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CN111304169B - Culture medium additive, cell culture medium containing same and application thereof - Google Patents

Culture medium additive, cell culture medium containing same and application thereof Download PDF

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CN111304169B
CN111304169B CN201911221993.4A CN201911221993A CN111304169B CN 111304169 B CN111304169 B CN 111304169B CN 201911221993 A CN201911221993 A CN 201911221993A CN 111304169 B CN111304169 B CN 111304169B
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付建家
付立家
张兰
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Beijing Asia East Bio Pharmaceutical Co Ltd
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    • C12N2730/00011—Details
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    • C12N2730/10151—Methods of production or purification of viral material

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Abstract

The invention relates to a culture medium additive, a cell culture medium containing the same and application thereof, wherein the culture medium additive comprises proline, asparagine and histidine, and/or transferrin and insulin. The culture medium additive is added into DMEM without glutamine together with 20-30 mL/L newborn calf serum, and then the culture medium additive can be used for culturing CHO cells specifically expressing hepatitis B virus surface antigens. The concentration of the newborn calf serum used by the cell culture medium provided by the invention is as low as 2%, the newborn calf serum can be directly used for culturing CHO cells by being matched with specific components without domestication of the CHO cells, and meanwhile, the expression level of hepatitis B virus surface antigen in the CHO cells is increased by 16-40% in 13 days.

Description

Culture medium additive, cell culture medium containing same and application thereof
Technical Field
The invention relates to the field of cell culture, in particular to a culture medium additive, a cell culture medium containing the same and application thereof.
Background
Hepatitis B is a disease caused by Hepatitis B Virus (HBV), and is an infectious virus, and Hepatitis B virus Surface Antigen (HBsAg) is used as a vaccine for Hepatitis B virus, and is a recombinant protein having a large demand. The expression system of recombinant HBsAg mainly comprises yeast cells and Chinese hamster ovary Cells (CHO).
Chinese hamster ovary Cells (CHO) transformed by hepatitis B virus S protein gene can secrete and express recombinant HBsAg after being cultured. Compared with recombinant vaccines produced by yeast cells, the recombinant vaccine has better immunogenicity and positive conversion rate. The biological product expressed by animal cells, particularly the CHO expressed product, has good safety, can be used in the production of hepatitis B vaccines, has no serious side reaction, particularly the CHO-C28 cell strain, and has no safety accident after being used for producing the hepatitis B vaccines for nearly 30 years.
Because the traditional production process adopts a large rotary bottle to culture recombinant CHO cells adherent to the wall under the condition of serum, the cost of the vaccine expressed by heavier yeast is high, and the market share is relatively small. There are many companies and institutions currently attempting to perform serum-free acclimation of recombinant CHO cells expressing HBsAg in culture to improve recombinant protein production and reduce production costs. However, the serum-free optimization of the recombinant CHO cells expressing the hepatitis B surface antigen has no obvious effect at present and is still lower than the yield of the traditional production process. In addition, the cell serum-free acclimation process may cause instability of the genetically engineered cell strain, resulting in reduced protein yield.
Disclosure of Invention
The invention aims to provide a culture medium additive, a cell culture medium containing the culture medium additive and application of the culture medium, wherein the culture medium can support recombinant CHO cells to efficiently express a recombinant hepatitis B vaccine, so that the problems of high production cost, low expression quantity and the like of the hepatitis B vaccine are solved.
In a first aspect, the invention provides a media supplement comprising proline, asparagine and histidine, and/or transferrin and insulin.
In a second aspect, the invention provides a cell culture medium containing the above medium additive, wherein the cell culture medium further comprises 20-30 mL/L newborn bovine serum.
The cell culture medium I comprises:
20-30 mL/L newborn bovine serum, 0.1-0.2 mg/mL proline, 0.1-0.2 mg/mL asparagine and 0.1-0.2 mg/mL histidine;
preferably 20-30 mL/L newborn calf serum, 0.1-0.12 mg/mL proline, 0.1-0.12 mg/mL asparagine and 0.1-0.12 mg/mL histidine.
Amino acids are essential nutrients in cell culture, and the requirements of cells for different amino acids are obviously different. It is not only a basic unit constituting a protein required for cell growth, but also a raw material for synthesizing a target protein. After the three amino acids are added, the expression level of the hepatitis B virus surface antigen of the CHO cell is improved, and the CHO cell can normally survive under the concentration of 2% newborn calf serum.
The cell culture medium II comprises:
20-30 mL/L newborn bovine serum, 6-10 mu g/mL transferrin and 3-5 mu g/mL insulin;
preferably 20-30 mL/L newborn bovine serum, 6-8 mu g/mL transferrin and 3-4 mu g/mL insulin.
Transferrin is a glycoprotein that binds iron ions, and can prevent the production of free radicals in the extracellular environment and prevent the cells from being damaged. The iron ions transported into the cells are combined with part of ferritin, and the functions of maintaining the growth and proliferation of the cells are exerted. Insulin regulates the absorption of glucose and uridine by cells to synthesize RNA, proteins and lipids, and it also binds to insulin receptors on cell membranes to regulate various metabolic pathways in cells, increases the synthesis of fatty acids and glucose, and plays an important role in cell growth.
The invention uses transferrin and insulin in a specific proportion, and can also play a role in improving the expression level of hepatitis B virus surface antigen of cells, and simultaneously ensure that CHO cells can normally survive under the concentration of 2 percent newborn calf serum.
The cell culture medium III comprises:
20-30 mL/L newborn bovine serum, 0.1-0.2 mg/mL proline, 0.1-0.2 mg/mL asparagine, 0.1-0.2 mg/mL histidine, 6-10 mu g/mL transferrin and 3-5 mu g/mL insulin;
preferably 20-30 mL/L newborn bovine serum, 0.1-0.12 mg/mL proline, 0.1-0.12 mg/mL asparagine, 0.1-0.12 mg/mL histidine, 6-8 mu g/mL transferrin and 3-4 mu g/mL insulin.
The two cell culture medium formulas are comprehensively used, the expression level of the hepatitis B virus surface antigen of the CHO cell is further increased, and the increase amplitude exceeds the sum of the two formulas.
Further, the above cell culture media were used together with a glutamine-free DMEM medium.
Further, the above cell culture medium can be used for culturing CHO cells, particularly CHO cells that can specifically express hepatitis B virus surface antigen (HBsAg), such as CHO-C28 cells.
The invention further provides a method for culturing cells by using the cell culture medium, wherein the cells are CHO cells, and preferably CHO cells capable of specifically expressing hepatitis B virus surface antigen.
The invention further provides application of the cell culture medium and the cell culture method in improving the expression level of the hepatitis B virus surface antigen in cells, wherein the cells are preferably CHO-C28 cells.
The invention provides a culture medium additive, a cell culture medium containing the same and application thereof, and the culture medium additive has the following beneficial effects:
the concentration of the newborn calf serum used by the cell culture medium provided by the invention is as low as 20mL/L, the newborn calf serum can be directly used for culturing CHO cells by being matched with specific amino acid and other components, the CHO cells do not need to be domesticated, the expression level of hepatitis B virus surface antigen in the CHO cells can be obviously improved, and the expression level is increased by 16-40% in 13 days. The cell culture medium provided by the invention can save cost, improve the expression level of the hepatitis B virus surface antigen of CHO cells, and has higher application value.
Drawings
FIG. 1 is a graph showing the change in the expression level of hepatitis B virus surface antigen (HBsAg) in CHO-C28 cells in the culture media of control group, experimental group 1, experimental group 2, and experimental group 3 according to Experimental example 1 of the present invention;
FIG. 2 is a graph showing the change in the expression level of hepatitis B virus surface antigen (HbsAg) in CHO-C28 cells after changing the amino acid type from histidine to glycine according to Experimental example 2 of the present invention;
FIG. 3 is a graph showing the change in the expression level of hepatitis B virus surface antigen (HbsAg) in CHO-C28 cells after the change in the amino acid concentration according to Experimental example 2 of the present invention;
FIG. 4 is a graph showing the change in the expression level of hepatitis B virus surface antigen (HBsAg) in CHO-C28 cells after the change in the concentrations of the respective components of the medium according to Experimental example 2 of the present invention;
FIG. 5 is a graph showing the change in the expression level of hepatitis B virus surface antigen (HBsAg) in CHO-C28 cells after the change in the composition of the medium according to Experimental example 2 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
This example provides a cell culture medium prepared by adding the following components in glutamine-free DMEM medium:
20mL/L newborn calf serum, 0.1mg/mL proline, 0.1mg/mL asparagine and 0.1mg/mL histidine.
Example 2
This example provides a cell culture medium prepared by adding the following components in glutamine-free DMEM medium:
20mL/L newborn bovine serum, 6 μ g/mL transferrin and 3 μ g/mL insulin.
Example 3
This example provides a cell culture medium prepared by adding the following components in glutamine-free DMEM medium:
20mL/L newborn calf serum, 0.1mg/mL proline, 0.1mg/mL asparagine, 0.1mg/mL histidine, 6. mu.g/mL transferrin and 3. mu.g/mL insulin.
Comparative example 1
This comparative example was based on example 1, with the replacement of histidine to an equivalent amount of glycine.
Comparative example 2
This comparative example, based on example 1, the three amino acid concentrations were changed as follows: 0.5mg/mL proline, 0.5mg/mL asparagine, and 0.5mg/mL histidine.
Comparative example 3
This comparative example was conducted in example 2, and the replacement transferrin concentration was 3. mu.g/mL, and the replacement insulin concentration was 2. mu.g/mL.
Comparative example 4
This comparative example is based on example 3, replacing histidine with an equivalent amount of glycine.
Experimental example 1
This example examined the expression levels of hepatitis B virus surface antigen (HBsAg) in CHO cells in different media.
Control group: glutamine-free DMEM medium supplemented with 6% newborn bovine serum.
Experimental group 1: cell culture media as shown in example 1.
Experimental group 2: cell culture media as shown in example 2.
Experimental group 3: cell culture media as shown in example 3.
The cell culture steps are as follows:
recovering a CHO-C28 cell strain from the working cell bank, wherein the survival rate of the recovered cell is not lower than 85%. And after 24 hours, replacing the cell growth liquid (DMEM medium plus 10-16% fetal calf serum), then placing the culture liquid at 37 ℃ for continuous culture for 2-3 days, after the square flask cells form a compact monolayer, digesting and passaging the cells by using trypsin, wherein the passage ratio is 1: 3-1: 4, and replacing the growth liquid once every 2-3 days.
The HBsAg detection method comprises the following steps:
the monitoring of the HBsAg content follows the operation instructions of the hepatitis B antigen diagnostic kit. And HBsAg standard substance 0.2ng/mL, 0.6ng/mL, 1.8ng/mL, 5.4ng/mL is used to establish content determination curve.
The experimental procedure was as follows:
and (3) when the cells are passaged to the required generation (the last passage is 32 th), changing the cell growth liquid for 2-4 days, pouring out the cell culture liquid after changing the cell growth liquid for 1-2 times, starting to change the cell maintenance liquid, and adding a new culture medium. The maintenance solution is collected and replaced once every 2-3 days, and the HbsAg expression level of the CHO-C28 cells is detected every day, so that the results shown in the tables 1-3 are obtained:
TABLE 1 expression level of HbsAg of CHO-C28 cells in control and experiment 1 media
Figure BDA0002301107330000061
TABLE 2 expression level of HbsAg of CHO-C28 cells in culture Medium of control group and Experimental group 2
Figure BDA0002301107330000062
Figure BDA0002301107330000071
TABLE 3 expression level of HbsAg of CHO-C28 cells in control and experiment 3 media
Figure BDA0002301107330000072
FIG. 1 is a graph showing the change of the expression level of HbsAg in CHO-C28 cells cultured in the media of the control group and the experimental groups 1 to 3 (corresponding to examples 1 to 3) according to this example.
As shown in Table 1-Table 3 and FIG. 1, the culture medium of the experimental groups 1-3 in this example has a certain degree of increase in the HbsAg expression level compared to the culture medium of the control group, and the newborn bovine serum used in the experimental groups 1-3 is only 2% compared to the control group, which means that the experimental groups 1-3 achieve higher expression efficiency with less serum.
In 40 days as an example, the cell culture medium shown in example 1 increased the HbsAg expression level by 17%, the cell culture medium shown in example 2 increased the HbsAg expression level by 16%, and the cell culture medium shown in example 3 increased the HbsAg expression level by 42%, which is more than the sum of the two media added together.
Experimental example 2
This experimental example measured the expression level of hepatitis B virus surface antigen (HBsAg) of CHO cells in the culture media of comparative example 1, comparative example 2 and comparative example 3 in the same manner as in experimental example 1, and obtained the results shown in tables 4 to 7:
TABLE 4 HbsAg expression levels of CHO-C28 cells in the culture medium of comparative example 1
Figure BDA0002301107330000081
TABLE 5 HbsAg expression level of CHO-C28 cells in the culture medium of comparative example 2
Figure BDA0002301107330000082
TABLE 6 HBsAg expression level of CHO-C28 cells in the culture medium of comparative example 3
Figure BDA0002301107330000083
Figure BDA0002301107330000091
TABLE 7 HbsAg expression level of CHO-C28 cells in the culture medium of comparative example 4
Figure BDA0002301107330000092
FIG. 2 is a graph showing the change in the expression level of hepatitis B virus surface antigen (HBsAg) in CHO-C28 cells after the amino acid type change according to this example, wherein the control group is the same as that of example 1, specifically, the change in the function of the medium shown in example 1 after histidine was changed to the same amount of glycine.
FIG. 3 is a graph showing the change in the expression level of hepatitis B virus surface antigen (HbsAg) in CHO-C28 cells after the change in the amino acid concentration according to this example, wherein the control group is the same as the control group in example 1, specifically, the change in the culture function shown in example 1 after the change in the amino acid concentration.
As can be seen from FIGS. 2 and 3, the medium formulation of example 1 (20mL/L newborn bovine serum, 0.1mg/mL proline, 0.1mg/mL asparagine, and 0.1mg/mL histidine) had a more significant effect on the level of hepatitis B virus surface antigen (HbsAg) expression in CHO-C28 cells.
FIG. 4 is a graph showing the change in the expression level of hepatitis B virus surface antigen (HBsAg) in CHO-C28 cells after the change in the concentrations of the respective components of the medium, in which the control group is the same as that of example 1, specifically, the concentrations of transferrin and insulin in example 2 are reduced.
As can be seen from FIG. 4, the ability of the medium of example 2 to promote the increase in the expression level of hepatitis B virus surface antigen (HbsAg) in CHO-C28 cells was also reduced after the concentrations of transferrin and insulin were reduced.
FIG. 5 is a graph showing the change in the expression level of hepatitis B virus surface antigen (HbsAg) in CHO-C28 cells after the change in the medium composition, wherein the control group is the same as that of example 1, specifically, the histidine in example 3 is replaced with an equivalent amount of glycine.
As can be seen from FIG. 5, the ability of the medium of example 3 to promote the increase in the expression level of hepatitis B virus surface antigen (HbsAg) in CHO-C28 cells was also reduced after the amino acid composition was changed.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (7)

1. Cell culture medium, characterized in that it comprises, on the basis of glutamine-free DMEM medium, the following components in concentrations:
20mL/L newborn calf serum, 0.1mg/mL proline, 0.1mg/mL asparagine, 0.1mg/mL histidine, 6. mu.g/mL transferrin and 3. mu.g/mL insulin.
2. The cell culture medium of claim 1, wherein the cell culture medium is a CHO cell culture medium, and the CHO cell can specifically express hepatitis B virus surface antigen.
3. The cell culture medium of claim 2, wherein the CHO cells are CHO-C28 cells.
4. A method of cell culture comprising: culturing the cells with the cell culture medium of any one of claims 1-3.
5. The cell culture method according to claim 4, wherein the cells are CHO cells.
6. The cell culture method according to claim 5, wherein the CHO cell is a CHO cell that can specifically express hepatitis B virus surface antigen.
7. Use of the cell culture medium of any one of claims 1-3 or the cell culture method of claim 4 or 5 for increasing the expression level of hepatitis b virus surface antigen in cells that are CHO-C28 cells.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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CN1696283A (en) * 2004-05-12 2005-11-16 华东理工大学 A serum-free medium suitable for Chinese hamster ovary cell culture
CN102229908A (en) * 2011-06-03 2011-11-02 华东理工大学 Nutriment additive for high-density culture and product expression of animal cells
CN103074293A (en) * 2012-12-28 2013-05-01 杭州国牧生物科技有限公司 Culture medium for preparing influenza vaccine through MDCK cells and application method thereof

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
CN1362514A (en) * 2002-01-25 2002-08-07 华东理工大学 Serumless medium suitable for growth and maintenance of young hamster kidney cell
CN1696283A (en) * 2004-05-12 2005-11-16 华东理工大学 A serum-free medium suitable for Chinese hamster ovary cell culture
CN102229908A (en) * 2011-06-03 2011-11-02 华东理工大学 Nutriment additive for high-density culture and product expression of animal cells
CN103074293A (en) * 2012-12-28 2013-05-01 杭州国牧生物科技有限公司 Culture medium for preparing influenza vaccine through MDCK cells and application method thereof

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Title
Cell Culture Media for Recombinant Protein Expression in Chinese Hamster Ovary (CHO) Cells: History, Key Components, and Optimization Strategies;Frank V. Ritacco et al.;《Biotechnol Prog.》;20181231;全文 *
Developement of serum-free media in CHO-DG44 cells using a central composite statistical design;Ananth Parampalli et al.;《Cytotechnology》;20070705;全文 *
Evaluation of Medium Supplemented with Insulin–Transferrin–Selenium for Culture of Primary Bovine Calf Chondrocytes in Three-Dimensional Hydrogel Scaffolds;JOHN D. KISIDAY et al.;《TISSUE ENGINEERING》;20051231;全文 *

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