Virus production method and collection solution composition

Abstract

The present invention provides a method for producing a virus and a harvest solution composition. The method comprises the steps of: culturing cells, the cells being inoculated with a virus or transfected with a virus packaging component; and contacting the cultured cells with a harvest solution composition to harvest the virus in one step, the harvest solution composition comprising trypsin, a pH buffer, and optionally a nuclease, and having a pH in the range of greater than 7.5 and less than or equal to 10.5. The virus production method of the present invention has advantages such as simple operation, ease of scale-up, and stable yield, and provides an unexpectedly significant improvement in yield compared to conventional techniques. Furthermore, the integrity of virus particles can be ensured without impairing the biological activity of the virus. Therefore, it is highly suitable for mass production of viruses.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, and in particular to a method for producing a virus and a collection solution composition. More specifically, the present invention relates to a method for producing a virus by culturing cells and a collection solution composition for harvesting a virus subjected to cell culture. [Background technology]

[0002] The preparation of viral biological products such as oncolytic viral drugs, viral vaccines, or recombinant viral vectors usually involves the following steps: inoculating uninfected cells of an appropriate generation with the virus; harvesting the infected cells when they show a certain degree of cytotoxicity; lysing the cells using various methods to harvest the virus; then purifying the virus by density gradient centrifugation or chromatography to obtain a virus stock solution; adding a certain amount of buffer solution to obtain a semi-finished product; and finally lyophilizing or dispensing the semi-finished product to obtain a finished product.

[0003] Currently, cell factories or culture tanks are primarily used for mass cell cultivation to produce viral biological products. Much of the amplified virus resides within cells. Therefore, the virus harvesting process requires effective cell disruption and lysis to release the virus and ensure the integrity of the virus particles. However, different harvesting processes have different effects on cell lysis and virus particle integrity. The harvesting method directly affects the virus titer, and ensuring a high effective titer of the harvested virus stock is a key factor in determining the quality of the final product.

[0004] Vaccinia virus, herpes simplex virus, varicella-zoster virus, and adenovirus are intracellular viruses with strong cell-binding activity, so when harvesting the viruses, cells must be collected and lysed to obtain free virus particles. Currently, for recombinant viral vectors based on lentivirus, retrovirus, and adenovirus-related virus, only free virus outside the cells is collected during the actual packaging and production process, and the large amount of intracellular virus is not fully utilized.

[0005] Currently, the mass production of these viruses involves the freeze-thaw method, which lyses cells to extract the viruses within them. However, the freeze-thaw method has drawbacks, such as high energy consumption, a long process, multiple steps, difficulty in scaling up, and unstable yields, limiting the production scale of viral biological products. While hypotonic and chemical lysis methods are also used to extract viruses within cells, many of the viruses bound to the cell membrane are not fully released. Furthermore, many chemical lysis agents destroy the envelopes of enveloped viruses (such as vaccinia virus and herpes simplex virus), resulting in the loss of viral biological activity.

[0006] Therefore, there remains a need for a method that allows for efficient production of viruses by cell culture and a harvest solution that allows for efficient harvesting of viruses subjected to cell culture, which is essential for obtaining products such as high-quality viral vaccines and recombinant viral vectors. Summary of the Invention

[0007] To address one or more of the above-mentioned problems in the prior art, the present invention provides a method for producing a virus and a harvest solution composition.

[0008] Specifically, the present invention provides: (1) A method for producing a virus, the method comprising: Culturing cells, wherein the cells have been inoculated with a virus or transfected with viral packaging elements; and 1. A process for harvesting viruses in one step by contacting cultured cells with a harvest solution composition comprising trypsin, a pH buffer, and optionally a nuclease, wherein the pH of the harvest solution composition is in the range of greater than 7.5 to 10.5.

[0009] (2) The method according to (1), wherein the concentration of trypsin in the collecting solution composition is in the range of 0.01 to 0.12% (w / v), preferably in the range of 0.03 to 0.06% (w / v).

[0010] (3) The method according to (1) or (2), wherein the concentration of the nuclease in the collecting solution composition is in the range of 1 to 100 IU / ml, preferably in the range of 1 to 50 IU / ml, and more preferably in the range of 1 to 5 IU / ml.

[0011] (4) The method according to any one of (1) to (3), wherein the osmotic pressure of the collecting solution composition is in the range of 0 to 50 mOsmol / kg or 800 to 2500 mOsmol / kg, preferably in the range of 0 to 20 mOsmol / kg or 1785 to 2000 mOsmol / kg, and more preferably in the range of 1 to 20 mOsmol / kg.

[0012] (5) The method according to any one of (1) to (4), wherein the pH of the collecting solution composition is in the range of 8.5 to 9.5.

[0013] (6) The method according to any one of (1) to (5), wherein the pH buffer solution is selected from a Tris buffer solution and a sodium bicarbonate buffer solution.

[0014] (7) The method according to any one of (1) to (6), wherein the culture is carried out in a cell flask, a cell factory, or a culture tank, and the culture includes adherent culture and suspension culture.

[0015] (8) When the culture is an adherent culture, the amount of the collection solution composition is 35 μl or more of the collection solution composition / cm 2 of cells, preferably 70 μl or more of collection solution composition / cm 2When the culture is a suspension culture, the amount of the harvesting solution composition is 35 μl or more of the harvesting solution composition / 10 5 Cells, preferably 70 μl or more of the collection solution composition / 10 5 The method according to any one of (1) to (7), wherein the range is a cell.

[0016] (9) The method according to any one of (1) to (8), wherein the cultured cells are contacted with the collection solution composition for a time period ranging from 5 to 60 minutes.

[0017] (10) The method according to any one of (1) to (9), wherein the cells are selected from Vero cells, 293 cells, CEF cells, and HeLa cells.

[0018] (11) The method according to any one of (1) to (10), wherein the virus includes 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, coxsackievirus, Newcastle disease virus, and Maraba virus, and is preferably an enveloped virus.

[0019] (12) The method according to any one of (1) to (11), wherein the cells are 293 cells and the virus is vaccinia virus.

[0020] (13) The method according to any one of (1) to (5) and (7) to (12), wherein the collection solution composition comprises a Tris buffer solution, trypsin, and a nuclease, and the concentration of the Tris buffer solution is in the range of 1 to 50 mM, preferably in the range of 1 to 10 mM.

[0021] (14) A collection solution composition for collecting viruses subjected to cell culture, the collection solution composition comprising trypsin, a pH buffer, and optionally a nuclease, and the pH of the collection solution composition is in the range of more than 7.5 and not more than 10.5.

[0022] (15) The collecting solution composition according to (14), wherein the concentration of trypsin in the collecting solution composition is in the range of 0.01 to 0.12% (w / v), preferably in the range of 0.03 to 0.06% (w / v).

[0023] (16) The collecting solution composition according to (14) or (15), wherein the concentration of the nuclease in the collecting solution composition is in the range of 1 to 100 IU / ml, preferably in the range of 1 to 50 IU / ml, and more preferably in the range of 1 to 5 IU / ml.

[0024] (17) The collecting solution composition according to any one of (14) to (16), wherein the osmotic pressure of the collecting solution composition is in the range of 0 to 50 mOsmol / kg or 800 to 2500 mOsmol / kg, preferably in the range of 0 to 20 mOsmol / kg or 1785 to 2000 mOsmol / kg, and more preferably in the range of 1 to 20 mOsmol / kg.

[0025] (18) The collecting solution composition according to any one of (14) to (17), wherein the pH of the collecting solution composition is in the range of 8.5 to 9.5.

[0026] (19) The collecting solution composition according to any one of (14) to (18), wherein the pH buffer solution is selected from a Tris buffer solution and a sodium bicarbonate buffer solution.

[0027] (20) The collecting solution composition according to any one of (14) to (19), wherein the culture is carried out in a cell flask, a cell factory, or a culture tank, and the culture includes adherent culture and suspension culture.

[0028] (21) The collecting solution composition according to any one of (14) to (20), wherein the cells are selected from Vero cells, 293 cells, CEF cells, and HeLa cells.

[0029] (22) The fluid-collecting composition according to any one of (14) to (21), wherein the virus includes 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, coxsackievirus, Newcastle disease virus, and Maraba virus, and is preferably an enveloped virus.

[0030] (23) The collecting solution composition according to any one of (14) to (22), wherein the cells are 293 cells and the virus is vaccinia virus.

[0031] (24) The collecting solution composition according to any one of (14) to (18) and (20) to (23), comprising a Tris buffer solution, trypsin, and a nuclease, wherein the concentration of the Tris buffer solution is in the range of 1 to 50 mM, preferably in the range of 1 to 10 mM.

[0032] Compared with the prior art, the present invention has the following advantages and favorable effects:

[0033] First, the present invention proposes a one-step collection method for virus production, in which cultured cells are contacted with the collection solution composition of the present invention to collect viruses in one step. The inventors have obtained the collection solution composition of the present invention through extensive research. This collection solution composition can completely lyse cells and collect viruses without requiring other cell lysis methods or steps such as freezing and thawing, ultrasonic waves, or mechanical disruption.

[0034] The virus production method of the present invention has advantages such as simple operation, ease of scale-up, and stable yield, and unexpectedly achieves a significant improvement in yield compared to conventional techniques. Furthermore, it can ensure the integrity of virus particles without impairing the biological activity of the virus. Therefore, it is highly suitable for mass production of viruses.

[0035] In mass production, the use of the method and collection solution composition of the present invention can greatly simplify the production process, reduce production costs, and produce highly active virus products with high concentration and purity. [Brief explanation of the drawings]

[0036] [Figure 1] Figure 1 shows the results of virus harvest using various treatment solutions in one embodiment of the present invention, with the horizontal axis representing the various groups and the vertical axis representing the average yield per unit area (pfu / cm). DETAILED DESCRIPTION OF THE INVENTION

[0037] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be further described below by a detailed description of specific embodiments with reference to the accompanying drawings, but these are not intended to limit the present invention. Those skilled in the art can make various changes or improvements based on the basic idea of the present invention. These changes or improvements are intended to be included in the scope of the present invention as long as they do not deviate from the basic idea of the present invention.

[0038] The method for producing a virus of the present invention comprises culturing cells, the cells being inoculated with a virus or transfected with a virus packaging component, and contacting the cultured cells with a harvest solution composition of the present invention to harvest the virus in one step. The harvest solution composition of the present invention comprises trypsin, a pH buffer, and optionally a nuclease, and the pH of the harvest solution composition is in the range of greater than 7.5 and less than or equal to 10.5.

[0039] As used herein, "viral packaging elements" refers to the cis- and trans-acting elements required by a recombinant viral vector to package a lentivirus, retrovirus, adenovirus-related virus, and the like.

[0040] As used herein, "collecting viruses in one step" means that when cultured cells are contacted with the collection solution composition of the present invention, the collection solution composition completely lyses the cells and allows viruses to be collected, without the need for other cell lysis methods or steps such as freezing and thawing, ultrasonication, or mechanical disruption.

[0041] The inventors of the present invention have found that the simple use of hypotonic buffers in the prior art fails to achieve the goal of virus collection in one step, and the virus yield remains low. The inventors of the present invention have unexpectedly found that the use of the collection solution composition of the present invention enables virus collection 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 five times.

[0042] In the collecting solution composition, the trypsin concentration can be in the range of 0.01 to 0.12% (% is a mass-to-volume ratio (w / v), i.e., the number of grams of trypsin contained in 100 ml of the collecting solution composition is 0.01 to 0.12 g), for example, 0.03, 0.05, 0.07, 0.09, or 0.11% (w / v), preferably 0.03 to 0.06% (w / v). Trypsin may be commercially available, or may be conventional animal-derived trypsin, or may be recombinant trypsin.

[0043] When the collection solution composition contains a nuclease, the concentration of the nuclease can be in the range of 1 to 100 IU / ml, for example, 1, 3, 5, 10, 20, 40, 60, or 80 IU / ml, preferably in the range of 1 to 50 IU / ml, and more preferably in the range of 1 to 5 IU / ml. 2+(e.g., 1-10mM Mg 2+ , preferably 1 to 2 mM Mg 2+ ; e.g., 1 mM MgCl2) can be added.

[0044] The osmotic pressure of the collecting solution composition can be in the range of 0 to 50 mOsmol / kg (e.g., 1, 5, 10, 15, 30, 45 mOsmol / kg) or 800 to 2500 mOsmol / kg (e.g., 1000, 1250, 1500, 1700, 1900, 2100, 2300 mOsmol / kg). Preferably, the osmotic pressure of the collecting solution composition is in the range of 0 to 20 mOsmol / kg or 1785 to 2000 mOsmol / kg, more preferably in the range of 1 to 20 mOsmol / kg, and even more preferably in the range of 1 to 10 mOsmol / kg.

[0045] The pH of the collection solution composition is alkaline, more specifically, greater than 7.5 and less than 10.5, for example, 7.6, 8.0, 8.5, 9.0, 9.5, 10.0, or 10.5, and preferably 8.5 to 9.5. If the pH of the collection solution composition is 7.5 or less, cells cannot be completely lysed, resulting in a low virus yield. If the pH of the collection solution composition is greater than 10.5, virus activity is affected.

[0046] The pH buffer of the collection solution composition can be selected from Tris buffer and sodium bicarbonate buffer, preferably Tris buffer.

[0047] In one embodiment, the collection solution composition of the present invention comprises a Tris buffer solution, trypsin, and a nuclease. The concentration of the Tris buffer solution may be 1 to 50 mM, for example, 1, 3, 5, 8, 10, 20, 30, or 40 mM, and preferably 1 to 10 mM.

[0048] In the method of the present invention, when cells are cultured in adherent culture, the amount of the harvesting solution composition is 35 μl or more of the harvesting solution composition / cm 2 of cells, preferably 70 μl or more of the collection solution composition / cm 2of cells, e.g., 75, 100, 125, 150, 200 μl of collection solution composition / cm 2 Here, "square centimeter of cells (cm 2 The term "cells" refers to the sum of all adherent cells grown in one square centimeter. When cells are cultured in suspension, the amount of the harvesting solution composition is 35 μl or more of the harvesting solution composition / 10 5 Cells, preferably 70 μl or more of the collection solution composition / 10 5 Cells, e.g., 75, 100, 125, 150, 200 μl of collection solution composition / 10 5 It may be a cell.

[0049] In the present invention, the contact time between the cultured cells and the collection solution composition is usually 60 minutes or less, and the cells can be completely lysed within a contact time of usually 5 to 60 minutes (e.g., 10 minutes, 20 minutes, 30 minutes, 40 minutes, or 50 minutes). Cell lysis can be observed under a microscope. If no intact cells are observed under a microscope, the cells are considered to be completely lysed.

[0050] In the present invention, inoculation of a virus or virus package and cell culture can be performed using methods commonly used in the art. The virus inoculation dose can be 0.001 to 0.2 MOI, for example, 0.005, 0.01, 0.02, 0.03, 0.05, 0.08, 0.1, or 0.15 MOI. As used herein, the term "MOI" or "multiplicity of infection" refers to the ratio between the number of viruses and the number of cells, and refers to the number of viruses infected by each cell that undergoes viral infection. MOI = pfu / cell, i.e., cell number × MOI = total PFU.

[0051] In the present invention, cell culture is carried out using, for example, a cell flask (scale: 25 to 225 cm 2 cells / flask), cell factory (scale 500-800cm 2 cells / stage, e.g. 632cm 2 cells / stage), or culture tanks (scales from 0.5 to 500 cm 2The culture can be carried out in a medium (cell / culture vessel), including adherent culture and suspension culture. The medium can be one commonly used in the art, such as DMEM medium or MEM medium. The method of the present invention can further include a step of separating the cultured cells from the medium before contacting the cultured cells with the collection solution composition. Furthermore, the collected virus can be further purified by tangential flow centrifugation and / or column chromatography.

[0052] 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) and HeLa cells.

[0053] The Vero cell line was established from the kidney of a normal adult African green monkey by Y. Yasumura and Y. Kawakita at Chiba University, Japan. These cells are anchorage-dependent fibroblasts that can support the growth of a variety of viruses, including herpes simplex virus, vaccinia virus, Japanese encephalitis virus, poliovirus, and rabies virus. The WHO considers Vero cells safe for use within 150 generations, they are non-tumorigenic, and they are approved for the production of human viral vaccines.

[0054] The 293 cell line is a human embryonic kidney hypotriploid cell line transformed with Adenovirus type 5 (Ad5) strain 75, containing the E1 region of Ad5 and complementing the E1 region deletion. It was established in 1976 by F.L. Graham and J.S. Miley of McMaster University in Canada using DNA transfection techniques. There are many derivatives of 293 cells, including HEK293, Ad293, 293T / 17, and AAV-293. The 293 cell line is widely used for the production, gene expression, and protein expression of adenovirus, retrovirus, lentivirus, and vaccinia virus.

[0055] Chicken embryo fibroblasts (CEFs) were the earliest target for tissue culture. Tissue culture researchers have used chicken embryos for many of their studies. Chicken embryo fibroblasts (CEFs) are easily obtained, easily prepared, have good resistance, and are suitable for the growth and propagation of many viruses.

[0056] Therefore, it is widely used in vaccine production, virus culture, and cell and molecular biology research (see, for example, the following reference: 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): pp.1383-92).

[0057] HeLa cells are the first aneuploid epithelial cell line derived from human tissue by subculture. They were established in 1951 by G.O. Gey et al. from cervical cancer tissue of a 31-year-old black woman. Compared to other cancer cell lines, this cell line has extremely rapid proliferation, is highly sensitive to viruses such as poliovirus, adenovirus, and vaccinia, and exhibits significant cytopathic properties, making it valuable for virus research and production. It is also widely used in tumor research, biological experiments, and cell culture.

[0058] 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, and Semliki Forest virus. Vesicular stomatitis virus, poliovirus, reovirus, vesicular stomatitis virus, poliovirus, Seneca Valley virus, echoenterovirus, coxsackievirus, Newcastle disease virus, and Maraba virus. Enveloped viruses are also preferred. Examples of enveloped viruses include vaccinia virus, varicella-zoster virus, rotavirus, EV71 virus, coxsackievirus (CA16 virus), hepatitis A virus, herpes simplex virus, lentivirus, and retrovirus.

[0059] Viruses that can be produced by the present invention include oncolytic viruses that 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. 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. Wild-type viruses with oncolytic effects include, but are not limited to, reovirus, vesicular stomatitis virus, poliovirus, Seneca Valley virus, echoenterovirus, coxsackievirus, Newcastle disease virus, and Maraba virus.

[0060] A foreign gene can be incorporated into the genome of the oncolytic virus. Examples of foreign genes include foreign immune regulatory genes, foreign screening genes, and foreign reporter genes. The genome of the oncolytic virus may not incorporate any foreign gene.

[0061] Vaccinia virus (abbreviated as VV; also known as poxvirus), a member of the Poxviridae family, has played an important role in the history of humankind's fight against infectious diseases. Before 1980, vaccinia virus was widely used, primarily as a smallpox vaccine, and ultimately eradicated smallpox worldwide. The successful expression of foreign genes in vaccinia virus, coupled with the safety of population vaccination, led to the development of vaccinia virus as a gene expression vector, a prophylactic and therapeutic vaccine vector, and an immunotherapy vector.

[0062] Vaccinia virus has four distinct infection forms during its life cycle: intracellular mature virus (IMV), intracellular enveloped virus (IEV), cytoplasmic enveloped virus (CEV), and extracellular enveloped virus (EEV). In most vaccinia virus strains (WR, Copenhagen, Ankara, Tiantan, etc.), IMV accounts for over 90% of all progeny viruses. After being assembled in virus factories, these viruses remain intracellularly and are not released outside the cell until cell lysis.

[0063] Oncolytic vaccinia viruses include, but are not limited to, Pexa-vac (available from Jennerex Biotherapeutics Co., Ltd.), JX-963 (available from Jennerex Biotherapeutics Co., Ltd.), JX-929 (available from Jennerex Biotherapeutics Co., Ltd.), VSC20 (preparation methods 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 Corporation), TG6002 (available from Transgene Corporation), DDvv-IL21 (see PCT Publication No. WO2019 / 062234A1), and the like.

[0064] Varicella-zoster virus (VZV) is a double-stranded DNA virus belonging to the alpha herpesvirinae subfamily. Its primary infection is chickenpox. Reactivation of latent infection causes shingles. Vaccination is the most effective method for preventing VZV-induced chickenpox and shingles. To date, the only vaccine approved for preventing VZV-induced disease is the live attenuated varicella vaccine (Oka strain). Vaccination with this vaccine is recognized as the most effective method for preventing VZV-induced chickenpox.

[0065] Herpes simplex virus (HSV) belongs to the alphavirinae subfamily of the Herpesviridae family. Currently, this virus is classified into type 1 and type 2 based on antigenic differences. Herpes simplex virus has the advantages of being able to use human cells as host cells, infecting a wide variety of human cells, being inhibited by antiherpes drugs, and not integrating into the host cell genome during the proliferation and latency phases, resulting in a low risk of insertional mutagenesis. It is widely used in gene therapy for tumors or degenerative diseases of the nervous system. HSV possesses a lytic pathway and is primarily intracellular.

[0066] Oncolytic herpes simplex viruses include, but are not limited to, HSV-1 herpes simplex virus, HSV-2 herpes simplex virus; specific examples include (for example): Imlygic™ (available from Amgen Co., Ltd.), G207 (available from Medigene Co., Ltd.), HF10 (available from Takara Bio Co., Ltd.), Seprehvir (available from Virttu Biologics Co., Ltd.), Orien X010 (available from Beijing Oriengene Biologics Co., Ltd.), NV1020 (available from Catherax Co., Ltd.), and the like.

[0067] Lentivirus (LV) belongs to the retrovirus family and is an RNA virus. The overall size of lentivirus is approximately 100 nanometers. It has the following advantages: a large carrying capacity for gene fragments, high transfection efficiency, a wide host range, and stable long-term expression. Currently, it has become an ideal vector for target gene delivery and is used in clinical treatments. It is one of the common viruses used to construct CAR-T cells. Two CD19-targeting CAR-T drugs sold in the United States, Kymriah (available from Novartis) and Yescarta (available from Kate), use lentiviruses as vectors.

[0068] Since a total amount of virus of 1.0E+12TU is required for one gene therapy, improving lentivirus packaging and production levels can effectively promote the development of lentivirus-mediated gene therapy.

[0069] Currently, lentivirus production is carried out by transiently transfecting a cell matrix with a plasmid to produce a limited number of packaged viruses. A common construction method involves constructing a target gene vector on the pLenti-gene plasmid, carrying the three proteins required for viral packaging: Gag / Pol, Rev, and VSV-G (a substitute for HIV-1 Env). Then, the four plasmids are co-transfected into host cells in proportion to each other. After incubation, the supernatant is harvested and the virus is purified.

[0070] Retroviruses are single-stranded RNA viruses. Their genomes are approximately 10 Kb and contain three essential genes from 5' to 3': Gag (encoding the viral core protein), Pol (reverse transcriptase), and Env (envelope glycoprotein on the surface of the virus particle). Furthermore, they contain long terminal repeats (LTRs) and a packaging signal (ψ) cis-acting element.

[0071] Retrovirus production usually involves transfecting a cell substrate with a plasmid, constructing a stable toxin-producing cell line, and screening for the appropriate target gene. The integration site of a retroviral vector is usually located in an open and transcriptionally active region of the chromosome, significantly increasing the likelihood of obtaining a cell line that efficiently expresses the exogenous target gene. Furthermore, retroviruses are also a common viral vector for constructing CAR-T cells.

[0072] Adenoviruses (AdV) are non-enveloped, spherical particles whose DNA exists in a linear, double-stranded form. Adenoviruses have a non-enveloped nucleocapsid with a diameter of 70-80 nanometers, an icosahedron, and three-dimensional symmetry. Adenoviruses are relatively safe vectors. Over 50% of the population has antibodies against Ad5.

[0073] Due to their advantages (e.g., high gene transfer efficiency, transfection into a wide variety of cells, ease of preparation and purification, and the fact that they are not integrated into the host cell genome and are only transiently expressed after entering the host cell), adenoviruses have become increasingly important in the fields of gene therapy, gene immunization, and vaccine preparation, and are widely used in various fields such as vaccine development, immunotherapy, and gene therapy.

[0074] Oncolytic adenoviruses include, but are not limited to, human adenovirus type 5 or human chimeric adenovirus; specific examples include (for example): Onyx-015 (available from Onyx Pharmaceuticals Co., Ltd.), H101 (available from Shanghai Sunway Biotech Co., Ltd.), Ad5-yCD / mutTKSR39rep-hIL12 (available from Henry Ford Health System Co., Ltd.), CG0070 (available from Cold Genesys Co., Ltd.), DNX-2401 (available from DNAtrix Co.), OBP-301 (available from Oncolys BioPharma Co., Ltd.), ONCOS-102 (available from Targovax Oy Co., Ltd. / Oncos Therapeutics Co., Ltd.), ColoAd1 (available from PsiOxus Therapeutics Co., Ltd.), VCN-01 (available from VCN Biosciences Co., Ltd.), ProstAtak™ (available from Advantagene Co., Ltd.), and the like.

[0075] Adenovirus-associated virus (AAV) belongs to the Parvoviridae family and is a non-enveloped, single-stranded, linear DNA virus with a genomic DNA of less than 5 Kb. AAV has advantages such as high safety, low immunogenicity, a wide host range, stable expression, and the ability to simultaneously infect cleaved and uncleaved cells. It has very attractive potential in the biomedical industry, especially in the field of gene therapy. The majority of recombinant AAV remains within the producer cells.

[0076] In one embodiment, the present invention uses 293 cells to produce vaccinia viruses (including oncolytic poxviruses). The production method includes:

[0077] Inoculating 293 cells with a working seed of vaccinia virus at an MOI of 0.001 to 0.2 (e.g., 0.005, 0.01, 0.02, 0.03, 0.05, 0.08, 0.1, or 0.15);

[0078] A step of culturing the inoculated cells to denature the cells, preferably until the cells are completely denatured (usually for 48 to 96 hours). For example, a cell flask can be used for the culture, and the culture is, for example, an adherent culture; and

[0079] A step of contacting cultured cells with the collection solution composition of the present invention to collect viruses in one step.

[0080] The collection solution composition contains trypsin, a pH buffer, and optionally a nuclease. The pH of the collection solution composition is greater than 7.5 and equal to or less than 10.5, preferably 8.5 to 9.5. Preferably, the collection solution composition contains a Tris buffer, trypsin, and a nuclease.

[0081] The concentration of the Tris buffer solution can be 1 to 50 mM (e.g., 1, 3, 5, 8, 10, 20, 30, or 40 mM), preferably 1 to 10 mM. The concentration of trypsin is 0.01 to 0.12% (w / v) (e.g., 0.03, 0.05, 0.07, 0.09, or 0.11% (w / v)), preferably 0.03 to 0.06% (w / v). The concentration of the nuclease can be 1 to 100 IU / ml, e.g., 1, 3, 5, 10, 20, 40, 60, or 80 IU / ml, preferably 1 to 50 IU / ml, and more preferably 1 to 5 IU / ml. The osmotic pressure of the collection solution composition is preferably hypotonic, and the pH is preferably 8.5 to 9.5.

[0082] The amount of collection solution composition is 35 μl or more of collection solution composition / cm 2 of cells, preferably 70 μl or more of collection solution composition / cm 2 range of cells, e.g., 75, 100, 125, 150, 200 μl of collection solution composition / cm 2 The cells may be:

[0083] The contact time of the cultured cells with the collection solution composition is usually 60 minutes or less, and the cells can usually be completely lysed within a contact time of 5 to 60 minutes (for example, 10, 20, 30, 40, or 50 minutes).

[0084] The virus production method of the present invention employs a gentle means for obtaining viruses in cultured cells. This method does not involve steps such as cell lysis, freeze-thawing, ultrasonication, or mechanical disruption. This process is simple, easy to operate, and easily scaled up, making it highly suitable for large-scale virus production by cell culture.

[0085] Furthermore, compared to conventional methods commonly used in prior art (such as the freeze-thaw method), the yield is significantly improved by 5 to 10 times, and at the same time, the integrity of the virus particles can be ensured without compromising the biological activity of the virus, making it possible to mass-produce the virus.

[0086] The following examples further illustrate the present invention but should not be construed as limiting it. Any modifications or substitutions made to the methods, steps, or conditions of the present invention that do not depart from the spirit and essence of the invention are within the scope of the present invention.

[0087] All numerical values herein should be understood as modified by the word "about." Numerical ranges expressed as closing values include all values and portions within that range (e.g., 1 to 100 includes 1, 10, 30, 50, 100, etc.). Due to space limitations, only a few examples are specifically listed, but the present application is considered to expressly list all possible combinations of values (including the stated minimum and maximum values). [Example]

[0088] Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the reagents used are commercially available. Unless otherwise specified, the concentration (%) of each reagent refers to the volume percentage concentration (%(v / v)) of the reagent.

[0089] The DMEM, MEM medium, serum, and reagents used were all commonly used commercially available products. A collection solution composition (also referred to as a treatment liquid or collection solution) was prepared according to the instructions in Preparation Example 1.

[0090] The materials used in the following examples are described below: 1. Tris base (obtained from Biosharp; catalog number: 77-86-1) Preparation of 400 mmol / L Tris base mother solution: 1.938 g of Tris base was weighed and dissolved in a small amount of water for injection, and the volume was adjusted to 40 ml with water for injection. The 400 mmol / L Tris base mother solution was diluted with water for injection to prepare 1 mM Tris.

[0091] 2. NaHCO3 (obtained from Gibco; catalog number: 25080-094) A 7.5% (w / v) NaHCO3 solution was prepared using ultrapure water.

[0092] 3. DMEM (Gibco; Catalog Number: C11965500CP) Methylcellulose (Sangon Biotech; product number: A600615-0250) 4% methylcellulose: 8.0 g of methylcellulose was weighed out, 160 ml of ultrapure water was added, and the mixture was shaken. The volume was then adjusted to 200 ml using a measuring cylinder, sterilized at 121°C for 20 minutes, cooled to room temperature, and stored at 4°C. The mixture was shaken daily until the methylcellulose was completely dissolved.

[0093] Semi-solid medium: 360 ml of DMEM medium, 15 ml of FBS, and 125 ml of 4% methylcellulose were taken, shaken to make the medium uniform, and stored at 4°C.

[0094] 4. Trypsin (obtained from Gibco; catalog number A12177-01) Before use, trypsin was diluted with the treatment solution to a final concentration of 0.01 to 0.12% (w / v).

[0095] 5. Nuclease (obtained from Merck; catalog number: 70746-10KUN) The nuclease was diluted with the treatment solution to a final concentration of 1 to 100 IU / ml before use. For example, 1 mM MgCl2 can be added to activate the nuclease.

[0096] 6. Composition of PBS buffer: NaCl 137 mM, KCl 2.7 mM, NaHPO 10 mM, KHPO 2 mM, pH 7.2

[0097] The composition of the buffer solution "potassium dihydrogen phosphate and disodium hydrogen phosphate" is potassium dihydrogen phosphate 0.29% (g / ml), disodium hydrogen phosphate 0.026% (g / ml), and pH 7.2 to 7.4.

[0098] Potassium dihydrogen phosphate was obtained from Shanghai Sinopharm Co., Ltd. (catalog number: 7778-77-0); disodium hydrogen phosphate was obtained from Hushi Co., Ltd. (catalog number: 10020318); all of analytical purity.

[0099] 7. Vaccinia virus working seed: The vaccinia virus is the recombinant oncolytic poxvirus DDvv-hIL21 disclosed in PCT Publication No. WO 2019 / 062234 A1, and its preparation method is described in Preparation Examples 1 and 2 of this PCT application.

[0100] 9.293 [HEK-293] cells were obtained from ATCC (catalog number: ATCC™ CRL-1573). Vero cells were obtained from ATCC (catalog number: ATCC™ CCL-81). HeLa cells were obtained from ATCC (catalog number: ATCC™ CCL-2). SLF-1 cell line was obtained from the Chinese Academy of Sciences Institute of Microbiology (CGMCC) (deposit number: CGMCC No. 4875).

[0101] The test methods used in the following examples are described below: 1. Counting by trypan blue staining method The cells were washed with PBS, digested with trypsin, and suspended in PBS. Trypan blue staining solution was then added at a final concentration of 0.04% (w / v). The cells were then counted under a microscope. Dead cells were stained blue, while live cells were colorless and transparent. The number of live cells was used as the final data.

[0102] 2. Measurement of Viral Titer (1) Measurement of virus titer by plaque-forming unit (pfu) method Vero cells were seeded into a 6-well plate. When the cells had spread to more than 80% of the 6-well plate, the cell culture medium was gently aspirated. 500 μl of the virus sample to be tested, diluted with cell maintenance medium, was added. Five wells were set up in parallel for each dilution, and one well containing cell maintenance medium served as a negative control.

[0103] The plate was gently shaken and placed at 37°C for 2 hours, then gently shaken once every 30-60 minutes. The liquid in the wells was aspirated, and 2 ml of DMEM medium was added to wash out any unadsorbed free virus. 3-4 ml of semi-solid medium was then added, and the plate was incubated statically in a 37°C, 5% CO2 incubator for 2-3 days. Viral plaque formation was confirmed during the incubation period. (The following steps do not require sterile conditions.)

[0104] The 6-well plate was removed, the semi-solid medium was discarded, and 1 ml of 1% crystal violet staining solution was added. The plate was then left to stand at room temperature for 30 minutes to 3 hours. The staining solution was gently rinsed off with running water, and the number of plaques per well was counted (or counted after drying). The virus titer was calculated according to the following formula:

[0105] Viral titer (pfu / ml) = average number of viral plaques × dilution factor / amount of virus added (ml)

[0106] (2) Titer measurement by tissue culture median infectious dose (TCID50) method 293 cells in DMEM culture medium at approximately 1 × 10 5 A suspension of 100 μl / ml was prepared and inoculated into a 96-well plate at 100 μl per well. (At the same time, 10-fold dilutions of virus were prepared for infecting the cells.) 100 μl of the same virus dilution was added to the first 10 wells of each row, and an equal volume of 2% BCS DMEM was added to the 11th and 12th wells as a negative control.

[0107] The plates were placed in a CO2 incubator at 37°C for 10 days, and then observed under an inverted fluorescent microscope to assess and record the cytopathic effect (CPE) in each row. The evaluation criteria considered a positive result as long as only a small number of cells showed CPE. If it was difficult to assess CPE or cell death, the wells were compared with negative controls.

[0108] The titer was calculated according to the following formula: T = 10 1+d(S-0.5) IU / ml where d = Log10 dilution factor, S = sum of positive ratios from the first dilution. The difference in titers obtained from two parallel experiments must be ≤10 0.7 It would be.

[0109] 3. Measuring the Number of Viral Particles The virus was thoroughly lysed in a virus lysis solution, and the OD260 of the sample was measured using the virus stock solution as a blank.

[0110] After treating the virus sample with SDS (sodium dodecyl sulfate), the number of virus particles was determined by measuring the A260nm value (UV-SDS method): 250μl of virus was taken, an equal volume of 0.2% SDS solution was added, and the mixture was shaken to mix uniformly and placed in a 56°C water bath for 10 minutes. After cooling to room temperature, the mixture was briefly centrifuged. Equal volumes of the virus stabilization solution and 0.2% SDS solution were mixed, and the resulting solution was used as a blank control. The absorbance at 260nm and 280nm was measured. Two experiments were performed in parallel.

[0111] Calculation formula: Number of virus particles = A260nm x dilution factor x 1.1 x 10 12

[0112] Preparation Example 1 1. Preparation of the collection treatment liquid (also called collection solution) The collection solution was prepared in a sterile environment as follows:

[0113] (1) According to Table 1, the corresponding amounts of reagents were added to water for injection and dissolved thoroughly. (2) The solution sufficiently dissolved in step (1) was diluted to the target volume with water for injection. (3) The pH was adjusted to the corresponding value. (4) Sterilized by filtration and stored at room temperature for later use. (5) The osmotic pressure of each solution was measured using an automatic freezing point osmometer. The results are as follows: Osmolarity of 1mM Tris: 1mOsmol / kg Osmolality of 7.5% NaHCO3: 1785 mOsmol / kg

[0114] [Table 1]

[0115] 2.Cell culture Preparation of chicken embryo fibroblasts (CEFs): Seven- to nine-day-old SPF chicken embryos were collected. The surface of the eggs was wiped with a cotton ball containing iodine and 75% alcohol. After collection, the head, limbs, and internal organs were removed.

[0116] The remaining tissue fragments were cut and digested with trypsin at room temperature for 15-20 minutes. Complete DMEM medium was added, purged repeatedly with an automatic pipette, and allowed to settle for 5 minutes. The tissue was diluted with DMEM, and the processed tissue was filtered three times through four layers of sterile gauze. Finally, single cell counts were performed.

[0117] The treated CEF cells were cultured overnight at 37°C. The morphology and quality of the cells were observed under a microscope. The cells were counted using trypan blue staining and confirmed with a viable cell counter. The viability of the prepared CEF cells was calculated. Microscopic observation showed that the morphology was intact and in good condition.

[0118] 293, Vero, and HeLa cells were cultured using standard methods. The procedure was as follows: Adherent cells at 80% to 90% confluence were removed from a 37°C, 5% CO2 incubator. The surface of the cell culture flask was sprayed with 75% alcohol.

[0119] The cell culture flask was then rotated and placed in a biological safety cabinet, and the medium was aspirated using a sterile pipette. A volume of prewarmed PBS was added to wash the flask once, and the PBS was aspirated. 0.25% (w / v) trypsin was added to the cell culture flask, which was then shaken to thoroughly distribute the trypsin over the cell surface. The bottle cap was then closed, and the cell culture flask was placed in a 37°C, 5% CO2 incubator for digestion.

[0120] The cells were observed under a microscope to see if they had rounded up. If they had not, the digestion was continued until the cells were completely rounded up. The reaction was stopped by adding a certain amount of medium containing 10% FBS.

[0121] The cells on the flask were gently blown off with a pipette and resuspended by repeated pipetting. After resuspension, the cells were transferred to a new cell culture flask, supplemented with 10% FBS medium, and cultured in a 37°C, 5% CO2 incubator. The cells were observed daily until they reached 80%-90% confluence. They were then passaged again.

[0122] Example 2: Testing the cytolytic effect of treatment solutions T75 cell culture flasks (each flask has a cell culture area of 75 cm 2 ) were used to culture 293, Vero, and HeLa cells. After the cells grew into monolayers, they were used to test the cytolytic effect of the harvesting solution.

[0123] (1) Preparation of treatment liquid: The treatment liquid was prepared according to the formulation described in Preparation Example 1.

[0124] (2) Test of cell disruption effect (cell lysis state after trypsin digestion) When 293, Vero, and HeLa cells grew into a monolayer, the initial growth medium was discarded. The cells were completely digested with 1 ml of 0.25% (w / v) trypsin at 37°C for 2 minutes. Digestion was stopped by adding 5–10 ml of 10% FBS + DMEM medium. After uniform mixing, the cells were centrifuged at 300 g for 10 minutes.

[0125] The supernatant was discarded. The cell pellet was uniformly mixed with 8 ml of the treatment solution from Groups A to E of Preparation Example 1 and allowed to stand at room temperature (maximum 120 minutes). The time was recorded, and changes in cell morphology were observed under a microscope. A small amount of the cell suspension was sampled and observed under a microscope for cell disruption.

[0126] When no intact cells were visible under a microscope (considered to be completely lysed), the suspension was centrifuged at 300 g for 10 minutes, and the presence of a precipitate was confirmed after centrifugation. Each group of experiments was repeated more than three times.

[0127] (3) Cell disruption test (in situ cell lysis) When 293 cells, Vero cells, and HeLa cells grew into a monolayer, the initial growth medium was discarded. The cell surfaces of the adherent cells were washed once or twice with 2 to 5 ml of treatment solutions A to E of Preparation Example 1. Then, 8 ml of the same fresh treatment solution was added.

[0128] The cells were allowed to stand at room temperature. The time was recorded, and changes in cell morphology were observed under a microscope. When no intact cells were observed under a microscope (considered to be completely lysed), the suspension was centrifuged at 300 g for 10 minutes, and the presence of precipitates was confirmed after centrifugation. Each group of experiments was repeated more than three times.

[0129] The test results are shown in Tables 2 and 3, respectively.

[0130] [Table 2]

[0131] [Table 3]

[0132] In this example, the following results were obtained in both the trypsin digestion and in situ cell lysis tests:

[0133] The treatment solutions for Groups A and B were able to completely lyse cells within 15 minutes, and the complete lysis time for Group B cells was shorter than that for Group A cells. Cell debris was small, there was no obvious precipitate, and the supernatant after centrifugation of the cell suspension was not viscous. The effect of removing nucleic acids was obvious. The rate of lysed cells was 100%. There was no significant difference between 293 cells, Vero cells, and HeLa cells.

[0134] The treatment solution in Group C (control group) had a low cell lysis effect. After 120 minutes of lysis, many cell morphologies were still visible under a microscope. After centrifuging the cell suspension, a clear precipitate was visible. The supernatant was viscous, indicating that some cells had lysed and intracellular materials such as nucleic acids had been released.

[0135] The treatment solutions in groups D and E (control group) were able to lyse cells by extending the lysis time. Microscopic observation showed that the cell debris was large. After centrifuging the cell suspension, no obvious precipitate was observed, and the supernatant was not viscous. The effect of nucleic acid removal was obvious. The percentage of lysed cells was less than 100%. There was no significant difference between 293 cells, Vero cells, and HeLa cells.

[0136] As shown in Tables 2 and 3, by comparing cell lysis after trypsin digestion and in situ cell lysis, it can be seen that the lysis effect of the collection solution in each group is basically the same. When cells are first digested with trypsin, the lysis time is shorter than that of in situ lysis. Therefore, the effect of treatment with the collection solution after trypsin digestion is slightly better than that of in situ lysis.

[0137] The treatment solutions of Groups A and B could effectively lyse 293, Vero, and HeLa cells within 30 minutes, regardless of whether the cells were digested with trypsin or lysed in situ, demonstrating that the harvesting solution of the present invention is applicable to 293, Vero, and HeLa cells.

[0138] Example 3: Effect of virus culture and treatment on virus titer Vaccinia virus culture and harvest: 293 cells, HeLa cells, and CEF cells were seeded into T75 cell culture flasks. Once the cells had adhered and grown to over 80% confluence, a working seed of vaccinia virus was inoculated into each cell at an MOI of 0.02. The virus-inoculated cells in the cell culture flasks were placed at 37.0±1.0°C for 2 hours to allow for adsorption. After adsorption was complete, the cells were supplemented with virus maintenance solution containing 2% FBS and placed in an incubator at 37.0±1.0°C. The cells were cultured until complete cytopathogenesis occurred (usually 48-96 hours). The culture medium was then discarded, and treatment solutions A to E described in Preparation Example 1 were added, followed by treatment for 10-60 minutes. The cells were then observed under a microscope.

[0139] After the cells were completely lysed (maximum lysis time was 60 minutes), the virus solution was collected and the lysis time and cell disruption status were examined. The virus solution was centrifuged at 300 g for 10 minutes, and the supernatant was then collected and the virus titer was measured. The presence or absence of precipitate after centrifugation and the viscosity of the supernatant were recorded.

[0140] A freeze-thaw control group (abbreviated as freeze-thaw group) was also set up as follows: Completely cytopathic cells were pipetted, and the cells and infection medium were transferred to a 15 ml sterile centrifuge tube and rapidly frozen in a -80°C refrigerator for 120 minutes. Then, the tubes were rapidly thawed in a 37°C water bath for 10 minutes. This process was repeated three times. The virus solution was centrifuged at 750 g for 10 minutes, and the supernatant was collected to detect the virus titer. Each experiment was repeated three times, and the average values were used for statistical analysis.

[0141] The virus titer was measured by plaque assay (i.e., plaque-forming unit method). Depending on the test results, the collection effect of the treatment solution of each group and its influence on the virus titer were evaluated. The results are shown in Table 4 below.

[0142] [Table 4]

[0143] The results of this experiment show that: Both Group A and Group B treatment solutions were able to completely lyse cells within 10 minutes. The cell debris was small, there was no obvious precipitate, and the supernatant after centrifugation of the cell suspension was not viscous. The effect of removing nucleic acids was obvious. The rate of lysed cells was 100%. There was no significant difference between 293 cells and HeLa cells.

[0144] The treatment solution in Group C (control group) had a low cell lysis effect. After 60 minutes of lysis, many cell morphologies were still visible under a microscope, and after centrifuging the cell suspension, obvious precipitates were visible. The supernatant was relatively viscous, indicating that some cells had been lysed and intracellular materials such as nucleic acids had been released.

[0145] The treatment solutions in groups D and E (control group) were able to lyse cells by extending the lysis time. Microscopic observation showed that the cell debris was large. After centrifuging the cell suspension, no obvious precipitate was observed, and the supernatant was not viscous. The effect of nucleic acid removal was obvious. The percentage of lysed cells was less than 100%. There was no significant difference between 293 cells and HeLa cells.

[0146] In group F (freeze-thaw control group), the cell morphology was still visible under a microscope after three freeze-thaw cycles, and cell debris was large. There was no obvious precipitate, and the supernatant after centrifugation of the cell suspension was viscous with clumps of floating material. This indicates that the cells had lysed after three freeze-thaw cycles, releasing intracellular materials such as nucleic acids. The percentage of lysed cells was less than 100%. There was no significant difference between 293 cells and HeLa cells.

[0147] The treatment solutions of Groups A and B were able to effectively lyse 293 and HeLa cells in situ within 10 minutes, demonstrating that the harvesting solution of the present invention is applicable to both 293 and HeLa cells. (A viral titer deviation of ≦30% pfu / ml is considered an acceptable detection error range. The same applies below.) Antitumor activity testing in vitro and in vivo indicated that the biological activity of the prepared oncolytic virus was not impaired.

[0148] Example 4: Effect of different processing methods on virus collection 293 cells were cultured. When the cells had adhered and grown to over 80% confluence, they were inoculated with a working seed of vaccinia virus at an MOI of 0.02. The virus-inoculated cells in a T75 cell flask were incubated at 37.0±1.0°C for 2 hours for adsorption. After adsorption was complete, the cells were replenished with virus maintenance solution containing 2% FBS and placed in an incubator at 37.0±1.0°C for 48 hours.

[0149] After the cells were completely degenerated, the intracellular virus and the supernatant (extracellular virus) were collected. Eight ml of harvesting solution G and harvesting solution H were added to the intracellular virus, respectively, and the mixture was treated for 10 minutes. The state of the cells was observed under a microscope.

[0150] After the cells were completely lysed, the virus solution was collected. The virus solution was centrifuged at 300 g for 10 minutes, and the supernatant was collected for virus titer detection. A freeze-thaw control group was also set up as follows: completely cytopathic cells were pipetted, and the cells and infection medium were transferred to a 15 ml sterile centrifuge tube, rapidly frozen in a -80°C refrigerator for 120 minutes, and then thawed in a 37°C water bath for 10 minutes. This process was repeated three times.

[0151] The virus solution was centrifuged at 750 g for 10 minutes, and the supernatant was then collected and the virus titer was determined. The virus titer collected after treatment with the treatment solution was evaluated. Each group was repeated three times, and the average value was used for statistical analysis.

[0152] The results of this experiment are shown in Figure 1. This figure shows that for harvests in which cells were completely lysed and virus was released, the results varied greatly depending on the treatment of intracellular viruses. The results varied greatly due to the different treatments of intracellular viruses. The yield of the freeze-thaw group was low; the yield of treatment solution G (1 mM Tris + 0.06% (w / v) trypsin + 5 IU / ml nuclease) was approximately six times higher than that of the freeze-thaw group; the yield of treatment solution H (7.5% NaHCO3 + 0.06% (w / v) trypsin + 5 IU / ml nuclease) was approximately five times higher than that of the freeze-thaw group.

[0153] Example 5: Effect of different dosages of treatment solution on virus collection 293 cells were seeded in T75 cell culture flasks. When the cells had adhered and grown to over 80% confluence, they were inoculated with a working seed of vaccinia virus at an MOI of 0.02. The virus-inoculated cells in the cell culture flasks were incubated at 37.0 ± 1.0°C for 0–2 hours for adsorption.

[0154] After the adsorption was completed, the cells were supplemented with a virus maintenance solution containing 2% FBS, placed in an incubator at 37.0±1.0°C, and culture was continued. After the cells were completely degenerated, the virus was collected. The culture solution was discarded, and the treatment solution of Group A described in Preparation Example 1 was added to the cells in an amount corresponding to the area of the cultured cells (i.e., 35 μl / cm). 2 , 70 μl / cm 2 , 150 μl / cm 2 ) were added according to the

[0155] The treatments were carried out for 10 and 30 minutes, respectively. The state of the cells was observed under a microscope. The virus solution was collected and the state of cell disruption was recorded. The virus solution was centrifuged at 750 g for 10 minutes, and the supernatant was then collected and tested for virus titer. The presence or absence of precipitate after centrifugation and the viscosity of the supernatant were recorded.

[0156] A freeze-thaw control group was also set up as follows: the completely cytopathic cells were pipetted, and the cells and infection medium were transferred to a 15 ml sterile centrifuge tube and rapidly frozen in a -80°C refrigerator for 120 minutes. The tubes were then thawed in a 37°C water bath for 10 minutes. This process was repeated three times.

[0157] The virus solution was centrifuged at 750 g for 10 minutes, and the supernatant was collected and the virus titer was detected by plaque assay. Depending on the results of each test, the effects of different treatment solution dosages and treatment times on the collection effect and virus titer were evaluated. The results are shown in Table 5 below.

[0158] [Table 5]

[0159] The results of this experiment show that: The treatment volume and treatment time have a significant effect on the amount of cell lysis and virus yield. Treatment solution for Group A: 35, 70, 150 μl / cm 2 When used to treat intracellular viruses, cells were effectively lysed within 30 minutes, with a 100% cell lysis rate and no viscosity in the supernatant. The cell lysis effect was also positively correlated with the dosage and treatment time of the treatment solution. 2 When the treatment time was 10 minutes, the virus infectivity was the highest, more than six times that of the freeze-thaw group.

[0160] Example 6: Cultivation and Harvesting of Various Exemplary Viruses Varicella virus culture and collection: SLF-1 cells were seeded into a T25 cell culture flask. Once the cells had grown into a monolayer, a working seed of the Varicella-Zoster virus Oka strain (obtained from ATCC, number VR-795) was inoculated into the SLF-1 cells at an MOI of 0.001-0.1. The virus-inoculated cells in the cell flask were allowed to adsorb for 2 hours at 35.0±1.0°C.

[0161] After adsorption was complete, the virus MEM maintenance solution was replenished, and the cells were then placed in an incubator at 35.0±1.0°C and cultured for 48 to 96 hours. The culture medium was discarded, and the harvesting solutions A to E described in Preparation Example 1 were added. The cells were left to stand at room temperature for 10 minutes and then observed under a microscope. When the cells swelled significantly, they were shaken to separate, and the virus solution was then collected. The cell swelling time, cell detachment rate, cell disruption, and virus titer were measured. A freeze-thaw harvesting control group was also set up. Based on the results, the harvesting effect of each harvesting solution and its effect on virus titer were evaluated.

[0162] HSV culture and harvest: Vero cells were placed in a 37°C, 5% CO2 incubator for growth and cultivation. After the cells had grown into a monolayer, they were infected with the virus at an MOI of 0.001 to 0.1. When the cells had rounded up but had not yet fallen off, the infected cells and supernatant were harvested. For 10 to 60 minutes of treatment, treatment solutions A to E described in Preparation Example 1 were added, respectively. The state of the cells was observed under a microscope.

[0163] After the cells were completely lysed (maximum lysis time was 60 minutes), the virus solution was collected, and the time until cell lysis and cell disruption were recorded. The virus solution was centrifuged at 300 g for 10 minutes, and the supernatant was then collected and the virus titer was measured. The presence or absence of precipitate after centrifugation and the viscosity of the supernatant were recorded. A freeze-thaw control group and a supernatant control group were also set up. The virus titer was measured by plaque assay. The collection effect of the treatment solution in each group at various time points and its influence on the virus titer were evaluated according to the results.

[0164] Adenovirus culture and collection: 293 cells were placed in a 37°C, 5% CO2 incubator for growth and culture. After the cells had grown into a monolayer, they were infected with the virus at an MOI of 1 to 100. When the cells had rounded up but had not yet fallen off, the infected cells and supernatant were collected. For 10 to 60 minutes of treatment, treatment solutions A to E described in Preparation Example 1 were added, respectively. The state of the cells was observed under a microscope.

[0165] After the cells were completely lysed (maximum lysis time was 60 minutes), the virus solution was collected, and the time until cell lysis and cell disruption were recorded. The virus solution was centrifuged at 300 g for 10 minutes, and the supernatant was then collected and the virus titer was measured. The presence or absence of precipitate after centrifugation and the viscosity of the supernatant were recorded. A freeze-thaw control group and a supernatant control group were also set up. The virus titer was measured using the TCID50 method. The collection effect of the treatment solution in each group at various time points and its influence on the virus titer were evaluated according to the results.

[0166] Lentivirus culture and harvest: 293 cells were grown and cultured in a 37°C, 5% CO2 incubator. Four types of plasmids (i.e., three proteins required for viral packaging: Gag / Pol, Rev, and VSV-G (a substitute for HIV-1 Env) were independently placed on three plasmids, and a target gene vector was constructed on the pLenti-Gene plasmid, yielding four types of plasmids) were added to the cells for infection. When the cells became rounded but had not yet fallen off, the infected cells and supernatant were harvested. For 10-60 minutes of treatment, treatment solutions A to E described in Preparation Example 1 were added. The cells were observed under a microscope.

[0167] After the cells were completely lysed (maximum lysis time was 60 minutes), the virus solution was collected, and the time until cell lysis and cell disruption were recorded. The virus solution was centrifuged at 300 g for 10 minutes, and the supernatant was then collected and the virus titer was measured. The presence or absence of precipitate after centrifugation and the viscosity of the supernatant were recorded. A freeze-thaw control group and a supernatant control group were also set up. The collection effect of the treatment solution in each group at various time points and their influence on the virus titer were evaluated according to the results.

[0168] Example 7: Scale-up experiment of virus production process After resuscitation, 293 cells were grown and cultured in DMEM medium containing 10% fetal bovine serum. When the cells reached 80-90% confluence, they were seeded into cell factories and cultured in a CO2 incubator for approximately 48 hours. Afterwards, the cells were digested and a cell suspension was prepared.

[0169] The cell suspension was seeded into a cell culture vessel (obtained from NBS, CelliGen Plus / 310, surface area approximately 22 m2). The cells were perfusion cultured, and after 6-8 days, they were infected with vaccinia virus at an MOI of 0.02. The cells were infected with the virus for approximately 48-72 hours, after which the virus was harvested. By using the treatment solution for group G described in Preparation Example 1, 1.3 x 10 13 PFU / culture tank of virus was collected.

[0170] Example 8: Process scale-up experiment using cell factories After resuscitating, 293 cells were cultured in DMEM medium containing 10% fetal calf serum in three 10-layer cell factories (Corning "CellSTACK Chamber, 10 STACK, POLYSTYRENE, STERILE, 1 / 6"; total area of the three 10-layer cell factories: 6360 x 3 = 19080 cm). 2 When the cells reached 80-90% confluence, they were infected with vaccinia virus at an MOI of 0.02 using an infection medium of DMEM containing 2% serum.

[0171] Approximately 72 hours after viral infection, the cells were knocked off and transferred together with the medium to a sterile centrifuge bottle. The mixture was centrifuged at 2000 rpm at 4°C for 30 minutes while balancing. The supernatant was then discarded, and the cell pellet was transferred to 3 L of treatment solution (approximately 157 μl / cm) equilibrated to room temperature for lysis and collection. 2 The cells were treated with 100 μg of PBS.

[0172] The treatment solution consisted of 1 mM Tris, 0.03% recombinant trypsin, 5 U / ml nuclease, pH 9.0, and 1 mM MgCl2. 10 PFU of virus (yield per unit area): 4.09 x 10 6 PFU / cm 2 ) can be collected.

[0173] Example 9: Process scale-up experiment using a fermenter (5 L) 293 cells were resuscitated in three T225 cell culture flasks (Corning, catalog number: 431082) and grown and cultured in DMEM medium containing 10% fetal calf serum. When the cells reached 80%-90% confluence, they were transferred to a 10-layer cell factory (Corning "CellSTACK Chamber, 10 STACK, POLYSTYRENE, STERILE, 1 / 6"; total area: 6360 cm). 2 After culturing for approximately 72 hours in a CO2 incubator, the cells were digested to prepare a cell suspension.

[0174] Using the cell suspension, the inoculation volume was 470 ml, and the total number of cells was 1.78 × 10 9 The cell culture vessel (BioFlo 320, purchased from Eppendorf, 5L) has a surface area of approximately 150,000 cm 2 The cells were perfused and then infected with vaccinia virus at an MOI of 0.025 after 6-8 days. Approximately 72 hours after infection, the cells were lysed and the virus was collected in situ along with the treatment solution.

[0175] 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 volume of the treatment solution was approximately 13.5 L (approximately 90 μl / cm 2 The number of cells was 4.41 × 10 11 PFU / virus yield per unit area of culture vessel: 2.94 x 10 6 PFU / cm 2 ) can be collected.

[0176] Example 10: Process scale-up experiment using a fermenter (5 L) 293 cells were resuscitated in three T225 cell culture flasks (Corning, catalog number: 431082) and grown and cultured in DMEM medium containing 10% fetal calf serum. When the cells reached 80%-90% confluence, they were transferred to one cell factory (Corning "CellSTACK Chamber, 10 STACK, POLYSTYRENE, STERILE, 1 / 6"; total area: 6360 cm). 2 After culturing for approximately 72 hours in a CO2 incubator, the cells were digested to prepare a cell suspension.

[0177] Using the cell suspension, the seeding volume was 430 ml, and the total number of cells was 1.97 × 10 9 The cell culture vessel (BioFlo 320, purchased from Eppendorf, 5L) has a surface area of approximately 150,000 cm 2 The cells were perfused and then infected with vaccinia virus at an MOI of 0.06 after 6-8 days. Approximately 5 days after infection, the cells were lysed and the virus was collected in situ along with the treatment solution.

[0178] 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 volume of the treatment solution was approximately 15 L (approximately 100 μl / cm 2 The number of cells was 2.41 × 10 11 PFU / virus yield per unit area of culture vessel: 1.61 x 10 6 PFU / cm 2 ) can be collected.

[0179] Example 11: Process scale-up experiment using a fermenter (14 L) 293 cells were resuscitated in three T225 cell culture flasks (Corning, Catalog No.: 431082) and grown and cultured in DMEM medium containing 10% fetal bovine serum. When the cells reached 80% to 90% confluence, they were transferred to three 10-layer cell factories (Corning "CellSTACK Chamber, 10 STACK, POLYSTYRENE, STERILE, 1 / 6"; total area of the three 10-layer cell factories: 6360 x 3 = 19080 cm). 2 After culturing for approximately 72 hours in a CO2 incubator, the cells were digested to prepare a cell suspension.

[0180] Using a cell suspension, the inoculation volume was 1400 ml, and the total number of cells was 3.19 × 10 9 The cell culture vessel (BioFlo 320, purchased from Eppendorf, 14 L) has a surface area of approximately 450,000 cm 2 The cells were perfused and then infected with vaccinia virus at an MOI of 0.05 after 6-8 days. Approximately 72 hours after infection, the cells were lysed and the virus was collected in situ along with the treatment solution.

[0181] 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 volume of the treatment solution was approximately 50 L (approximately 110 μl / cm 2 The number of cells was 1.24 × 10 12 PFU / virus yield per unit area of culture vessel: 2.76 x 10 6 PFU / cm 2 ) can be collected.

[0182] From the above examples, it can be seen that the collection method and collection treatment solution of the present invention can greatly simplify the production process and are particularly suitable for mass production of viruses. By adopting the collection method and collection treatment solution of the present invention, the yield of the collected virus is high and the amount of impurities is reduced. This makes the subsequent purification process easier to carry out and results in higher purity and biological activity of the resulting virus.

[0183] It is understood that the above embodiments are merely exemplary embodiments used to explain the principles of the present invention, and the present invention is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and essence of the present invention, and these changes and modifications also fall within the protection scope of the present invention.

Claims

1. 1. A method for producing a virus, comprising the steps of: Culturing cells, wherein the cells have been inoculated with a virus or transfected with viral packaging elements; and A step of collecting viruses in one step by contacting cultured cells with a collection solution composition, wherein the collection solution composition comprises trypsin, a pH buffer, and a nuclease, and the pH of the collection solution composition is in the range of 8.5 to 9.5, the concentration of trypsin in the collection solution composition is in the range of 0.03 to 0.06% (w / v), the concentration of nuclease in the collection solution composition is in the range of 5 to 50 IU / ml, and the osmotic pressure of the collection solution composition is in the range of 1 to 20 mOsmol / kg or 1785 to 2000 mOsmol / kg.

2. 2. The method of claim 1, wherein the pH buffer is selected from Tris buffer and sodium bicarbonate buffer.

3. The method according to claim 1, wherein the culture is carried out in a cell flask, a cell factory, or a culture vessel, and the culture is an adherent culture or a suspension culture.

4. If the culture is an adherent culture, the amount of the harvesting solution composition is 35 μl or more of the harvesting solution composition / cm ２ When the culture is a suspension culture, the amount of the harvesting solution composition is 35 μl or more of the harvesting solution composition / 10 ５ The method of claim 1, wherein the range is a cell.

5. If the culture is an adherent culture, the amount of the harvesting solution composition is 70 μl or more of the harvesting solution composition / cm ２ The method of claim 1, wherein the range of cells is 100-1500.

6. When the culture is a suspension culture, the amount of the harvesting solution composition is 70 μl or more of the harvesting solution composition / 10 ５ The method of claim 1, wherein the range is a cell.

7. 10. The method of claim 1, wherein the cultured cells are contacted with the harvesting solution composition for a time period ranging from 5 to 60 minutes.

8. 2. The method of claim 1, wherein the cells are selected from Vero cells, 293 cells, CEF cells, and HeLa cells.

9. 2. The method of claim 1, wherein the virus comprises 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, coxsackievirus, Newcastle disease virus, and Maraba virus.

10. The method of claim 1 , wherein the virus comprises an enveloped virus.

11. 2. The method of claim 1, wherein the cells are 293 cells and the virus is vaccinia virus.

12. The method according to any one of claims 1 and 3 to 11, wherein the collection solution composition comprises a Tris buffer, trypsin, and a nuclease, and the concentration of the Tris buffer is in the range of 1 to 50 mM.

13. The method according to any one of claims 1 and 3 to 11, wherein the collection solution composition comprises a Tris buffer, trypsin, and a nuclease, and the concentration of the Tris buffer is in the range of 1 to 10 mM.

14. A collection solution composition for collecting viruses subjected to cell culture, the collection solution composition comprising trypsin, a pH buffer, and a nuclease, the collection solution composition having a pH in the range of 8.5 to 9.5, a trypsin concentration in the range of 0.03 to 0.06% (w / v), a nuclease concentration in the collection solution composition in the range of 5 to 50 IU / ml, and an osmotic pressure in the range of 1 to 20 mOsmol / kg or 1785 to 2000 mOsmol / kg.

15. 15. The collecting solution composition of claim 14, wherein the pH buffer is selected from a Tris buffer and a sodium bicarbonate buffer.

16. 15. The harvesting solution composition according to claim 14, wherein the culture is carried out in a cell flask, a cell factory, or a culture tank, and the culture is an adherent culture or a suspension culture.

17. 15. The collection solution composition of claim 14, wherein the cells are selected from Vero cells, 293 cells, CEF cells, and HeLa cells.

18. 15. The collection solution composition of claim 14, wherein the virus comprises 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, coxsackievirus, Newcastle disease virus, and Maraba virus.

19. 15. The collection solution composition of claim 14, wherein the virus comprises an enveloped virus.

20. 15. The collection solution composition of claim 14, wherein the cells are 293 cells and the virus is vaccinia virus.

21. The collecting solution composition according to any one of claims 14 and 16 to 20, wherein the collecting solution composition comprises a Tris buffer solution, trypsin, and a nuclease, and the concentration of the Tris buffer solution is in the range of 1 to 50 mM.

22. The collecting solution composition according to any one of claims 14 and 16 to 20, wherein the collecting solution composition comprises a Tris buffer solution, trypsin, and a nuclease, and the concentration of the Tris buffer solution is in the range of 1 to 10 mM.

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