grown to a monolayer was discarded, and the adherent cells were washed with 2-5 ml of the AE group treatment solution in Preparation Example 1 for 1-2 times, and then 8 ml of the same fresh treatment solution was added, and the cells were allowed to stand at room temperature. The time was counted and the cell morphology was observed under a microscope. When no intact cells were observed under the microscope (considered to be completely lysed), the suspension was centrifuged at 300g for 10 minutes to see if there was precipitation after centrifugation. Each group of experiments was repeated three times or more.

[0139] The test results are shown in Table 2 and Table 3 respectively.

[0140] Table 2 Cell lysis after trypsin digestion

[0141]

[0142] Table 3 In situ cell lysis

[0143]

[0144]

[0145] In this example, both the cell lysis test after trypsin digestion and the in situ cell lysis test showed that:

[0146] Both treatment solutions A and B can completely lyse cells within 15 minutes, and the complete lysis time of cells in group B is shorter than that in group A; the cell fragments are small, and after centrifugation of the cell suspension, there is no obvious precipitation and the supernatant is non-sticky, and the cell nucleic acid removal effect is obvious. The cell lysis ratio is 100%; there is no significant difference among 293, Vero, and HeLa cells;

[0147] The cell lysis effect of treatment solution C group (control group) was poor. After lysis for 120 minutes, many cell morphologies were still visible under the microscope. After the cell suspension was centrifuged, obvious precipitation was visible. The supernatant was relatively sticky, indicating that some cells had been lysed and intracellular substances such as nucleic acids were released.

[0148] As the lysis time of treatment solution D and E groups (control group) increases, the cells can be basically lysed. Under the microscope, it can be seen that the cell fragments are large. After the cell suspension is centrifuged, there is no obvious precipitation and the supernatant is not sticky. The cell nucleic acid removal effect is obvious. The cell lysis ratio does not reach 100%; there is no significant difference among 293, Vero, and HeLa cells;

[0149] Comparing the cell lysis after trypsin digestion and the cell in situ lysis in Table 2 and Table 3, it can be found that the lysis effect of each group of harvesting solution is basically the same. The time of first digesting cells with trypsin is shorter than that of in situ lysis of cells. The effect of treating with harvesting solution after trypsin digestion is slightly better than that of in situ lysis. Whether 293, Vero, and HeLa cells are digested with trypsin or lysed in situ, the treatment solution A and B groups can effectively lyse within 30 minutes, indicating that the harvesting solution of the present invention is applicable to 293, Vero, and HeLa cells.

[0150] Example 3 Effect of virus culture and treatment fluid on virus titer

[0151] Cultivation and harvesting of vaccinia virus: 293, HeLa, and CEF cells were inoculated into T75 cell culture bottles, and when the cells grew to a confluence greater than 80%, the vaccinia virus working seeds were inoculated into each cell at an MOI of 0.02, and the cell bottles inoculated with the virus were placed at 37.0±1.0°C for adsorption for 2 hours; after the adsorption, a virus maintenance solution containing 2% FBS was added, and the cells were placed in a 37.0±1.0°C incubator for continued cultivation. After the cells were completely diseased (generally 48-96h), the culture solution was discarded, and the AE group treatment solution described in Preparation Example 1 was added respectively, and the treatment was carried out for 10-60min. The cell state was observed under a microscope, and after the cells were completely lysed (up to 60min of lysis), the virus solution was collected, and the cell lysis time and cell fragmentation were recorded. The virus solution was centrifuged at 300g for 10min, and the supernatant was taken to detect the virus titer, and whether there was precipitation after centrifugation and whether the supernatant was viscous, etc. were recorded. At the same time, a freeze-thaw control group (hereinafter referred to as the freeze-thaw group) was set up in the following manner: the cells after complete lesions were blown down with a pipette, and the cells and infection medium were transferred to a 15 ml sterile centrifuge tube. They were placed in a -80°C refrigerator for rapid freezing for 120 minutes, and then quickly placed in a 37°C water bath for 10 minutes for thawing, and repeated 3 times. The virus solution was centrifuged at 750g for 10 minutes and the supernatant was taken to detect the virus titer. Each group of experiments was repeated more than three times, and the average value was taken for statistical analysis.

[0152] The virus titer was determined by plaque method (i.e., plaque forming unit method), and the harvesting effect of each group of treatment fluids and their influence on the virus titer were evaluated based on the test results. The results are shown in Table 4 below.

[0153] Table 4 Treatment fluid harvest vaccinia virus test results

[0154]

[0155] The results of this experiment show:

[0156] Treatment solutions A and B can completely lyse cells within 10 minutes. The cell fragments are small, and after centrifugation of the cell suspension, there is no obvious precipitation and the supernatant is non-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;

[0157] The cell lysis effect of treatment solution C group (control group) was poor. After lysis for 60 minutes, many cell morphologies were still visible under the microscope. After the cell suspension was centrifuged, obvious precipitation was visible. The supernatant was relatively sticky, indicating that some cells had been lysed and intracellular substances such as nucleic acids were released.

[0158] Treatment liquid D and E groups (control group) can basically lyse cells as the lysis time increases. Under the microscope, it can be seen that the cell fragments are large. 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 does not reach 100%; there is no significant difference between 293 and HeLa cells;

[0159] After freezing and thawing three times, the F group (freeze-thaw control group) still had cell morphology and large cell fragments under the microscope, but after centrifugation of the cell suspension, there was no obvious precipitation, and the supernatant was sticky and had flocculent floating objects, indicating that after freezing and thawing three times, the cells were lysed and released intracellular substances such as nucleic acids. The cell lysis ratio did not reach 100%; there was no significant difference between 293 and HeLa cells.

[0160] The treatment solutions A and B can effectively lyse 293 and HeLa cells in situ within 10 minutes, indicating that the harvest solution of the present invention is applicable to 293 and HeLa cells. (Virus titer value deviation ≤ 30% pfu / ml is considered an acceptable detection error range, the same below.) After in vitro and in vivo anti-tumor activity tests, the biological activity of the prepared lytic virus was not impaired.

[0161] Example 4 Effects of different treatment methods on harvested viruses

[0162] Culture 293 cells, and when the cells grow to a confluence of more than 80%, inoculate the cells with vaccinia virus working seeds at an MOI of 0.02, and place the T75 cell bottle inoculated with the virus at 37.0±1.0℃ for adsorption for 2 hours; after the adsorption, add a virus maintenance solution containing 2% FBS, and continue to culture in a 37.0±1.0℃ incubator for 48 hours. After the cells are completely diseased, harvest the intracellular virus and supernatant (extracellular virus). Add 8ml of harvest treatment solution G and harvest treatment solution H to the intracellular virus, treat for 10min, observe the cell state under a microscope, and collect the virus solution after the cells are completely lysed. Centrifuge the virus solution at 300g for 10min and take the supernatant to detect the virus titer. At the same time, set up a freeze-thaw control group in the following way: blow down the cells after complete disease with a pipette, and transfer the cells and infection medium to a 15ml sterile centrifuge tube. Place in -80℃ freezer for 120min, then quickly thaw in 37℃ water bath for 10min, repeat 3 times, centrifuge the virus solution at 750g for 10min, and take the supernatant to detect the virus titer. Evaluate the virus titer harvested after treatment with the treatment solution. Repeat each group of experiments for more than three times, and take the average value for statistical analysis.

[0163] The results of this experiment are as follows Figure 1 As shown in the figure, the cells were harvested when they were completely lysed and the viruses were released. The results obtained varied greatly due to the different ways of treating the intracellular viruses, among which: the yield of the freeze-thaw treatment was low; the yield of the treatment solution G group (1mM Tris+0.06% (w / v) trypsin+5IU / ml nuclease) was about 6 times that of the freeze-thaw group; the yield of the treatment solution H group (7.5% NaHCO3+0.06% (w / v) trypsin+5IU / ml nuclease) was about 5 times that of the freeze-thaw group.

[0164] Example 5 Effect of different amounts of treatment solution on the harvested virus

[0165] Inoculate 293 cells into T75 cell culture flasks. When the cells grow to a confluence of >80%, inoculate the cells with vaccinia virus working seeds at an MOI of 0.02. Place the virus-inoculated cell flasks at 37.0±1.0℃ for adsorption for 0-2 hours. After adsorption, add virus maintenance solution containing 2% FBS and continue to culture in a 37.0±1.0℃ incubator. Harvest the virus after the cells are completely diseased. Discard the culture solution, add the treatment solution of group A described in Preparation Example 1, and adjust the concentration of the solution according to the culture cell area at 35μl / cm 2 , 70μl / cm 2 , 150μl / cm 2, respectively, at 10 and 30 minutes of treatment, observe the cell state under a microscope, collect the virus liquid, and record the cell breakage. Centrifuge the virus liquid at 750g for 10 minutes, 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, set up a freeze-thaw control group in the following way: blow down the cells after complete lesions with a pipette, and transfer the cells and infection medium to a 15ml sterile centrifuge tube. Place in a -80℃ refrigerator and freeze quickly for 120 minutes, then quickly put in a 37℃ water bath to thaw for 10 minutes, repeat 3 times, centrifuge the virus liquid at 750g for 10 minutes, take the supernatant to detect the virus titer. The virus titer was determined by the plaque method. According to the results of various tests, the effects of treating the harvested virus with different amounts and different times of treatment solution on the harvest effect and titer were evaluated. The results are shown in Table 5 below.

[0166] Table 5 Experimental results of different treatment liquid dosage and treatment time compared with freeze-thaw group

[0167]

[0168] The results of this experiment show:

[0169] The treatment dosage and treatment time have a significant impact on the cell lysis effect and virus yield. The dosage is 35, 70, 150 μl / cm 2 The intracellular viruses harvested by treating with the treatment solution of group A were able to effectively lyse the cells within 30 minutes, with a cell lysis rate of 100% and the supernatant was non-sticky. The cell lysis efficiency was positively correlated with the amount of treatment solution and the treatment time. The amount of treatment solution was 150 μl / cm 2 When the treatment time was 10 minutes, the virus titer was the highest, more than 6 times that of the freeze-thaw group.

[0170] Example 6 Cultivation and Harvesting of Various Exemplary Viruses

[0171] Culture and harvest of varicella virus: SLF-1 was inoculated into a T25 cell culture flask, and when the cells grew to a monolayer, the Oka strain of varicella zoster virus (from ATCC, numbered VR-795) working seeds were inoculated into SLF-1 cells at an MOI of 0.001-0.1, and the cell flask inoculated with the virus was placed at 35.0±1.0°C for adsorption for 0.5-2 hours; after the adsorption, the virus MEM maintenance solution was added, and the culture was continued in a 35.0±1.0°C incubator for 48-96 hours, the culture solution was discarded, and the AE group harvest solution described in Preparation Example 1 was added respectively, and after standing at room temperature for 10 minutes, the cell state was observed under a microscope, and when the cells were significantly swollen, the cells were shaken to fall off, and the virus solution was collected. Various tests such as cell swelling time, cell shedding ratio, cell fragmentation and virus titer were performed. At the same time, a freeze-thaw harvest control group was set. According to the results of various tests, the harvest effect of each group of harvest solutions and the effect on virus titer were evaluated.

[0172] Cultivation and harvesting of HSV: Vero cells were placed in a 37°C, 5% CO2 incubator for amplification and culture. After the cells grew to a monolayer, they were infected with an MOI value of 0.001-0.1. When the cells became round but had not yet fallen off, the infected cells and supernatant were harvested, and the AE group treatment solution described in Preparation Example 1 was added respectively, and the treatment was carried out for 10-60 minutes. The cell state was observed under a microscope, and after the cells were completely lysed (maximum lysis of 60 minutes), the virus liquid was collected, and the cell lysis time and cell fragmentation were recorded. After the virus liquid was centrifuged at 300g for 10 minutes, the supernatant was taken to detect the virus titer, and it was recorded whether there was precipitation after centrifugation and whether the supernatant was viscous. At the same time, a freeze-thaw harvest control group and a supernatant control group were set up. The virus titer was determined by the plaque method, and the harvest effect of each group of treatment solutions at different time points and the effect on the virus titer were evaluated according to the test results.

[0173] Culture and harvest of adenovirus: 293 cells were cultured in a 37°C, 5% CO2 incubator for amplification. After the cells grew to a monolayer, they were infected with an MOI value of 1-100. When the cells became round but had not fallen off, the infected cells and supernatant were harvested, and the AE group treatment solution described in Preparation Example 1 was added respectively, and the treatment was performed for 10-60 minutes. The cell state was observed under a microscope. After the cells were completely lysed (maximum lysis of 60 minutes), the virus solution was collected, and the cell lysis time and cell fragmentation were recorded. After the virus solution was centrifuged at 300g for 10 minutes, the supernatant was taken to detect the virus titer, and it was recorded whether there was precipitation after centrifugation and whether the supernatant was viscous. At the same time, a freeze-thaw harvest control group and a supernatant control group were set up. The virus titer was determined according to the TCID50 method, and the harvest effect of each group of treatment solutions at different time points and the effect on the virus titer were evaluated.

[0174] Cultivation and harvesting of lentivirus: 293 cells were amplified and cultured in a 37°C, 5% CO2 incubator. After adding four plasmids (i.e., the three proteins Gag / Pol, Rev, and VSV-G (replacing HIV-1 Env) required for virus packaging were placed on three plasmids independently, and the vector of the target gene was constructed on the plasmid pLenti-gene to obtain four plasmids), the cells were infected, and the infected cells and supernatant were harvested when the cells became round but had not fallen off. The AE group treatment solution described in Preparation Example 1 was added respectively, and the treatment was carried out for 10-60 minutes. The cell state was observed under a microscope. After the cells were completely lysed (up to 60 minutes of lysis), the virus solution was collected, and the cell lysis time and cell fragmentation were recorded. After the virus solution was centrifuged at 300g for 10 minutes, the supernatant was taken to detect the virus titer, and whether there was precipitation after centrifugation and whether the supernatant was viscous, etc. At the same time, a freeze-thaw harvest control group and a supernatant control group were set up. According to the test results, the harvest effect of each group of treatment solutions at different time points and the effect on the virus titer were evaluated.

[0175] Example 7 Scale-up test of virus production process

[0176] After the 293 cells were revived, they were expanded and cultured in DMEM culture medium containing 10% calf serum. When the cell confluence was 80%-90%, the cell factory was inoculated. After culturing in a CO2 incubator for about 48 hours, the cells were digested to prepare a cell suspension, which was used to inoculate a cell tank (obtained from NBS, model CelliGen Plus / 310, with a surface area of ​​about 22 square meters); the cells were perfused and cultured, and infected with vaccinia virus at an MOI of 0.02 after 6-8 days. The cells were harvested about 48-72 hours after the virus infection, wherein 1.3×10 13 PFU / jar of virus.

[0177] Example 8 Cell factory process scale-up experiment

[0178] After 293 cells were revived, they were expanded to three 10-layer cell factories (brand: Corning, model: "CellSTACK Chamber, 10STACK, POLYSTYRENE, STERILE, 1 / 6"). The total area of ​​the three 10-layer cell factories was 6360*3=19080cm. 2 ). When the cell confluence is 80%-90%, the cells are infected with vaccinia virus at MOI=0.02, and the infection medium is 2% serum DMEM culture medium. About 72 hours after virus infection, the cells are patted off and transferred to a sterile centrifuge bottle together with the culture medium, balanced, and centrifuged at 4°C and 2000rpm for 30 minutes. The supernatant is discarded and the cell pellet is washed with 3L of treatment solution (about 157μl / cm2 Cells) were lysed and harvested, and the treatment solution consisted of 1mM Tris, 0.03% recombinant trypsin, 5U / ml nuclease, pH 9.0, and 1mM MgCl2 was added. 7.80×10 10 PFU of virus (yield per unit area: 4.09×10 6 pFU / cm 2 ).

[0179] Example 9 Fermentation tank (5L) process scale-up experiment

[0180] 293 cells were revived into three T225 cell culture flasks (Brand: Corning, Catalog No.: 431082), and cultured with DMEM containing 10% calf serum. When the cell confluence reached 80%-90%, they were inoculated into a 10-layer cell factory (Brand: Corning, Model: "CellSTACK Chamber, 10 STACK, POLYSTYRENE, STERILE, 1 / 6", total area: 6360 cm 2 ), and after culturing in a CO2 incubator for about 72 hours, the cells were digested to prepare a cell suspension, which was used to inoculate a cell tank (purchased from Eppendorf, model BioFlo 320, 5L cell tank with a surface area of ​​about 150000cm 2 ); inoculation volume 470 ml, total cell volume: 1.78×10 9 Cells were perfused and cultured for 6-8 days, and then infected with vaccinia virus at an MOI of 0.025. About 72 hours after infection, the cells were harvested by in situ lysis using a treatment solution. The treatment solution consisted of 1 mM Tris, 0.03% recombinant trypsin, 5 U / ml nuclease, pH 9.3, and 1 mM MgCl2; a total of about 13.5 L (about 90 μl / cm 2 cells). 4.41×10 11 PFU / tank of virus (yield per unit area: 2.94×10 6 pFU / cm 2 ).

[0181] Example 10 Fermentation tank (5L) process scale-up experiment

[0182] 293 cells were revived and transferred to three T225 cell culture flasks (Brand: Corning, Catalog No.: 431082), and cultured with DMEM containing 10% calf serum. When the cell confluence reached 80%-90%, one cell factory (Brand: Corning, Model: "CellSTACK Chamber, 10 STACK, POLYSTYRENE, STERILE, 1 / 6", total area: 6360 cm) was inoculated. 2 ), and after culturing in a CO2 incubator for about 72 hours, the cells were digested to prepare a cell suspension, which was used to inoculate a cell tank (purchased from Eppendorf, model BioFlo 320, 5L cell tank with a surface area of ​​about 150000cm 2 ); inoculation volume 430 ml, total cell volume: 1.97×10 9 Cells were perfused and cultured for 6-8 days, and then infected with vaccinia virus at an MOI of 0.06. About 5 days after infection, the cells were harvested by in situ lysis using a treatment solution. The treatment solution consisted of 1 mM Tris, 0.03% recombinant trypsin, 5 U / ml nuclease, pH 9.3, and 1 mM MgCl2. The total treatment solution was about 15 L (about 100 μl / cm 2 cells). 2.41×10 11 PFU / tank of virus (yield per unit area: 1.61×10 6 PFU / cm 2 ).

[0183] Example 11 Fermentation tank (14L) process scale-up experiment

[0184] 293 cells were revived in three T225 cell culture flasks (brand: Corning, catalog number: 431082), and cultured with DMEM containing 10% calf serum. When the cell confluence reached 80%-90%, three 10-layer cell factories (brand: Corning, model: "CellSTACK Chamber, 10 STACK, POLYSTYRENE, STERILE, 1 / 6" were inoculated. The total area of ​​the three 10-layer cell factories was 6360*3=19080cm 2 ), and after culturing in a CO2 incubator for about 72 hours, the cells were digested to prepare a cell suspension, which was used to inoculate a cell tank (purchased from Eppendorf, model BioFlo 320, 14L tank with a surface area of ​​about 450000cm 2 ); inoculation volume 1400 ml, total cell volume: 3.19×10 9Cells were perfused and cultured for 6-8 days, and then infected with vaccinia virus at an MOI of 0.05. About 72 hours after infection, the cells were harvested by in situ lysis using a treatment solution. The treatment solution consisted of 1 mM Tris, 0.03% recombinant trypsin, 5 U / ml nuclease, pH 9.3, and 1 mM MgCl2. The total treatment solution was about 50 L (about 110 μl / cm 2 cells). 1.24×10 12 pFU / tank of virus (yield per unit area: 2.76×10 6 PFU / cm 2 ).

[0185] As can be seen from the above embodiments, by adopting the method of the present invention and the treatment solution for harvesting, the production process can be greatly simplified, so that it is particularly suitable for large-scale production of viruses. The virus yield obtained by the method of the present invention and the treatment solution for harvesting is high and the impurities are low, which further makes the subsequent purification process easier to implement, and the purity and biological activity of the obtained virus are also higher.

[0186] It is to be understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present invention, but the present invention is not limited thereto. For those skilled in the art, various modifications and variations can be made without departing from the spirit and essence of the present invention, and these modifications and variations are also within the scope of protection of the present invention.

Claims

1. A method for producing a virus, the method comprising: Cultivating cells, wherein the cells are cells that have been inoculated with viruses or transfected with virus packaging elements; as well as The cultured cells are contacted with a harvesting fluid composition to harvest the virus in one step, wherein the harvesting fluid composition comprises: pancreatin, pH buffer and nuclease, and the pH of the harvesting fluid composition is greater than or equal to 9.0 and not more than 10.5; wherein in the harvesting fluid composition, the concentration of the pancreatin is 0.01-0.12% (w / v).

2. The method according to claim 1, characterized in that: In the harvest fluid composition, the concentration of pancreatic enzyme is 0.03-0.06% (w / v).

3. The method according to claim 1, characterized in that In the harvest fluid composition, the concentration of the nuclease is 1-100 IU / ml.

4. The method according to claim 1, characterized in that: In the harvest fluid composition, the concentration of the nuclease is 1-50 IU / ml.

5. The method according to claim 1, characterized in that In the harvest fluid composition, the concentration of the nuclease is 1-5 IU / ml.

6. The method according to claim 1, characterized in that The osmotic pressure of the harvest fluid composition ranges from greater than 0 to less than or equal to 50 mOsmol / kg or 800-2500 mOsmol / kg.

7. The method according to claim 1, characterized in that The osmotic pressure of the harvest fluid composition ranges from greater than 0 to less than or equal to 20 mOsmol / kg or 1785-2000 mOsmol / kg.

8. The method according to claim 1, characterized in that The osmotic pressure of the harvest fluid composition is in the range of 1-20 mOsmol / kg.

9. The method according to claim 1, characterized in that: The pH of the harvest fluid composition is 9.0-9.

5.

10. The method according to claim 1, characterized in that The pH buffer is selected from Tris salt buffer and sodium bicarbonate buffer.

11. The method according to claim 1, characterized in that: The culture is carried out in a cell bottle, a cell factory or a fermenter, and the culture methods include adherent culture and suspension culture.

12. The method according to claim 1, characterized in that When the culture method is adherent culture, the amount of the harvesting liquid composition is greater than or equal to 35 μl of the harvesting liquid composition / cm 2 cells; when the culture method is suspension culture, the amount of the harvesting liquid composition is greater than or equal to 35 μl of the harvesting liquid composition / 10 5 cells.

13. The method according to claim 1, characterized in that When the culture method is adherent culture, the amount of the harvesting liquid composition is greater than or equal to 70 μl of the harvesting liquid composition / cm 2 cells; when the culture method is suspension culture, the amount of the harvesting liquid composition is greater than or equal to 70 μl of the harvesting liquid composition / 10 5 cells.

14. The method according to claim 1, characterized in that The cultured cells are in contact with the harvesting liquid composition for 5-60 minutes.

15. The method according to claim 1, characterized in that The cells are selected from Vero cells, 293 cells, CEF cells, and Hela cells.

16. The method according to claim 1, characterized in that The viruses include: vaccinia virus, varicella-zoster virus, EV71 virus, hepatitis A virus, herpes simplex virus, lentivirus, retrovirus, adenovirus, adeno-associated virus, measles virus, Semliki Forest virus, reovirus, vesicular stomatitis virus, polio virus, Seneca Valley virus, echovirus, coxsackievirus, Newcastle disease virus and Maraba virus.

17. The method according to claim 1, characterized in that The virus is an enveloped virus.

18. The method according to claim 1, characterized in that The virus is rotavirus.

19. The method according to claim 1, characterized in that The cells are 293 cells, and the virus is vaccinia virus.

20. The method according to any one of claims 1-9, 11-19, characterized in that: The harvest fluid composition comprises Tris salt buffer, trypsin and nuclease, and the concentration of the Tris salt buffer is 1-50 mM.

21. The method according to claim 20, characterized in that The concentration of the Tris saline buffer is 1-10 mM.

22. A harvesting fluid composition for harvesting virus cultured in cells, characterized in that: The harvesting fluid composition comprises: pancreatin, pH buffer and nuclease, and the pH of the harvesting fluid composition is greater than or equal to 9.0 and not more than 10.5; wherein in the harvesting fluid composition, the concentration of the pancreatin is 0.01-0.12% (w / v).

23. The harvesting fluid composition according to claim 22, characterized in that In the harvest fluid composition, the concentration of pancreatic enzyme is 0.03-0.06% (w / v).

24. The harvesting fluid composition according to claim 22, characterized in that In the harvest fluid composition, the concentration of the nuclease is 1-100 IU / ml.

25. The harvesting fluid composition according to claim 22, characterized in that In the harvest fluid composition, the concentration of the nuclease is 1-50 IU / ml.

26. The harvesting fluid composition according to claim 22, characterized in that In the harvest fluid composition, the concentration of the nuclease is 1-5 IU / ml.

27. The harvesting fluid composition according to claim 22, characterized in that The osmotic pressure of the harvest fluid composition ranges from greater than 0 to less than or equal to 50 mOsmol / kg or 800-2500 mOsmol / kg.

28. The harvesting fluid composition according to claim 22, characterized in that The osmotic pressure of the harvest fluid composition ranges from greater than 0 to less than or equal to 20 mOsmol / kg or 1785-2000 mOsmol / kg.

29. The harvesting fluid composition according to claim 22, characterized in that The osmotic pressure of the harvest fluid composition is in the range of 1-20 mOsmol / kg.

30. The harvesting fluid composition according to claim 22, characterized in that The pH of the harvest fluid composition is 9.0-9.

5.

31. The harvesting fluid composition according to claim 22, characterized in that The pH buffer is selected from Tris salt buffer and sodium bicarbonate buffer.

32. The harvest fluid composition according to any one of claims 22-30, characterized in that The harvest fluid composition comprises Tris salt buffer, trypsin and nuclease, and the concentration of the Tris salt buffer is 1-50 mM.

33. The harvesting fluid composition according to claim 32, characterized in that The concentration of the Tris saline buffer is 1-10 mM.

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