阅读说明：本技术 调节cho-k1表达系统所分泌抗体的酸性峰含量的方法 (Method for regulating acid peak content of antibody secreted by CHO-K1 expression system ) 是由 肖尚 翁源灿 于 2018-06-25 设计创作，主要内容包括：本发明涉及一种调节CHO-K1表达系统所分泌抗体的酸性峰含量的方法,将CHO-K1细胞接种到培养基中进行细胞培养,在细胞培养过程中添加调节剂,继续培养以使CHO-K1细胞分泌具有合适酸性峰含量的目的抗体,其中,调节剂选自酪氨酸和半胱氨酸中的至少一种。这种方法在细胞培养工艺流程即能够根据需要,调节酪氨酸或半胱氨酸的添加量即可提高或降低抗体酸性峰含量,且调节剂不影响细胞的生长,抗体表达量高。(The invention relates to a method for regulating the acid peak content of an antibody secreted by a CHO-K1 expression system, which comprises the steps of inoculating CHO-K1 cells into a culture medium for cell culture, adding a regulator in the cell culture process, and continuing the culture to ensure that the CHO-K1 cells secrete a target antibody with proper acid peak content, wherein the regulator is selected from at least one of tyrosine and cysteine. The method can adjust the addition amount of tyrosine or cysteine according to the requirement in the cell culture process flow, so as to improve or reduce the acid peak content of the antibody, and the regulator does not influence the growth of cells and has high antibody expression level.)

1. A method for improving the content of an acidic peak of an antibody secreted by a CHO-K1 expression system is characterized by comprising the following steps:

inoculating CHO-K1 cells into the culture medium for cell culture, and

cysteine and a feed medium are added in the cell culture process, and the culture is continued so that the CHO-K1 cell secretes the target antibody with proper acidic peak content.

2. The method of claim 1, wherein the medium is: CD optiCHO^TMAGT^TMCulture Medium, Cellvento^TMA mixture of one or more of CD-220 medium, Dynamis medium and Advance medium.

3. The method of claim 1, wherein the feed medium is: effect Feed^TMA+、Effendent Feed^TMB + or Cell boost7a feed medium.

4. The method according to claim 3, wherein the feed medium is added once a day to once every three days, and the feed medium is added at a time of 1% to 5% of the total volume of the medium.

5. The method according to claim 1, wherein cysteine is added during the cell culture in an amount of from 0.01 mmol/(L-day) to 1.2 mmol/(L-day).

6. The method of claim 5, wherein the cysteine is added in an amount of 0.14 mmol/(L-day) to 1.2 mmol/(L-day).

7. The method according to any one of claims 1 to 6, wherein the sugar concentration in the cell culture solution is maintained at 2 g/L to 6 g/L during the cell culture.

8. The method according to any one of claims 1 to 6, wherein the CHO-K1 cell is a cell carrying a GS gene and having an exogenous gene expression vector inserted downstream of the GS gene.

9. The method of claim 8, wherein said exogenous gene expression vector is selected from at least one of a nivolumab expression vector, a bevacizumab expression vector, and an adalimumab expression vector.

10. A method for improving the content of an acidic peak of an antibody secreted by a CHO-K1 expression system is characterized by comprising the following steps:

inoculating CHO-K1 cells into a culture medium for cell culture, wherein the culture medium contains a Dynamis culture medium and an Advance culture medium, and the volume ratio of the Dynamis culture medium to the Advance culture medium is 1-2: 1-2; and

after culturing in the culture medium for 1 to 3 days, adding cysteine and Cell boost7a feed culture medium into the culture medium, maintaining the sugar concentration in a Cell culture solution at 2 to 6 g/L, and continuously culturing for 10 to 15 days to improve the acid peak content of the target antibody secreted by the CHO-K1 Cell, wherein the addition amount of the cysteine is 0.01 mmol/(L. day) -1.2 mmol/(L. day), the Cell boost7a feed culture medium is added once a day to three days, and the Cell boost7a feed culture medium added each time accounts for 1 to 5 percent of the total volume of the culture medium.

Technical Field

The invention relates to the field of cell culture methods, in particular to a method for regulating the content of an acidic peak of an antibody secreted by a CHO-K1 expression system.

Background

Monoclonal antibodies have the characteristics of high specificity, low side effect and excellent effect, and are increasingly widely applied in treatment and diagnosis. Among them, CHO cells (Chinese Hamster ovary cells) are the most widely used host cells for producing monoclonal antibody protein drugs, and nearly 70% of therapeutic proteins on the market are produced by expression of CHO cells. Compared with other cell expression systems, the CHO cell has the advantages of post-translational modification similar to human, clear historical background, stable cell, rapid growth and the like.

However, because monoclonal antibodies have complex macromolecular structures, a large amount of modifications, such as glycosylation, oxidation, deamidation and the like, can be generated in the production and storage processes of the monoclonal antibodies, and the monoclonal antibodies are also called as charge isomers, a series of peaks can be obtained by adopting a CEX-HP L C or CIEF method for analysis, and the peaks are mainly divided into three types of acidic peaks, main peaks and basic peaks.

One method is to add glutamine into the culture medium to reduce the content of acidic peaks, however, glutamine has a very adverse effect on the stability of the later process, and after glutamine is added in the culture process, glutamine is easily degraded to generate a large amount of NH4+, thereby having an adverse effect on the growth and expression of cells. In addition, in the CHO expression system, the addition of glutamine can only reduce the content of the acidic peak of the secreted antibody, and cannot increase the content of the acidic peak of the antibody as needed. In some antibody biological similar drugs, the content of the acidic peak is often reduced compared with the original drug, but in clinical use, the maintenance of the similar acidic peak of the similar drug and the original drug is very necessary for safety, so how to increase the content of the acidic peak of the antibody is a very difficult problem.

Disclosure of Invention

Based on the above, it is necessary to provide a method capable of adjusting the acidic peak content of the antibody secreted from the CHO-K1 expression system as required and increasing the expression level of the antibody.

A method for regulating the acidic peak content of an antibody secreted by a CHO-K1 expression system, comprising the following steps:

inoculating CHO-K1 cells into the culture medium for cell culture, and

adding a regulator in the cell culture process, and continuing the culture to make the CHO-K1 cell secrete the target antibody with proper acidic peak content, wherein the regulator is selected from at least one of tyrosine and cysteine.

In one embodiment, wherein the medium is: CD optiCHO^TMAGT^TMCulture Medium, Cellvento^TMA mixture of one or more of CD-220 medium, Dynamis medium, or Advance medium.

In one embodiment, the method further comprises adding a feed medium during the cell culture, wherein the feed medium is: effect Feed^TMA+、Effendent Feed^TMB + or Cell boost7a feed medium.

In one embodiment, the feeding medium is added in a manner of 1 to 3 days per time, and the feeding medium is added in each time in a range of 1 to 5% of the total volume of the medium.

In one embodiment, the operation of adding the regulator during the cell culture process is specifically as follows:

tyrosine is added in the cell culture process, the addition amount of the tyrosine is 0.01 mmol/(L-day) to 1.2 mmol/(L-day), or,

cysteine is added during the cell culture process, and the addition amount of the cysteine is 0.01 mmol/(L. multidot. day) to 1.2 mmol/(L. multidot. day), or,

tyrosine and cysteine are added in the cell culture process, and the total addition amount of the tyrosine and the cysteine is 0.01 mmol/(L-day) to 1.2 mmol/(L-day).

In one embodiment, the sugar concentration in the cell culture fluid is maintained at 2 g/L-6 g/L during the cell culture process.

In one embodiment, the CHO-K1 cell is a cell that carries a GS gene and has an exogenous gene expression vector inserted downstream of the GS gene.

In one embodiment, the exogenous gene expression vector is selected from at least one of a nivolumab expression vector, a bevacizumab expression vector, and an adalimumab expression vector.

A method for reducing the content of acidic peaks of an antibody secreted by a CHO-K1 expression system comprises the following steps:

inoculating CHO-K1 cells into a culture medium for cell culture, wherein the culture medium contains a Dynamis culture medium and an Advance culture medium, and the volume ratio of the Dynamis culture medium to the Advance culture medium is 1-2: 1-2; and

after culturing in the culture medium for 1 to 3 days, adding tyrosine and Cell boost7a feed culture medium into the culture medium, maintaining the sugar concentration in a Cell culture solution at 2 to 6 g/L, and continuing culturing for 10 to 15 days to reduce the content of the acid peak of the target antibody secreted by the CHO-K1 Cell, wherein the tyrosine is added in an amount of 0.01 mmol/(L-day) to 1.2 mmol/(L-day), the Cell boost7a feed culture medium is added in an amount of 1 to 3 days per time, and the Cell boost7a feed culture medium added each time accounts for 1 to 5 percent of the total volume of the culture medium.

A method for improving the content of an acidic peak of an antibody secreted by a CHO-K1 expression system comprises the following steps:

inoculating CHO-K1 cells into a culture medium for cell culture, wherein the culture medium contains a Dynamis culture medium and an Advance culture medium, and the volume ratio of the Dynamis culture medium to the Advance culture medium is 1-2: 1-2; and

after culturing in the culture medium for 1 to 3 days, adding cysteine and Cell boost7a feed medium into the culture medium, maintaining the sugar concentration in the Cell culture solution at 2 to 6 g/L, and continuing culturing for 10 to 15 days to improve the acid peak content of the target antibody secreted by the CHO-K1 Cell, wherein the addition amount of the cysteine is 0.01 mmol/(L-day) to 1.2 mmol/(L-day), the addition manner of the Cell boost7a feed medium is 1 to 3 days, and the Cell boost7a feed medium added each time accounts for 1 to 5 percent of the total volume of the culture medium.

For a long time before, the aim of cell culture studies has been mainly to increase the expression level of the protein of interest. With higher and higher expression levels, researchers have recently begun to focus on the effect of cell culture on antibody quality. However, cell culture is complex, and research is difficult, particularly, the nutrient components of the cell culture medium are very complex, and the influence of the components of the culture medium on the quality of the antibody is not thoroughly discovered in the current research. Glutamine is widely studied as a source of nitrogen commonly used in cell culture, and its action on other amino acids is rarely studied. However, the current stable CHO cell line screening system is a glutamine synthetase system (GS), and glutamine does not need to be added in cell culture, so that the current common method is not to regulate additional amino acid as a regulator. The invention carries out a great deal of research and study on the aspects of selection of the regulator and the like, breaks through the technical bias before, and unexpectedly discovers that the acid peak content of the antibody secreted by the CHO-K1 expression system can be regulated by inoculating the CHO-K1 cells into a culture medium for culture and adding tyrosine or cysteine in the cell culture process. The method can adjust the addition amount of tyrosine or cysteine according to the requirement in the cell culture process flow, so as to improve or reduce the acid peak content of the antibody, and the regulator does not influence the growth of cells and has high antibody expression level.

Drawings

FIG. 1 is a graph showing the results of statistics of the expression levels of antibodies secreted from the cells of each group in examples 1 to 15;

FIG. 2 is a statistical chart of the results of the density of viable cells of each group of examples 1 to 5;

FIG. 3 is a statistical chart of the results of the density of viable cells of each group of examples 6 to 10;

FIG. 4 is a statistical chart of the results of the density of viable cells of each group in examples 11 to 15;

FIG. 5 is a statistical chart showing the content of acidic peaks of the antibody secreted by the cells of each group of examples 11 to 15;

FIG. 6 is a graph showing the main effect of the acidic peak of the antibody.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.

One embodiment of the method for regulating the acidic peak content of the antibody secreted by the CHO-K1 expression system comprises the following steps S110 to S120.

S110, inoculating CHO-K1 cells into a culture medium for cell culture.

Specifically, the CHO-K1 cell is a cell carrying a GS gene and having a foreign gene expression vector inserted downstream of the GS gene. CHO-K1 cells are capable of expressing exogenous proteins such as monoclonal antibodies and the like.

Specifically, the CHO-K1 cell is a cell carrying a GS gene and having an exogenous gene expression vector inserted downstream of the GS gene.

Further, the exogenous gene expression vector is at least one selected from the group consisting of a nivolumab expression vector, a bevacizumab expression vector, and an adalimumab expression vector.

Specifically, the medium may be CD optiCHO^TMAGT^TMCulture Medium, Cellvento^TMA mixture of one or more of CD-220 medium, Dynamis medium, or Advance medium.

Preferably, the culture medium contains a Dynamis culture medium and an Advance culture medium, and the volume ratio of the Dynamis culture medium to the Advance culture medium is 1-2: 1-2. Dynamis and Advance culture medium mainly contains lipid, inorganic salt, vitamins, etc. The Dynamis culture medium and the Advances culture medium provide necessary nutrients for cell growth and guarantee for cell growth. The culture medium is a Dynamis culture medium, and the Advance culture medium is matched with a regulator, so that the acid peak content of the antibody can be better regulated.

In the present embodiment, the Dynamis medium is specifically Dynamis^TMAGT^TMMedium, available from Saimer Feishale, Inc., having a designation A2617504. CD optiCHO^TMAGT^TMAlso available from zemer feishel corporation under the designation a14285 EF. The advanced culture medium is specifically EX-Advanced CHO Fed-batch Medium available from Merck under the trade designation 24366C. Cellvento O^TMCD-220 medium was also purchased from Merck under the designation 1.01885.0010.

S120, adding a regulator in the cell culture process, and continuing the culture to make the CHO-K1 cell secrete the target antibody with proper acidic peak content, wherein the regulator is selected from at least one of tyrosine and cysteine.

In the process of cell culture, a regulator is additionally added, the regulation of the acid peak of the antibody is realized in the cell culture stage, the target antibody with proper acid peak content is prepared from the source, and the purification and separation process in the later stage is simplified.

Specifically, the modulator is selected from at least one of tyrosine and cysteine. Through a large number of research experiments, the acidic peak of the target antibody can be reduced by adding tyrosine into a specific culture medium and continuously culturing the CHO-K1 cells. Cysteine was added to a specific medium, and the acidic peak of the target antibody was increased by further culturing the CHO-K1 cells. Furthermore, the addition of tyrosine and cysteine does not substantially affect cell growth, resulting in an increase in the expression level of the antibody.

In one embodiment, tyrosine is added during the cell culture process in an amount of 0.01 mmol/(L. multidot. day) to 1.2 mmol/(L. multidot. day). This tyrosine is further added in an amount of 0.06 mmol/(L. multidot. day) to 1.2 mmol/(L. multidot. day).

In another embodiment, cysteine is added during the cell culture in an amount of 0.01 mmol/(L. multidot. day) to 1.2 mmol/(L. multidot. day). This addition amount of cysteine is 0.06 mmol/(L. multidot. day) to 1.2 mmol/(L. multidot. day).

In another embodiment, tyrosine and cysteine are added during the cell culture process, and the total amount of tyrosine and cysteine added is 0.01 mmol/(L-day) to 1.2 mmol/(L-day). furthermore, when they are added in a mixed manner, the molar ratio of tyrosine to cysteine is 1-2: 1-2.

In one embodiment, the method further comprises adding a feed medium during the cell culture process, wherein the feed medium is added in a mode of 1-3 days, and the feed medium added in each time accounts for 1-5% of the total volume of the medium. During the culture process, the added feed medium is matched to improve the expression amount of the antibody and the stability of the process.

In particular, the Feed medium may be an effective Feed^TMA+、Effendent Feed^TMB + and Cellboost7 a.

In this embodiment, the feed medium is Cell boost7a, available from Hyclone under the accession number ABA 212153A. Effect Feed^TMA+、Effendent Feed^TMAll B + were purchased from siemer feishal, inc, with the respective cargo numbers: a25023-04 and A25030-04.

In one embodiment, the method also comprises the step of maintaining the sugar concentration in the cell culture solution to be 2 g/L-6 g/L during the cell culture process, wherein the sugar is specifically glucose, and the sugar concentration in the cell culture solution is maintained during the culture process, so that the expression amount of the antibody and the process stability are improved.

Specifically, after the regulator is added, a sample is taken every day for cell counting and sugar content is detected, and if the sugar concentration in the culture medium is not enough, the sugar is supplemented, so that the normal growth of the cells is ensured.

In one embodiment, the method for reducing the content of the acidic peak of the antibody secreted by the CHO-K1 expression system comprises the following steps of inoculating CHO-K1 cells into a culture medium for Cell culture, wherein the culture medium contains a Dynamis culture medium and an Advance culture medium, the volume ratio of the Dynamis culture medium to the Advance culture medium is 1-2: 1-2, after culturing for 1-3 days in the culture medium, tyrosine and a Cell boost7a feed medium are added into the culture medium, the sugar concentration in a Cell culture solution is maintained at 2 g/L-6 g/25, the culture is continued for 10-5915 days to reduce the content of the acidic peak of the target antibody secreted by the CHO-K1 cells, wherein the tyrosine is 0.01 mmol/(2-3875-L-L days), the feed medium is added in a mode of 1 day/3875-1.2 mmol/(L-5-days), the content of the feed medium added per time is 0.01-3 days, the feed medium added is 1-7-mesh antibody added in the CHO-K1 cells, and the culture medium is added in a culture medium, the culture medium has no influence on the total growth of the Cell culture medium, and the Cell culture medium, the effective expression parameters are reduced, and the added amount of the feed medium is increased by the added.

The application also provides a method for improving the content of an acid peak of an antibody secreted by a CHO-K1 expression system, which specifically comprises the following steps of inoculating CHO-K1 cells into a culture medium for Cell culture, wherein the culture medium contains a Dynamis culture medium and an Advance culture medium, the volume ratio of the Dynamis culture medium to the Advance culture medium is 1-2: 1-2, after culturing for 1-3 days in the culture medium, adding cysteine and a Cell boost7a feed medium into the culture medium, maintaining the sugar concentration in a Cell culture solution at 2 g/L-6 g/L, and continuing culturing for 10-15 days to improve the content of the acid peak of the target antibody secreted by the CHO-K1 cells, wherein the adding mode of the cysteine is 0.01 mmol/(L-1.2 mmol/(L-day), the adding mode of the feed medium is 1-3 days, the adding amount of the cysteine is 0.01 mmol/(L-day), the adding mode of the feed medium is 1-3 days, the adding amount of the feed medium per feed medium is high, the content of the feed medium in the Cell culture medium is not high, and the content of the antibody in the feed medium is not effective in the culture medium, and the culture medium is not effective in the process of the addition of the Cell culture medium, and the addition of the additive is not effective in the Cell culture medium, the Cell growth process, the additive, the feed medium is not effective in the Cell growth of the culture medium, and the additive.

The method for regulating the acid peak content of the antibody secreted by the CHO-K1 expression system has at least the following beneficial effects: the regulator is additionally added in the cell culture process flow, the content of an acid peak of the antibody can be improved or reduced by regulating the addition amount of tyrosine or cysteine according to needs, the regulator is matched with a culture medium to culture a CHO-K1 cell, the content of the acid peak of the antibody secreted by the CHO-K1 cell is regulated, a target antibody with proper acid peak content is prepared from a source, and the later purification and separation process is simplified. And tyrosine and cysteine do not influence the growth of cells, and the expression level of the antibody is high. On the basis of ensuring the yield, the proportion of the acidic peak in the antibody is adjusted, so that the proportion of the acidic peak meets the expected requirement, and the quality of the antibody is ensured.

Furthermore, a certain amount of Cell boost7a feed medium is added in the Cell culture process, and the added regulator is matched with the Cell boost7a feed medium, so that the expression amount and the process stability of the antibody are improved.

Furthermore, the sugar concentration in the cell culture solution is maintained in the cell culture process, and the expression amount of the antibody and the stability of the process are improved.

The following are specific examples.