Process optimization method for perfusion culture of oncolytic herpes virus by using bioreactor

Abstract

The invention mainly provides a method for producing oncolytic viruses by using a bioreactor. Through perfusion culture, the Vero cell quantity during virus infection is optimized, so that the purposes of increasing the virus yield, reducing the batch difference and finally producing the oncolytic virus can be achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals. Specifically, the present invention provides a process optimization method for perfusion culture of oncolytic herpes virus using a bioreactor. Background Art

[0002] Oncolytic viruses selectively infect tumor cells by inactivating or defective tumor suppressor genes in target cells, replicate in large quantities in tumor cells and eventually destroy them, while regulating the tumor microenvironment to stimulate immune responses. Herpes simplex virus (HSV), especially herpes simplex virus type 1 (HSV-1), is one of the most widely studied DNA viruses as OV. In October 2015, the FDA approved the first oncolytic virus product T-Vec, which uses herpes simplex virus type 1 (HSV-1) as a carrier, for the treatment of melanoma, showing good therapeutic efficacy and safety.

[0003] Traditional virus production processes such as roller bottles and cell factories have disadvantages such as complex operation procedures, high risk of contamination, inability to adjust cell / virus culture conditions, large space occupation and high staff requirements. Large-scale culture technology using bioreactors has increasingly important practical significance. Summary of the invention

[0004] The present invention mainly provides a method for producing oncolytic viruses using a bioreactor. Through perfusion culture, the amount of Vero cells during virus infection is optimized, so as to increase the virus yield, reduce the batch difference, and ultimately be used for the purpose of oncolytic virus production.

[0005] The present invention is based on the conditions of comprehensive adaptation to the oncolytic virus replication and amplification system, and adopts a perfusion culture method to provide new nutrients for the growth of Vero cells, increase cell density, and obtain a virus harvest liquid with a higher virus titer. In addition, the carrier sheet in the harvesting process can better filter some cell fragments, reducing the difficulty of later purification and removal of process-related impurities (HCD, HCP).

[0006] To this end, the present invention provides a method for producing an oncolytic virus, comprising:

[0007] 1) using a bioreactor for perfusion culture of Vero cells, wherein a sheet carrier is provided in the bioreactor, and the Vero cells are inoculated and perfused cultured on the sheet carrier;

[0008] 2) infecting the Vero cells with an oncolytic virus and performing virus culture;

[0009] 3) Harvesting oncolytic virus;

[0010] Preferably, the oncolytic virus is herpes simplex virus (HSV).

[0011] In some embodiments, the method further has one or more technical features selected from the following (i)-(iii):

[0012] (i) In step 1), the seeding density of the Vero cells is 4E+05cells / mL~8E+05cells / mL, such as 4E+05cells / mL~4.5E+05cells / mL, 4.5E+05cells / mL~5E+05cells / mL, 5E+05cells / mL~5.5E+05cells / mL, 5.5E+05cell s / mL~6E+05cells / mL, 6E+05cells / mL~6.5E+05cells / mL, 6.5E+05cells / mL~7E+05cells / mL, 7E+05cells / mL~7.5E+05cells / mL or 7.5E+05cells / mL~8E+05cells / mL, such as 5E+05cells / mL;

[0013] (ii) in step 2), when the culture density of the Vero cells is 1 to 2E+07 cells / mL, infecting the Vero cells with an oncolytic virus and performing virus culture;

[0014] (iii) The density of the sheet-like carrier is 25-35 g / L, such as 25-27 g / L, 27-29 g / L, 29-31 g / L, 31-33 g / L or 33-35 g / L, such as 28 g / L.

[0015] In some embodiments, the method further has one or more technical features selected from the following (i)-(v):

[0016] (i) In step 1), during the perfusion culture, the glucose concentration is controlled at 1.5-2.0 g / L, such as 1.5-1.6 g / L, 1.6-1.7 g / L, 1.7-1.8 g / L, 1.8-1.9 g / L or 1.9-2.0 g / L;

[0017] (ii) in step 1), during the perfusion culture, the pH is 7.1 to 7.3, for example, 7.1 to 7.2 or 7.2 to 7.3;

[0018] (iii) in step 1), during the perfusion culture, the temperature is 35-40°C, for example, 35-36°C, 36-37°C, 37-38°C, 38-39°C or 39-40°C, for example, 37°C;

[0019] (iv) in step 1), during the perfusion culture, the dissolved oxygen is 45% to 50%, for example, 45% to 46%, 46% to 47%, 47% to 48%, 48% to 49% or 49% to 50%;

[0020] (v) In step 1), during the perfusion culture, the stirring speed is 60-80 rpm, such as 60-65 rpm, 65-70 rpm, 70-75 rpm or 75-80 rpm.

[0021] In some embodiments, the method further has one or more technical features selected from the following (i)-(iv):

[0022] (i) In step 2), when the virus is cultured, the pH is 7.3 to 7.5, for example, 7.3 to 7.4 or 7.4 to 7.5;

[0023] (ii) in step 2), the virus is cultured at a temperature of 32-37°C, such as 32-33°C, 33-34°C, 34-35°C, 35-36°C or 36-37°C, such as 34°C;

[0024] (iii) in step 2), during the virus culture, the dissolved oxygen is 45% to 50%, for example, 45% to 46%, 46% to 47%, 47% to 48%, 48% to 49% or 49% to 50%;

[0025] (iv) In step 2), during the virus culture, the stirring speed is 60-80 rpm, such as 60-65 rpm, 65-70 rpm, 70-75 rpm or 75-80 rpm.

[0026] In some embodiments, the method further has one or more technical features selected from the following (i)-(iii):

[0027] (i) In step 1), during the perfusion culture, the culture medium is a DMEM culture medium containing 10% serum;

[0028] (ii) in step 2), the virus culture medium is serum-free DMEM medium;

[0029] (iii) The volume of the culture medium during the perfusion culture in step 1) is the same as the volume of the culture medium during the virus culture in step 2).

[0030] In some embodiments, in step 2), when the Vero cells are infected with an oncolytic virus, the oncolytic virus is inoculated at an MOI of 0.001 to 0.01, such as 0.001 to 0.002, 0.002 to 0.004, 0.004 to 0.005, 0.005 to 0.006, 0.006 to 0.008, or 0.008 to 0.01.

[0031] In some embodiments, the oncolytic virus is herpes simplex virus type 1 (HSV-1).

[0032] In some embodiments, the oncolytic virus is recombinant HSV-1.

[0033] In some embodiments, the recombinant HSV-1 comprises a modification selected from at least one of the following:

[0034] a. Delete ICP34.5;

[0035] b. Insert a nucleic acid encoding at least one exogenous polypeptide selected from the following: IL12, IL15 / 15RA, PD-L1.

[0036] In some embodiments, in step 3), the oncolytic virus is harvested after culturing for 70 to 90 hours (e.g., 70 to 72 hours, 72 to 74 hours, 74 to 75 hours, 75 to 76 hours, 76 to 78 hours, 78 to 80 hours, 80 to 82 hours, 82 to 84 hours, 84 to 85 hours, 85 to 86 hours, 86 to 88 hours, or 88 to 90 hours, e.g., 80 hours).

[0037] In some embodiments, the method further has one or more technical features selected from the following (i)-(ii):

[0038] (i) the sheet carrier is a Disks sheet carrier;

[0039] (ii) The bioreactor is a basket bioreactor, such as a 10 L basket bioreactor.

[0040] In some embodiments, the sheet-shaped carrier is pre-soaked and washed with a buffer.

[0041] In some embodiments, the buffer is PBS buffer.

[0042] In some embodiments, the pH of the PBS buffer is 7.2-7.6, such as 7.2-7.3, 7.3-7.4, 7.4-7.5 or 7.5-7.6, such as 7.2.

[0043] In some embodiments, in step 3) of the method, the virus produced by Vero cells is harvested using an extract, wherein the extract comprises sucrose, Tris hydrochloric acid and sodium chloride, wherein the proportion of sucrose is 1%-5%, for example, 1%-2%, 2%-3%, 3%-4%, 4%-5%, the proportion of Tris hydrochloric acid is 2%-8%, for example, 2%-3%, 3%-4%, 4%-5%, 5%-6%, 6%-7%, 7%-8%, and the proportion of sodium chloride is 10%-15%, for example, 10%-11%, 11%-12%, 12%-13%, 13%-14%, 14%-15%.

[0044] In some embodiments, the extract is incubated with the sheet-shaped carrier in step 1) for 30-60 minutes, such as 30-40 minutes, 40-50 minutes, or 50-60 minutes.

[0045] Beneficial Effects

[0046] 1. Oncolytic herpes virus is a type of natural or recombinant virus that can selectively infect and replicate in large quantities, ultimately dissolving tumor cells without damaging normal cells. It mainly achieves the purpose of tumor targeted therapy by selectively killing tumor cells and inducing the body to produce specific anti-tumor immune responses.

[0047] The innovative purpose of the present invention is to optimize the amplification process of oncolytic virus in perfusion culture by using a bioreactor to the greatest extent, to obtain a higher titer of oncolytic virus by increasing the cell density in the reactor, and to stabilize the process to achieve good repeatability and high stability, which can ultimately be used for the production of oncolytic virus products.

[0048] 2. The present invention mainly fixes the Disks sheet carrier in the bioreactor, cultivates Vero cells by optimizing the perfusion process parameters, and selects the appropriate time point for perfusion according to the cell growth rate, nutrient consumption and metabolite inhibition to ensure a reasonable culture environment and a low level of metabolite inhibition. Perfusion increases the cell mass and realizes large-scale production of oncolytic viruses, while achieving the purpose of increasing virus yield, reducing the difficulty of downstream purification and removal of process-related impurities (HCD, HCP), and reducing batch differences.

[0049] The cell perfusion system can keep cells or enzymes in the reactor and maintain a high Vero cell density (1-2E+07 cells / ml); without perfusion, the cell density is generally 8.0E+06 cells / ml.

[0050] 3. Traditionally, viruses are harvested by breaking cells with a high-pressure homogenizer, which easily produces tiny fragments of sheet carriers, thereby introducing impurities into the virus harvest solution. The present invention uses an extraction solution to harvest viruses, which can minimize the use of additional equipment, such as crushers, homogenizers, etc., and reduce the cost of GMP equipment validation and maintenance. At the same time, the possibility of introducing other sources of contamination can be minimized. At the same time, the extraction solution can use biological methods to separate viruses from cells, while not affecting the breakage of the carrier sheet on which cell growth depends, reducing the impact of exogenous impurities on virus purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The effect of perfusion on cell growth density is shown.

[0052] Figure 2 The effect of perfusion on viral titer is shown.

[0053] Figure 3 The effect of perfusion on impurities is shown.

[0054] Figure 4 The effect of extract virus harvest on the purity of the virus harvest is shown. DETAILED DESCRIPTION

[0055] Example 1

[0056] In order to make the present invention easier to understand, the present invention is explained below with specific implementation examples. It should be understood that: the implementation examples are only used to illustrate the present invention rather than to limit the present invention, and specific experimental methods not mentioned in the implementation examples below are carried out according to conventional experimental methods.

[0057] Implementation Case 1

[0058] Cells: African green monkey kidney cells Vero (ATCC)

[0059] Oncolytic virus: Recombinant HSV-1 oncolytic virus, self-constructed

[0060] Bioreactor: 10L basket bioreactor (Shanghai Ritai Pharmaceutical Equipment Engineering Co., Ltd.)

[0061] Carrier: Disks sheet carrier (Shanghai Golos Biotechnology Co., Ltd.)

[0062] Cell culture medium: DMEM medium (10% serum) (Gibco, USA)

[0063] Virus infection medium: serum-free DMEM medium (Gibco, USA)

[0064] MOI: 0.001~0.01

[0065] Glucose content determination: M-100 biochemical analyzer (Shenzhen Silman Company) direct determination

[0066] Cell seed: After cell recovery, subculture at a density of 1.0 to 1.5E+05 cells / ml and expand to 4 to 8E+05 cells / ml

[0067] Virus seeds: build your own working seed library (WVB)

[0068] Cultivate cells in bioreactor: Add 25-35g / L Disks sheet carrier and pH 7.2 phosphate buffer PBS into the reactor, discard PBS and add 5L DMEM for sterility monitoring. After the test is qualified, inoculate Vero cells (inoculation amount is about 4-8E+05 cells / ml) and start perfusion culture. The culture parameters are set as: pH 7.1-7.3, temperature 37°C, dissolved oxygen 45%-50%, stirring speed 60-80rpm, and control the glucose concentration at 1.5-2.0g / L. Take samples regularly every day to measure the glucose consumption, and calculate the cell density in the reactor based on the glucose consumption.

[0069] Virus infection and harvesting: Virus infection can begin when the cell culture density reaches 1-2E+07 cells / ml. Inoculate the virus according to the MOI and replace it with serum-free DMEM culture medium for virus culture. The culture parameters are set as follows: pH 7.3-7.5, temperature 34°C, dissolved oxygen 45%-50%, stirring speed 60-80rpm, and the virus can be harvested after culturing for about 70-90 hours. Use an extract to harvest the virus. The extract formula is 1%-5% sucrose, 2%-8% Tris hydrochloric acid and 10%-15% sodium chloride. After the extract is incubated with the Disks sheet carrier for 45±15 minutes, the extract and the virus culture medium are collected together, that is, the virus harvest liquid. As can be seen from Table 2, compared with the method of harvesting viruses by breaking cells with a high-pressure homogenizer, the use of an extract to harvest viruses has a higher titer, which significantly helps to increase virus yield. In addition, by Figure 4 It can be seen that using extraction liquid to harvest viruses can obtain higher virus titers, relatively reduce impurities, and obtain virus harvest liquid with higher purity.

[0070] Method for determining the titer of oncolytic virus: The virus sample is diluted in a gradient manner and added to a six-well plate filled with Vero cells. After static adsorption, the virus solution is discarded, and a culture medium containing methylcellulose is added for 72 to 96 hours. The culture medium is discarded, and the sample is fixed with formaldehyde and stained with 2% crystal violet solution for 30 minutes. After rinsing, the plaques are counted. The number of virus particles contained in the sample is then calculated based on the dilution.

[0071] Table 1 Bioreactor process flow chart

[0072]

[0073]

[0074] Table 2 Effect of different virus harvesting methods on virus yield (PFU / ml)

[0075] High pressure homogenizer crushing 2.0E+07 2.4E+07 Extract 4.4E+07 3.0E+07

Claims

1. A method for producing an oncolytic virus, comprising: 1) using a bioreactor for perfusion culture of Vero cells, wherein a sheet carrier is provided in the bioreactor, and the Vero cells are inoculated and perfused cultured on the sheet carrier; 2) infecting the Vero cells with an oncolytic virus and performing virus culture; 3) Harvesting oncolytic virus; Preferably, the oncolytic virus is herpes simplex virus (HSV).

2. The method of claim 1, wherein: The method also has one or more technical features selected from the following (i)-(iii): ( i) In step 1), the seeding density of the Vero cells is 4E+05cells / mL~8E+05cells / mL, such as 4E+05cells / mL~4.5E+05cells / mL, 4.5E+05cells / mL~5E+05cells / mL, 5E+05cells / mL~5.5E+05cells / mL, 5.5E+05cells / mL~6E+05cells / mL, 6E+05cells / mL~6.5E+05cells / mL, 6.5E+05cells / mL~7E+05cells / mL, 7E+05cells / mL~7.5E+05cells / mL or 7.5E+05cells / mL~8E+05cells / mL, such as 5E+05cells / mL; (ii) in step 2), when the culture density of the Vero cells is 1 to 2E+07 cells / mL, infecting the Vero cells with an oncolytic virus and performing virus culture; (iii) The density of the sheet-like carrier is 25-35 g / L, such as 25-27 g / L, 27-29 g / L, 29-31 g / L, 31-33 g / L or 33-35 g / L, such as 28 g / L.

3. The method according to any one of claims 1 to 2, wherein: The method also has one or more technical features selected from the following (i)-(v): ( i) in step 1), during the perfusion culture, the glucose concentration is controlled at 1.5-2.0 g / L, such as 1.5-1.6 g / L, 1.6-1.7 g / L, 1.7-1.8 g / L, 1.8-1.9 g / L or 1.9-2.0 g / L; (ii) in step 1), during the perfusion culture, the pH is 7.1 to 7.3, for example, 7.1 to 7.2 or 7.2 to 7.3; (iii) in step 1), during the perfusion culture, the temperature is 35-40°C, for example, 35-36°C, 36-37°C, 37-38°C, 38-39°C or 39-40°C, for example, 37°C; (iv) in step 1), during the perfusion culture, the dissolved oxygen is 45% to 50%, for example, 45% to 46%, 46% to 47%, 47% to 48%, 48% to 49% or 49% to 50%; (v) In step 1), during the perfusion culture, the stirring speed is 60-80 rpm, such as 60-65 rpm, 65-70 rpm, 70-75 rpm or 75-80 rpm.

4. The method according to any one of claims 1 to 3, wherein The method also has one or more technical features selected from the following (i)-(iv): ( i) in step 2), when the virus is cultured, the pH is 7.3 to 7.5, for example, 7.3 to 7.4 or 7.4 to 7.5; (ii) in step 2), the virus is cultured at a temperature of 32-37°C, such as 32-33°C, 33-34°C, 34-35°C, 35-36°C or 36-37°C, such as 34°C; (iii) in step 2), during the virus culture, the dissolved oxygen is 45% to 50%, for example, 45% to 46%, 46% to 47%, 47% to 48%, 48% to 49% or 49% to 50%; (iv) In step 2), during the virus culture, the stirring speed is 60-80 rpm, such as 60-65 rpm, 65-70 rpm, 70-75 rpm or 75-80 rpm.

5. The method according to any one of claims 1 to 4, wherein The method also has one or more technical features selected from the following (i)-(iii): ( i) in step 1), during the perfusion culture, the culture medium is a DMEM culture medium containing 10% serum; (ii) in step 2), the virus culture medium is serum-free DMEM medium; (iii) The volume of the culture medium during the perfusion culture in step 1) is the same as the volume of the culture medium during the virus culture in step 2).

6. The method according to any one of claims 1 to 5, wherein In step 2), when the Vero cells are infected with an oncolytic virus, the oncolytic virus is inoculated at an MOI of 0.001 to 0.01, for example, 0.001 to 0.002, 0.002 to 0.004, 0.004 to 0.005, 0.005 to 0.006, 0.006 to 0.008 or 0.008 to 0.

01.

7. The method according to any one of claims 1 to 6, wherein The oncolytic virus is herpes simplex virus type 1 (HSV-1); Preferably, the oncolytic virus is recombinant HSV-1; Preferably, the recombinant HSV-1 comprises at least one modification selected from the group consisting of: a. Delete ICP34.5; b. Insert a nucleic acid encoding at least one exogenous polypeptide selected from the following: IL12, IL15 / 15RA, PD-L1.

8. The method according to any one of claims 1 to 7, wherein In step 3), the oncolytic virus is harvested by culturing for 70 to 90 hours (e.g., 70 to 72 hours, 72 to 74 hours, 74 to 75 hours, 75 to 76 hours, 76 to 78 hours, 78 to 80 hours, 80 to 82 hours, 82 to 84 hours, 84 to 85 hours, 85 to 86 hours, 86 to 88 hours or 88 to 90 hours, e.g., 80 hours).

9. The method according to any one of claims 1 to 8, wherein The method also has one or more technical features selected from the following (i)-(ii): (i) the sheet carrier is a Disks sheet carrier; (ii) The bioreactor is a basket bioreactor, such as a 10 L basket bioreactor.

10. The method according to any one of claims 1 to 9, wherein The sheet carrier is pre-infiltrated and cleaned with a buffer solution; Preferably, the buffer is PBS buffer; Preferably, the pH of the PBS buffer is 7.2-7.6, such as 7.2-7.3, 7.3-7.4, 7.4-7.5 or 7.5-7.6, such as 7.

2.

11. The method of claim 1, wherein in step 3), the virus produced by Vero cells is harvested using an extract, the extract comprising sucrose, Tris hydrochloric acid and sodium chloride, wherein the proportion of sucrose is 1%-5%, for example, 1%-2%, 2%-3%, 3%-4%, 4%-5%, the proportion of Tris hydrochloric acid is 2%-8%, for example, 2%-3%, 3%-4%, 4%-5%, 5%-6%, 6%-7%, 7%-8%, and the proportion of sodium chloride is 10%-15%, for example, 10%-11%, 11%-12%, 12%-13%, 13%-14%, 14%-15%.

12. The method of claim 11, wherein the extract is incubated with the sheet-shaped carrier in step 1) for 30-60 minutes, such as 30-40 minutes, 40-50 minutes, or 50-60 minutes.

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