阅读说明：本技术 一种高效表达重组猫干扰素ω2突变体的哺乳动物细胞培养工艺 (Mammalian cell culture process for efficiently expressing recombinant cat interferon omega 2 mutant ) 是由 代燕平 王雯茜 高小平 罗弟祥 于 2020-03-31 设计创作，主要内容包括：本发明提供了一种高效表达重组猫干扰素-ω2(rFeIFN-ω2)突变体的哺乳动物细胞悬浮培养工艺,该培养工艺在生物反应器中进行,包括：(1)培养初始温度设定为36.5℃,溶氧40％、pH7±0.3,于培养第6天降低温度至33℃；(2)于培养第3、6、8、10、12天补充5％、5％、5％、5％、3％无血清补料培养基1,于培养第4、7、9、11天分别流加2％的无血清补料培养基2；于培养第5、8天分别流加6g/L的水解物,维持溶氧40％、pH7±0.3,于第18天结束培养。本发明培养工艺流程简单,细胞在高密度生长条件下保持活率时间长,目标蛋白表达可达2.5g/L以上,且抗病毒活性佳,生产成本低,适合大规模产业化应用。(The invention provides a mammalian cell suspension culture process for efficiently expressing recombinant cat interferon-omega 2 (rFeIFN-omega 2) mutants, which is carried out in a bioreactor and comprises the following steps: (1) setting the initial culture temperature to 36.5 ℃, dissolving oxygen at 40%, and reducing the temperature to 33 ℃ on the 6 th day of culture, wherein the pH is 7 +/-0.3; (2) supplementing 5%, 3% of serum-free supplemented medium 1 on days 3, 6, 8, 10, and 12, and feeding 2% of serum-free supplemented medium 2 on days 4, 7, 9, and 11; 6g/L of the hydrolysate was fed on days 5 and 8, respectively, dissolved oxygen was maintained at 40%, pH 7. + -. 0.3, and the culture was terminated on day 18. The invention has simple culture process flow, long survival rate of cells under high-density growth condition, high antiviral activity and low production cost, and the target protein expression can reach more than 2.5g/L, thereby being suitable for large-scale industrialized application.)

1. A mammalian cell culture process for efficiently expressing a recombinant feline interferon-omega 2 (rFeIFN-omega 2) mutant is characterized by comprising the following specific steps:

(1) inoculating the conventionally prepared seed cells into a bioreactor containing a serum-free basal medium for suspension culture;

(2) the cell seeding density is 5-8 × 10⁵cellsPer mL, preferably 5X 10⁵cells/mL；

(3) The working volume of the basic culture medium in the bioreactor is 1-500L, the rotating speed is set to be 10-200 r/min, and specifically,

the working volume of the medium in a 7 liter bioreactor is 1-5L, preferably 3L; setting the rotation speed of 100 and 200 revolutions per minute, preferably 150 revolutions per minute;

the working volume of the medium in a 50 liter bioreactor is 10-40L, preferably 30L; setting the rotating speed to be 80-120 revolutions per minute, preferably 90 revolutions per minute;

the working volume of the medium in the 250 liter bioreactor is 40-200L, preferably 150L; setting the rotating speed to be 10-80 revolutions per minute, preferably 50 revolutions per minute;

(4) setting the initial culture cell temperature at 36-38 deg.c, preferably 36.5 deg.c;

(5) setting the dissolved oxygen condition at 30-80%, preferably 40%;

(6) controlling the pH value to be 5-7.5, preferably 7 +/-0.3;

(7) supplementing 3-10% of serum-free supplemented medium 1, preferably 5% on the 3 rd day of culture, and simultaneously supplementing 1-5 mM of glutamine, preferably 2 mM; supplementing glucose solution in the culture process to ensure that the content of glucose in the culture solution is not lower than 2g/L, and controlling the cell viability to be more than 70% in the culture process;

(8) supplementing 1-5% of serum-free supplemented medium 2, preferably 2% on the 4 th day of culture;

(9) supplementing 1-10 g/L of hydrolysate on the 5 th day of culture, preferably 6 g/L;

(10) supplementing 5% of serum-free supplemented medium 1 again on the 6 th day of culture, and adjusting the temperature to 33 ℃ for continuous culture;

(11) supplementing 2% of serum-free supplemented medium 2 again at the 7 th day of culture, and maintaining the temperature at 33 ℃ for continuous culture;

(12) supplementing 5% serum-free supplemented medium 1 and 6g/L hydrolysate again at 8 days of culture, and maintaining the temperature at 33 deg.C for further culture;

(13) supplementing 2% of serum-free supplemented medium 2 again at the 9 th day of culture, and maintaining the temperature at 33 ℃ for continuous culture;

(14) supplementing 5% of serum-free supplemented medium 1 again at 10 days of culture, and maintaining the temperature at 33 ℃ for continuous culture;

(15) supplementing 2% of serum-free supplemented medium 2 again on the 11 th day of culture, and continuing the culture at the temperature of 33 ℃;

(16) the culture was continued at 33 ℃ by supplementing the culture with 3% of serum-free feed medium 1 again on day 12.

2. The process of claim 1, wherein the mammalian cell is a Chinese hamster ovary cell, preferably a CHO DG44 cell.

3. The cell culture process according to claim 1, wherein the serum-free basal medium consists of one or more of ActiPro medium with concentration of 10-30g/L and SFM4CHO medium with concentration of 10-20 g/L; the additive is composed of one or more of F68 with concentration of 0.5-2g/L, glucose with concentration of 3-5g/L, and glutamine with concentration of 4-8mM, and has pH value of 7-7.5.

4. The Cell culture process according to claim 1, wherein the serum-free feed medium 1 comprises one or more of Cell Boost 7a at a concentration of 100-200 g/L and Cell Boost 7b at a concentration of 50-100 g/L; the serum-free supplemented medium 2 consists of EX-CELL Advanced CHO Feed 1 with the concentration of 50-100 g/L.

5. The cell culture process according to claim 1, wherein the hydrolysate is composed of one or more of Hypep 7504 with a concentration of 50-200 g/L and PF Plus ACF hydrolysate with a concentration of 50-200 g/L.

Technical Field

The invention relates to a mammalian cell culture process for efficiently expressing an rFeIFN-omega 2 mutant, belonging to the technical field of biological pharmacy.

Background

In 1985, interferon omega was first cloned from sendai virus-induced Namalwa cells and has now been identified as present in cats, pigs, horses, rabbits, bats, cattle and sheep. In 1992, the feline interferon gene was isolated by Nakamura, and then rFeiIFN-. omega.drugs were successfully developed and became the only approved formulation of feline interferon Omega, called Virbagen Omega, which has been widely used in Japan and European and American countries. The traditional Chinese medicine composition is mainly used for treating feline immunodeficiency virus, feline leukemia virus infectious diseases and canine parvovirus infectious diseases, and simultaneously has good treatment effect on infectious diseases such as feline viral rhinotracheitis, feline calicivirus, canine distemper virus, infectious hepatitis virus, rabies virus, pseudorabies virus, infectious gastroenteritis virus, canine coronavirus, canine herpes virus and the like.

In recent years, with the intensive research on Feline INTERFERON-omega, Chinese scholars Liuwen et al isolated 13 Novel Feline INTERFERONs-omega from cats in 2005, wherein-omega 2 obtained through a yeast expression system showed the strongest antiviral activity (Liuwen et al: Feline omega INTERFERON and its coding gene and application 2005, grant publication No. CN 1730491A) (Yang et al: Cloning and Characterization OF a Novel Feline IFN-omega. JOURNAL OF INTERFERON & CYTOKINE RESEARCH.2007; 27: 119-. In 2015, Huxiaoyuan and the like perform amino acid site-directed mutagenesis on cat omega 2 interferon by comparing 13 subtype gene sequences and amino acid sequences of cat omega interferon, clone genes encoding cat omega 2 interferon mutants into baculovirus transfer vectors to be recombined with baculovirus to infect insect hosts, express exogenous genes to obtain cat omega 2 interferon mutant proteins, and improve the antiviral activity of cat omega 2 interferon by more than 45% through mutagenesis (Huxiaoyuan and the like: cat omega 2 interferon mutants, a preparation method and application thereof 2015, an authorization notice number: CN 108276486A). If the cat omega 2 interferon mutant protein can be industrially produced, the method has good application prospect. Currently, FeINF-omega is successfully expressed mainly in escherichia coli, yeast and a silkworm baculovirus system, but rFeINF-omega expressed by escherichia coli is poor in activity, and core alpha 1,3 fucose with high proportion exists in an N-glycan structure of a product expressed by the silkworm baculovirus expression system, so that the product has potential immunogenic reaction (Minagawa S et al. novel recombinant viral expression N-polysaccharides with reduced produced by product of silkworm baculovirus expression system BMC Vet Res.2018; 14: 260).

Mammalian cell expression systems can direct the correct folding of proteins, providing multiple post-translational processing modification capabilities such as complex N-type glycosylation and accurate O-type glycosylation, and thus the expression products are closest to natural higher biological protein molecules in terms of molecular structure, physicochemical properties and biological functions. The feline belongs to mammals, and rFeINF-omega is a glycoprotein, and a foreign protein produced by post-translational modification of mammalian cells is higher than a prokaryotic expression system and eukaryotic expression systems such as yeast and insect cells in terms of activity, is closer to a natural protein, and can overcome the immunogenicity risk caused by N-glycan chain core alpha 1-3 fucose peculiar to an insect or plant cell expression system. However, mammalian cell expression systems are costly and inefficient. Therefore, the invention develops a process method which is simple and easy to control and low in cost, the yield of the rFeINF-omega 2 mutant protein produced by the process can reach more than 2.5g/L, and the antiviral activity of the product is obviously superior to that of other expression systems.

Disclosure of Invention

The invention aims to provide a mammalian cell culture process for efficiently expressing an rFeINF-omega 2 mutant, which solves the problems of low efficiency and high cost of mammalian cell expression. Specifically, a production culture process with simple and easy control operation, low cost, high expression product yield and high yield is provided for the industrialization of the rFeINF-omega 2 mutant protein, and the main technical scheme is as follows:

inoculating the mammalian cells expressing the rFeINF-omega 2 mutant into a bioreactor containing a serum-free basal medium for suspension culture, controlling the temperature, dissolved oxygen, pH and rotating speed in the culture process, feeding different serum-free supplemented media, hydrolysate and glutamine regularly in the culture process, monitoring the growth condition of the cells and supplementing a glucose solution.

Preferably, the mammalian cell is a chinese hamster ovary cell, preferably a CHO DG44 cell.

Preferably, the cell seeding density is 5-8X 10⁵Per mL, preferably 5X 10⁵/mL。

Preferably, the working volume of the basic culture medium of the bioreactor is 1-500L, the rotating speed is set to be 10-200 r/min, and specifically,

the working volume of the medium in a 7 liter bioreactor is 1-5L, preferably 3L; setting the rotation speed of 100 and 200 revolutions per minute, preferably 150 revolutions per minute;

the working volume of the medium in a 50 liter bioreactor is 10-40L, preferably 30L; setting the rotating speed to be 80-120 revolutions per minute, preferably 90 revolutions per minute;

the working volume of the medium in the 250 liter bioreactor is 40-200L, preferably 150L; setting the rotating speed to be 10-80 revolutions per minute, preferably 50 revolutions per minute;

preferably, the temperature is controlled by setting the initial culture temperature to be 36-38 ℃, preferably 36.5 ℃; the culture temperature was decreased to 33 ℃ from the 6 th day of the culture.

Preferably, the dissolved oxygen control condition is 30% -80%, preferably 40%;

preferably, the pH control range is 5-7.5, preferably 7 +/-0.3;

preferably, the feeding mode of the serum-free feeding medium 1 is that 5%, 3% of the serum-free feeding medium is respectively fed on days 3, 6, 8, 10 and 12; the feeding mode of the serum-free feed medium 2 is that 2 percent of the serum-free feed medium is respectively fed on days 4, 7, 9 and 11;

preferably, the hydrolysis material is fed in a mode of feeding 6g/L of hydrolysis material on days 5 and 8 respectively;

preferably, the serum-free basal medium consists of one or more of ActiPro medium with the concentration of 10-30g/L and SFM4CHO medium with the concentration of 10-20 g/L; and additives, which are composed of one or more of F68 with the concentration of 0.5-2g/L, glucose with the concentration of 3-5g/L and glutamine with the concentration of 4-8mM and the pH value of 7-7.5.

Preferably, the serum-free supplemented medium 1 consists of one or more of Cell Boost 7a with the concentration of 100-200 g/L and Cell Boost 7b supplemented with the concentration of 50-100 g/L; the serum-free supplemented medium 2 consists of EX-CELL Advanced CHO Feed 1 with the concentration of 50-100 g/L.

Preferably, the hydrolysate is composed of one or more of Hypep 7504 with the concentration of 50-200 g/L and PF Plus ACF hydrolysate with the concentration of 50-200 g/L;

the invention has the beneficial effects that: provides a simple and high-efficiency mammalian cell culture process for expressing the rFeINF-omega mutant, so that the expression product has high yield, low cost and good antiviral activity, and is suitable for large-scale industrial application.

Drawings

FIG. 1 shows the curves of the number of days and cell density of rFeINF- ω mutant protein-expressing cells cultured in a bioreactor

FIG. 2 shows the curves of the number of days of culture and the cell viability of rFeINF-omega mutant protein-expressing cells in a bioreactor

FIG. 3 shows the SDS-PAGE examination result of culture supernatant samples of rFeINF-omega mutant protein-expressing cells in a bioreactor (reduction)

FIG. 4 shows the expression level of rFeINF-omega mutant protein expressing cells in culture supernatant samples in a bioreactor

Detailed Description

The following examples are provided to further understand and explain the present invention and are not to be construed as limiting the invention.

Example 1 recombinant Cat interferon-omega 2 mutant protein expressing cell expansion culture

Resuscitating mammalian cells expressing recombinant feline interferon-omega 2 mutant at 5X 10⁵Inoculating cells/ml density into a cell shake flask, placing the cell shake flask into a shaking table for shaking culture, and setting parameters of the shaking table as follows: 36.5 ℃ and 5% CO₂140 rpm/min; after continuous culture for about 3 days, the cell density reaches 2-3X 10⁶cells/ml at 5X 10⁵Subculturing is carried out at a cell/ml density until the number of cells is sufficient to inoculate the bioreactor, and seed cell liquid is obtained.

Example 2 recombinant Cat interferon-omega 2 mutant protein expressing cell bioreactor culture

Mixing seed cell liquid at 5 × 10⁵cells/ml are inoculated into a bioreactor (the working volume is 3L), an ActiPro culture medium is adopted for culture, and the set technological parameters are as follows: culturing at 36.5 deg.C, dissolved oxygen content of 40%, pH of 7 + -0.3, and stirring speed of 150 rpmDay 6, the temperature was reduced to 33 ℃; feeding 5%, 3% Cell Boost 7a/Cell Boost 7b (10:1 ratio) on days 3, 6, 8, 10, 12 respectively, feeding 2% EX-CELL Advanced CHO Feed 1 on days 4, 7, 9, 11 respectively, feeding 6g/L of Hypep 7504 hydrolysate on days 5 and 8 respectively, and feeding 1% 200mM glutamine on day 3; taking cell supernatant every day to perform biochemical index measurement, and supplementing 1% 400g/L glucose solution when the glucose concentration is lower than 2g/L to keep the glucose concentration above 2 g/L. Cells were cultured continuously until the peak density reached 3.27X 10 at day 8⁷cells/ml (figure 1), the survival rate is maintained at a constant level after the culture under the high-density growth condition for 14 days, the survival rate is slightly reduced after the culture for 15 days, and the cell survival rate is maintained above 80 percent after the culture for 18 days (figure 2), and the culture is stopped to obtain cell stock solution.

Example 3 yield assessment of recombinant feline interferon-omega 2 mutant proteins

Supernatant samples from 12 th, 14 th, 16 th and 18 th days of culture in the bioreactor were subjected to SDS-PAGE and Coomassie blue staining to identify that a clear rFeIFN-omega 2 mutant protein band existed at about 20kDa and the expression level of the rFeIFN-omega 2 mutant protein increased with the increase of culture time (FIG. 3). While the supernatant samples were purified by affinity chromatography and then the purified rfefn- ω 2 mutant proteins were quantified by BCA, the results also showed elevated protein expression levels with increasing culture time, up to 2.61g/L at day 18 of harvest (figure 4). Another evaluation of the specific activity of the purified product against VSV virus showed that the biological specific activity of the rFeIFN-omega 2 mutein produced by the inventive culture process against VSV>3×10⁸IU/mg。