A composition and its application in inhibiting retrovirus inactivation

Abstract

The present invention belongs to the technical field of indicator viruses, and specifically relates to a composition and its application in inhibiting retroviral inactivation. The composition provided by the present invention includes 15 mM glycine betaine and 50 mM fructose. Fructose can increase the half-life and cell specific productivity of retroviruses, and glycine betaine can neutralize the negative effects of high osmotic pressure environment on cell growth. The present invention compounds glycine betaine and fructose in a certain ratio, which can improve the stability of retroviruses, inhibit retroviral inactivation, reduce the inactivation rate of retroviruses, increase cell specific productivity, increase the half-life of the virus, harvest a retroviral stock solution with high stability and long half-life, reduce time and cost, and thus improve the safety and effectiveness of retroviral production.

Description

Technical Field

[0001] The invention belongs to the technical field of indicator viruses, and in particular relates to a composition and an application thereof in inhibiting the inactivation of retroviruses. Background Art

[0002] In order to ensure the safety of viruses in biotechnology products derived from human or animal cell lines, it is necessary to verify the removal or inactivation of viruses in biotechnology products. In the virus removal or inactivation step, the indicator virus (Virus Spike) is added to the sample for inactivation, and then the change value of the infectious virus is detected by relevant indicator cells, or other detection methods are used to detect the residual amount of virus in the sample to estimate the effectiveness of the process. Many biotechnology therapeutic products, such as monoclonal antibodies, recombinant proteins, vaccines, and gene therapy viral vectors, are produced in Chinese hamster ovary (CHO) cells. CHO cells contain endogenous retroviruses that are defective or non-infectious. Therefore, experimental verification of retrovirus removal or inactivation is required during the production of these biotechnology therapeutic products.

[0003] Xenotropic murine leukemia virus (X-MuLv) is an important indicator virus and is widely used in virus clearance or inactivation validation of CHO-produced biotech therapeutic products. In the virus clearance process, in order to avoid the addition of viruses causing dilution effects or changing the properties of the product, the indicator virus titer is required to be as high as possible (>8.0 log 10 ). However, the X-MuLv virus has a low titer and is unstable between batches, so its production and purification are particularly important. However, its production and efficiency are limited by its inherent instability (low half-life), which can have an adverse effect on virus production, resulting in a low virus titer. Currently, the industry is working hard to inactivate X-MuLv and increase the titer of X-MuLv virus, but there are few related literature reports on the results. Therefore, how to inhibit the inactivation of X-MuLv virus, reduce the inactivation rate of X-MuLv virus, improve the stability of X-MuLv virus, reduce time and cost, and thus improve the effectiveness of virus production is particularly important in this field. Summary of the invention

[0004] The purpose of the present invention is to provide a composition and its application in inhibiting retrovirus inactivation, improving retrovirus stability, inhibiting retrovirus inactivation, improving cell specific productivity, harvesting retrovirus stock solution with high stability and long half-life, reducing time and cost, and improving the effectiveness of retrovirus production.

[0005] The present invention provides a composition comprising glycine betaine and fructose;

[0006] The concentration of glycine betaine in the composition is 15 mM;

[0007] The concentration of fructose in the composition is 50 mM.

[0008] Preferably, the solvent of the composition is a culture medium for retrovirus culture.

[0009] The present invention also provides the use of the composition described in the above technical solution in the preparation of retrovirus.

[0010] The present invention also provides the use of the composition described in the above technical solution in improving virus stability and / or inhibiting virus inactivation; the virus is a retrovirus.

[0011] Preferably, said improving virus stability comprises improving virus half-life;

[0012] The inhibition of virus inactivation includes reducing the virus inactivation rate and / or increasing the cell specific productivity.

[0013] Preferably, the retrovirus comprises xenotropic murine leukemia virus.

[0014] The present invention also provides a method for improving the stability of retrovirus and / or inhibiting the inactivation of retrovirus, comprising the following steps: culturing cells producing retrovirus using a cell culture medium containing glycine betaine and fructose, collecting the supernatant, and obtaining a retrovirus stock solution;

[0015] The concentration of glycine betaine in the cell culture medium is 15 mM, and the concentration of fructose is 50 mM.

[0016] Preferably, the ratio of the concentration of glycine betaine to the number of cells of the retrovirus-producing cells is 15 mM: 1×10 5 ~3×10 5 indivual.

[0017] Preferably, the retrovirus comprises xenotropic murine leukemia virus.

[0018] Preferably, the cells producing the heterotropic murine leukemia virus include mouse skin fibroblasts and / or astrocyte PG-4 passage cells.

[0019] Beneficial effects:

[0020] The present invention provides a composition, comprising glycine betaine and fructose; the concentration of glycine betaine in the composition is 15 mM; the concentration of fructose in the composition is 50 mM. Fructose can increase the half-life and cell specific productivity of retroviruses, but due to the addition of fructose, the osmotic pressure in the cell culture process of producing retroviruses will increase, which will have a negative effect on cell growth, leading to cell density, thereby reducing the virus titer and accelerating virus inactivation; while glycine betaine can neutralize the negative effect of high osmotic pressure environment on cell growth. The present invention compounds glycine betaine and fructose in a certain ratio, which can improve the stability of retroviruses, inhibit the inactivation of retroviruses, reduce the inactivation rate of retroviruses, improve the cell specific productivity, and improve the half-life of the virus. Thus, a retrovirus stock solution with high stability and long half-life can be harvested, time and cost can be reduced, and the safety and effectiveness of retrovirus production can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0022] Figure 1 This is a graph showing the comparison of cell density under culture conditions of different treatment groups in Example 1;

[0023] Figure 2 This is a graph showing the titer change of the virus solution in Example 3 under different treatment conditions; where ** p <0.01,## p <0.01;

[0024] Figure 3 This is a statistical result diagram of the inactivation rate of the virus solution in Example 3 under different treatment conditions; among which, ** p <0.01, # p <0.05. DETAILED DESCRIPTION

[0025] The present invention provides a composition, comprising glycine betaine and fructose; the concentration of glycine betaine in the composition is 15 mM; the concentration of fructose in the composition is 50 mM.

[0026] As an embodiment, the solvent of the composition of the present invention is a medium for retrovirus culture. The present invention has no strict requirements on the specific type of the medium for retrovirus culture, and it can be selected according to the type of retrovirus. For example, as an embodiment, when the retrovirus is a heterotropic mouse leukemia virus, the medium for retrovirus culture can be selected as McCoy's 5a complete medium; the McCoy's 5a complete medium includes a basal medium and 10% FBS, and the basal medium is McCoy's 5A (MC-5A) medium purchased from Beijing Yi Biological Shenzhou Technology Co., Ltd. The McCoy's 5A (MC-5A) medium of the present invention does not contain serum.

[0027] The composition of the present invention includes fructose; the concentration of fructose in the composition is 50 mM. The present invention utilizes fructose to increase the half-life of retrovirus and the specific productivity of cells. The present invention has no strict requirements on the source of the fructose, which can be purchased conventionally or prepared by itself. The fructose used in the embodiment of the present invention was purchased from Biyuntian, and the product number is ST2317.

[0028] The composition of the present invention includes glycine betaine; the concentration of glycine betaine in the composition is 15mM. The present invention utilizes glycine betaine to compensate for the adverse effects of fructose on cells during use and neutralize the negative effects of high osmotic pressure environment on cell growth. The present invention has no strict requirements on the source of the glycine betaine, which can be purchased conventionally or prepared by itself. The glycine betaine used in the embodiment of the present invention was purchased from Sigma, and the product number is 61962.

[0029] The present invention combines glycine betaine and fructose and strictly limits the compounding ratio, which can improve the stability of retrovirus to the greatest extent, inhibit the inactivation of retrovirus, reduce the inactivation rate of retrovirus, improve the cell specific productivity, increase the half-life of the virus, harvest the retrovirus stock solution with high stability and long half-life, reduce time and cost, and thus improve the safety and effectiveness of retrovirus production.

[0030] In view of this, the use of the composition described in the above technical scheme in the preparation of retroviruses also belongs to the protection scope of the present invention. The use of the composition described in the above technical scheme in improving virus stability and / or inhibiting virus inactivation also belongs to the protection scope of the present invention, and the virus is a retrovirus. As an embodiment, the improvement of virus stability includes increasing the half-life of the virus. As an embodiment, the inhibition of virus inactivation includes reducing the virus inactivation rate and / or increasing the cell specific productivity; as another embodiment, the inhibition of virus inactivation includes reducing the virus inactivation rate and increasing the cell specific productivity. As an embodiment, the retrovirus includes heterotropic mouse leukemia virus.

[0031] The present invention also provides a method for improving the stability of retrovirus and / or inhibiting the inactivation of retrovirus, comprising the following steps: culturing cells producing retrovirus using a cell culture medium containing glycine betaine and fructose, collecting the supernatant, and obtaining a retrovirus stock solution; the concentration of glycine betaine in the cell culture medium is 15 mM, and the concentration of fructose is 50 mM.

[0032] As an embodiment, the culture temperature is 37° C. As an embodiment, the ratio of the concentration of glycine betaine to the number of cells of the retrovirus-producing cells is 15 mM: 1×10 5 ~3×10 5 As another embodiment, the ratio of the concentration of glycine betaine to the number of cells of the retrovirus-producing cells is 15 mM: 2×10 5 The present invention utilizes a cell culture medium containing glycine betaine and fructose to culture cells producing retroviruses, strictly limits the ratio of glycine betaine to fructose, does not inhibit the later growth of cells, and significantly increases the cell specific productivity and the virus titer compared to using fructose or glycine betaine alone.

[0033] As an embodiment, the retrovirus includes heterotropic murine leukemia virus. The type of basal culture medium for culturing the cells producing the retrovirus in the present invention is not strictly required, and can be conventionally selected according to the type of cells. As an embodiment, the cells producing the heterotropic murine leukemia virus include mouse skin fibroblasts and / or astrocyte PG-4 passage cells; as another embodiment, the cells producing the heterotropic murine leukemia virus are mouse skin fibroblasts. As an embodiment, the culture medium for culturing the mouse skin fibroblasts and astrocyte PG-4 passage cells can be McCoy's 5a complete medium respectively; the McCoy's 5a complete medium includes a basal culture medium and 10% FBS, and the basal culture medium is McCoy's 5A (MC-5A) medium, which is purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. The McCoy's 5A (MC-5A) medium described in the present invention does not contain serum.

[0034] In order to further illustrate the present invention, a composition provided by the present invention and its application in inhibiting retrovirus inactivation are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Preparatory Example

[0036] Prepare 3.5M fructose mother solution: weigh 126 g fructose, add a final volume of 200 mL sterile pure water, wait for complete dissolution, filter and sterilize using a 0.2 μm syringe filter, and store at 2-8°C for later use.

[0037] Prepare 1 M glycine betaine stock solution: weigh 1.1715 g of glycine betaine, add a final volume of 10 mL of sterile pure water, and after it is completely dissolved, filter and sterilize using a syringe filter with a pore size of 0.2 μm, and store at 2~8°C for later use.

[0038] Example 1

[0039] Mouse skin fibroblasts (M. dunni) producing xenotropic murine leukemia virus (X-MuLv) were cultured at 2 × 10 5 The cells were inoculated into 6-well plates at a density of 100 cells / well and randomly divided into 8 treatment groups for the following treatments:

[0040] Treatment group 1 (control group): cells were cultured in a medium with an osmotic pressure of 313 mOsm / L; the medium was MC-5A medium supplemented with 10% FBS, i.e., McCoy's 5a complete medium;

[0041] Treatment group 2 (15 mM glycine betaine): cells were cultured in a medium containing 15 mM glycine betaine at an osmotic pressure of 333 mOsm / L; the medium was MC-5A medium supplemented with 10% FBS;

[0042] Treatment group 3 (50 mM fructose): cells were cultured in a medium with an osmotic pressure of 352 mOsm / L and 50 mM fructose; the medium was MC-5A medium supplemented with 10% FBS;

[0043] Treatment group 4 (50 mM fructose + 15 mM glycine betaine): cells were cultured in a medium with an osmotic pressure of 369 mOsm / L, containing 50 mM fructose and 15 mM glycine betaine; the medium was MC-5A medium supplemented with 10% FBS;

[0044] Treatment group 5 (75 mM fructose): cells were cultured in a medium with an osmotic pressure of 374 mOsm / L and 75 mM fructose; the medium was MC-5A medium supplemented with 10% FBS;

[0045] Treatment group 6 (75 mM fructose + 15 mM glycine betaine): cells were cultured in a medium with an osmotic pressure of 394 mOsm / L, containing 75 mM fructose and 15 mM glycine betaine; the medium was MC-5A medium supplemented with 10% FBS;

[0046] Treatment group 7 (140 mM fructose): cells were cultured in a medium with an osmotic pressure of 447 mOsm / L and 140 mM fructose; the medium was MC-5A medium supplemented with 10% FBS;

[0047] Treatment Group 8 (140 mM fructose + 15 mM glycine betaine): cells were cultured in a medium with an osmotic pressure of 466 mOsm / L, containing 140 mM fructose and 15 mM glycine betaine; the medium was MC-5A medium supplemented with 10% FBS;

[0048] (2) Step (1) Each treatment group was cultured at 37°C. Virus supernatant was harvested on days 0, 3, 5, 6, 7, 8 and 9 after inoculation of cells, and cells were digested for cell counting. The results are shown in Tables 1 and Figure 1 shown.

[0049] Table 1 Cell density of different treatment groups (10 5 / mL)

[0050]

[0051] Note: ns means no significant difference, * p <0.05,** p <0.01,*** p <0.001.

[0052] According to Table 1 and Figure 1 It can be seen that the addition of 50 mM, 75 mM, and 140 mM fructose increased the osmotic pressure of the culture medium. In the later stages of cell culture and virus amplification, cell growth was inhibited, but the cell density of cells cultured in the medium with 50 mM fructose and 15 mM glycine betaine added at the same time was not significantly different from that of the control group. Therefore, it was determined that 50 mM fructose + 15 mM glycine betaine in the virus amplification medium was the optimal culture condition.

[0053] Example 2

[0054] The PG-4 passage cells (PG-4 cells) producing heterotropic murine leukemia virus (X-MuLv) were seeded into 96-well plates at a density of 2000 cells / well. On the second day, the virus stock solutions harvested from the control group, 50 mM fructose group, and 50 mM fructose + 15 mM glycine betaine group in Example 1 were cultured for 10 min in MC-5A medium containing 10% FBS. -1 to 10 -6 The diluted virus solution was added to a 96-well plate at a rate of 100 μL / well, with 8 replicate wells per dilution gradient in each treatment group.

[0055] After one week of cell culture in each treatment group, the cytopathic effect (CPE) was determined, and the TCID was calculated according to the Karber method (Karber G. Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Naunyn Schmiedeb Arch Exp Pathol Pharmakol 1931;162(4):480e3). 50 The virus titer was determined by calculating the cell specific productivity (TCID of virus produced per cell per day). 50 Each treatment group was tested twice, and the results are shown in Table 2.

[0056] Table 2 X-MuLv virus titers and cell specific productivity amplified in different treatment groups

[0057]

[0058] As can be seen from Table 2, the titer of X-MuLv virus amplified in the medium containing 50 mM fructose and 15 mM glycine betaine was higher than that in the control group and the 50 mM fructose group, and the cell specific productivity was significantly increased.

[0059] Example 3

[0060] (1) The X-MuLv stock solutions harvested after 6 days of culture in Group 1 (control group), Group 3 (50 mM fructose group) and Group 4 (50 mM fructose + 15 mM glycine betaine group) in Example 1 were used as research objects. The cells were incubated at 37°C for 0 h, 24 h and 48 h, respectively. The virus titer was detected according to the method of Example 2. Each treatment group was tested twice. The results are shown in Tables 3 and 4. Figure 2 shown.

[0061] Table 3 Titer values ​​of virus solution under different treatment conditions (log 10 TCID 50 / mL)

[0062]

[0063] According to Table 3 and Figure 2 It can be seen that after incubation at 37°C for 48 h, the virus titer of X-MuLv amplified in the medium containing 50 mM fructose and 15 mM glycine betaine decreased the slowest, indicating that the virus stability of X-MuLv amplified using the virus amplification medium containing 50 mM fructose and 15 mM glycine betaine was significantly enhanced.

[0064] (2) Based on the conclusion of step (1), the virus inactivation rate (%) was calculated according to the following formula. Each treatment group was tested twice. The results are shown in Tables 4 and Figure 3 shown.

[0065] Virus inactivation rate (%) = (initial virus titer - virus titer after treatment) / initial virus titer × 100%;

[0066] Table 4 Inactivation rate of virus solution under different treatment conditions (%)

[0067]

[0068] According to Table 4 and Figure 3 It can be seen that after incubation at 37°C for 48 h, the inactivation rate of X-MuLv amplified in the medium containing 50 mM fructose and 15 mM glycine betaine was the lowest, indicating that the inactivation rate of X-MuLv virus can be significantly reduced by amplifying the virus using a virus amplification medium containing 50 mM fructose and 15 mM glycine betaine.

[0069] (3) With reference to the prior art (Le Doux JM, Davis HE, Morgan JR, Yarmush ML. Kinetics of retrovirus production and decay. Biotechnol Bioeng. 1999 Jun 20;63(6):654-62. PMID: 10397822), the half-life of the virus under different treatment conditions was statistically analyzed. Each treatment group was tested twice. The results are shown in Table 5.

[0070] Table 5 Half-life of virus solution under different treatment conditions (h)

[0071]

[0072] Note: ns means no significant difference, * p <0.05.

[0073] According to Table 5, the virus half-life was higher in the 50 mM fructose and 15 mM glycine betaine groups.

[0074] Based on the above content, it can be seen that using MC-5A medium containing 50 mM fructose + 15 mM glycine betaine to culture cells and amplify X-MuLv virus can enable fructose to play its role in stabilizing the half-life of the retrovirus under the condition that glycine betaine maintains the osmotic pressure of the cell culture medium.

[0075] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a composition for improving virus stability and / or inhibiting virus inactivation; the virus is a retrovirus, characterized in that: The composition includes glycine betaine and fructose; The concentration of glycine betaine in the composition is 15 mM; The concentration of fructose in the composition is 50 mM; Improving virus stability means increasing virus half-life; The inhibition of virus inactivation is to reduce the virus inactivation rate and / or increase the cell specific productivity; The retrovirus is xenotropic murine leukemia virus; The cells for producing the heterotropic mouse leukemia virus are mouse skin fibroblasts and / or astrocyte PG-4 passage cells.

2. The use according to claim 1, characterized in that: The solvent of the composition is a culture medium for retrovirus culture.

3. A method for improving retrovirus stability and / or inhibiting retrovirus inactivation, characterized in that: The steps include: Using a cell culture medium containing glycine betaine and fructose to culture cells producing retrovirus, collecting the supernatant to obtain a retrovirus stock solution; The concentration of glycine betaine in the cell culture medium is 15 mM, and the concentration of fructose is 50 mM; Improving virus stability means increasing virus half-life; The inhibition of virus inactivation is to reduce the virus inactivation rate and / or increase the cell specific productivity; The retrovirus is xenotropic murine leukemia virus; The cells for producing the heterotropic mouse leukemia virus are mouse skin fibroblasts and / or astrocyte PG-4 passage cells.

4. The method according to claim 3, characterized in that The ratio of the concentration of glycine betaine to the number of cells of the retrovirus-producing cells is 15 mM: 1×10 5 ~3×10 5 indivual.

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