Search

CN-121991903-A - Method for producing Coxsackie virus by using HEK293 cells

CN121991903ACN 121991903 ACN121991903 ACN 121991903ACN-121991903-A

Abstract

The invention belongs to the technical field of biology, and relates to a method for producing CVB1 by culturing HEK293 cells in vitro, which comprises (1) culturing host cells in a culture medium I, wherein the culture medium I comprises a basal medium of L-alanyl-L-glutamine, (2) adding a culture medium II into a cell culture in the step (1) to continuously culture the host cells, (3) adding a culture medium III into the cell culture in the step (2) to inoculate viruses, and continuously culturing the host cells after virus inoculation, and (4) obtaining a cell culture containing target viruses. The invention also relates to media for producing CVB1 viruses and their use for producing CVB1 viruses.

Inventors

  • Ji Chengfa
  • ZHANG LEI
  • XIONG FEI
  • Fu Wengun
  • YANG LISHENG
  • SHI WEI

Assignees

  • 杭州养生堂生物医药有限公司

Dates

Publication Date
20260508
Application Date
20260213

Claims (11)

  1. 1. A method of culturing cells in vitro to produce a virus of interest comprising: (1) Culturing a host cell in medium I, wherein the medium I is a basal medium comprising L-alanyl-L-glutamine; (2) Adding medium II to the cell culture of step (1), continuing to culture the host cell, wherein the medium II comprises the following components: Arginine, cysteine, histidine, isoleucine, leucine, lysine, valine, tyrosine, methionine, phenylalanine, threonine, tryptophan, choline chloride, folic acid, methionine, inositol, nicotinamide, calcium D-pantothenate, pyridoxal, vitamin B2, vitamin B1, thiamine, vitamin B12, L-alanyl-L-glutamine, cell BoostTM, sodium pyruvate, sodium dichloroacetate, naCl, KCl, KH 2 PO 4 , and Na 2 HPO 4 ; (3) Adding a medium III to the cell culture of the step (2), inoculating the virus, and continuously culturing the host cells after the virus inoculation, wherein the medium III comprises the following components: Arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, tyrosine, choline chloride, folic acid, inositol, nicotinamide, calcium D-pantothenate, pyridoxal, vitamin B2, thiamine, vitamin B12, L-alanyl-L-glutamine, cell BoostTM, sodium pyruvate, sodium dichloroacetate, naCl, KCl, KH 2 PO 4 , and Na 2 HPO 4 ; (4) Obtaining a cell culture comprising the virus of interest.
  2. 2. The method of claim 1, wherein step (1) comprises: Culturing host cells in medium I until the cell density of the host cells reaches (1-2). Times.10 6 cells/ml; preferably, the concentration of the L-alanyl-L-glutamine in the culture medium I is 1.8-2.2 mM; Preferably, the basal medium is selected from the group consisting of HEK293 CD medium, optipro x 293CD medium, x 2 x S001 medium, and any combination thereof; Preferably, the medium I is HEK293 CD medium comprising L-alanyl-L-glutamine at a concentration of 1.8-2.2 mM (e.g., 2 mM) in the medium I; Preferably, the cultivation is carried out at a cultivation temperature of 37.0.+ -. 1.0 ℃ and a pH of the medium I of 6.8-7.2; Preferably, the dissolved oxygen content of the culture medium I is 30-70% of air saturation.
  3. 3. The method of claim 1 or 2, wherein step (2) comprises: Adding medium II to the cell culture of step (1), continuing to culture the host cells until the cell density of the host cells reaches (2-4) ×10 6 cells/ml; Preferably, in the medium II, arginine is at a concentration of 2-4. g/L, cysteine is at a concentration of 0.5-1 g/L, histidine is at a concentration of 0.75-1.5 g/L, isoleucine is at a concentration of 1-2g/L, leucine is at a concentration of 1-2g/L, lysine is at a concentration of 1-2.5 g/L, valine is at a concentration of 1-2g/L, tyrosine is at a concentration of 0.7-1.2 g/L, methionine is at a concentration of 0.25-0.5 g/L, phenylalanine is at a concentration of 0.6-1 g/L, threonine is at a concentration of 0.9-1.5 g/L, tryptophan is at a concentration of 0.2-0.3 g/L, choline chloride is at a concentration of 15-30 mg/L, inositol is at a concentration of 25-75 mg/L, nicotinamide is at a concentration of 15-30D-35/L, vitamin B is at a concentration of 15-30D-35 is at a concentration of mg-3.3932 g/L, sodium acetate is at a concentration of mg-3.3932-3.5.3932 g/L, tryptophan is at a concentration of mg-35.5-3.5.35/L, and vitamin B is at a concentration of mg-3-3.35.5.3 g/L, vitamin is at a concentration of mg-3-3.5.35.5.35 g/L, the concentration of Na 2 HPO 4 is 250-350 mg/L; preferably, the volume ratio of medium II to the cell culture of step (1) is from 1:80 to 1:200; Preferably, the cultivation is performed at a cultivation temperature of 37.0.+ -. 1.0 ℃.
  4. 4. The method according to claim 3, wherein step (2) further comprises, after the cell density of the host cells reaches (2-4). Times.10 6 cells/ml, performing perfusion culture using medium I for 4 days at a perfusion rate of 0.4-0.6V/day, 0.65-0.85V/day, 1.15-1.35V/day, 1.9-2.1V/day, respectively, wherein V represents the total volume of the cell culture; preferably, the perfusion culture is performed at a culture temperature of 37.0 ℃ plus or minus 1.0 ℃.
  5. 5. The method of any one of claims 1-4, wherein step (3) comprises: Adding a culture medium III into the cell culture in the step (2), inoculating viruses according to the MOI of 0.01-0.05, and continuously culturing host cells after virus inoculation; Preferably, in the medium III, arginine is 2-4 g/L, cysteine is 0.5-1 g/L, histidine is 0.75-1.5 g/L, isoleucine is 1-2 g/L, leucine is 1-2 g/L, lysine is 1-2.5 g/L, methionine is 0.25-0.5 g/L, phenylalanine is 0.6-1 g/L, threonine is 0.9-1.5 g/L, tryptophan is 0.2-0.3 g/L, inositol is 25-75 mg/L, nicotinamide is 15-30 mg/L, valine is 1-2 g/L, tyrosine is 0.7-1.2 g/L, thiamine is 15-30 mg/L, and L-alanyl-L-250 mM,Cell BoostTM 5 is 15-35/L; preferably, the volume ratio of medium III to the cell culture of step (2) is from 1:80 to 1:200; Preferably, the inoculation temperature is 35.0 ± 1.0 ℃.
  6. 6. The method according to any one of claims 1 to 5, wherein in the step (3), the culturing temperature of the culture is 35.0.+ -. 1.0 ℃ and the culturing time is 24 to 48 hours.
  7. 7. The method of any one of claims 1-6, wherein the host cell is a mammalian cell; Preferably, the host cell is a HEK293 cell.
  8. 8. The method of any one of claims 1-7, wherein the virus is Coxsackievirus (Coxsackievirus); preferably, the virus is Coxsackievirus B1 (Coxsackievirus B1, CVB 1).
  9. 9. A culture medium which is medium II as defined in claim 1 or 3 or medium III as defined in claim 1 or 5.
  10. 10. A kit comprising any two or all three of medium I, medium II, medium III, wherein medium I is as defined in claim 1 or 2, medium II is as defined in claim 1 or 3, and medium III is as defined in claim 1 or 4.
  11. 11. Use of the medium of claim 8 or the kit of claim 9 for the preparation of a virus in a host cell; Preferably, the host cell is a mammalian cell; preferably, the host cell is a HEK293 cell; Preferably, the virus is a Coxsackievirus (Coxsackievirus); preferably, the virus is Coxsackievirus B1 (Coxsackievirus B1, CVB 1).

Description

Method for producing Coxsackie virus by using HEK293 cells Technical Field The invention belongs to the technical field of biology, and relates to a method for producing CVB1 by culturing HEK293 cells in vitro. The invention also relates to media for producing CVB1 viruses and their use for producing CVB1 viruses. Background Coxsackieviruses (CVs) can be divided into A, B groups according to genotype, group a contains 23 serotypes and group B has 6 serotypes. Coxsackievirus B1 (CVB 1) is a common human pathogen, belonging to enteroviruses, and is a non-enveloped single-stranded positive-strand RNA virus. The susceptible population is mainly infants, the adult infection rate is low, and the main clinical symptoms are common cold symptoms such as fever, headache, diarrhea and the like, and severe diseases such as occasional aseptic meningitis, myocarditis, hepatitis, pancreatitis, hand-foot-mouth disease and the like. Because of the lack of specific drugs for treating enteroviruses including CVB1, developing CVB1 vaccines is an important measure for preventing neonatal infection and reducing morbidity. It has been proved that natural CVB1 can kill various tumor cell lines, and because CVB1 has unique advantages of small genome, penetration of blood brain barrier, slight symptoms after adult infection, etc., the recombinant CVB1 obtained after artificial attenuation transformation can be developed into oncolytic virus medicines. If CVB1 is developed into vaccine or therapeutic oncolytic virus drugs, large-scale virus preparation methods are required to be developed. For the production process of the virus medicaments, not only the control strategy of each process parameter in the cell culture stage and the virus culture stage is required to be considered, but also the selection of the culture mode is required to be considered, so that the process is stable and has good repeatability, the virus yield is finally improved to the maximum extent, and the requirement of commercial amplified production is met. At present, most of the enterovirus-producing processes are based on the preparation of adherent cells with a serum-containing medium, and suspension cell culture and a virus-producing process are rarely adopted, namely, [1] such as Chen Guojun and the like is adopted to carry out the adherent culture of Vero cells with a serum-containing RPMI-1640 medium to prepare laboratory-grade CVB1 viruses, [2] such as Yang Ting and the like is adopted to carry out the adherent culture of KMB17 cells in a cell factory by using the serum-containing medium to produce CVA16, and CN112641798A reports that DMEM containing 10% FBS is firstly adopted to carry out the adherent culture of Vero cells in a culture bottle, then the culture medium is replaced by serum-free DMEM after the confluency rate is 90%, and then CVB3 viruses are inoculated to carry out the virus production and the like. Although the adherence process can better support cell growth, the subsequent amplification is limited and requires higher labor cost, the culture of cells by using microcarriers or sheet carriers is reported to produce enteroviruses, however, the transfer success rate of cells among carriers becomes a key factor influencing the process stability in the amplification process. Therefore, the process of attaching wall and microcarrier is gradually developed to the suspension process which can be more convenient for enlarging. In addition, in general, the adherence process or microcarrier culture process requires addition of new born or fetal bovine serum to the cell culture medium to promote cell growth and toxigenesis. However, the introduction of serum increases the cost of HCP, exogenous factors, mycoplasma or non-product virus removal during subsequent virus purification, additionally increasing the risk of quality control, and thus more and more processes begin to shift towards serum-free media or well-defined chemically defined media. At present, a plurality of domestic and foreign companies successively develop commercial serum-free culture mediums for various cells, can better meet the culture requirement of partial cell lines (strains) for production, and is used for antibody production or virus preparation. Based on the concept of mass source design (Quality by design, qbD), the chemically defined culture medium with definite components is used in the cell suspension culture and virus production process, so that the product quality can be improved from the source, the stability among batches is enhanced, the exogenous pollution is reduced, the purification pressure is lightened, and the commercial scale is enlarged. Because CVB1 has the characteristic of cracking cells during production, the continuous culture mode is not suitable, and only a basic batch culture or fed-batch culture mode can be selected, but in order to improve the yield of single-batch viruses, the cells are usually subjected to virus inoculation and virus production a