CN108823173A - Supplemented medium and preparation method thereof for Hybridoma Cell Culture - Google Patents
Supplemented medium and preparation method thereof for Hybridoma Cell Culture Download PDFInfo
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- CN108823173A CN108823173A CN201810818028.4A CN201810818028A CN108823173A CN 108823173 A CN108823173 A CN 108823173A CN 201810818028 A CN201810818028 A CN 201810818028A CN 108823173 A CN108823173 A CN 108823173A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
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- C12N2500/00—Specific components of cell culture medium
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- C12N2500/32—Amino acids
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- C12N2500/00—Specific components of cell culture medium
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- C12N2500/34—Sugars
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
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Abstract
The invention discloses a kind of supplemented mediums for Hybridoma Cell Culture, include following components in the water for injection of 1000ml unit volume：Glucose 10g, glutamine 3g, ox blood albumin 60g, valine 52mg, isoleucine 54mg, phenylalanine-3,4-quinone 5mg, leucine 59mg, tryptophan 9mg, threonine 53mg, methionine 17mg, lysine 91mg.Raw material proportioning science of the present invention, preparation method is simple, and the supplemented medium of preparation overcomes difference between batch existing for existing supplemented medium, has reached stationary flow and has added culture process, has stablized the purpose for improving antibody production.When using stream plus formula cultural method, as supplemented medium, after inoculated and cultured 72 hours, purified antibody production is not less than 300mg/L, and process stabilizing, and antibody production is stablized.
Description
Technical field
The present invention relates to Hybridoma Cell Cultures, more particularly, to a kind of feed-batch culture for Hybridoma Cell Culture
Base, the invention further relates to the preparation methods of the supplemented medium.
Background technique
Hybridoma technology is established within 1975, by the way that murine myeloma cell and the mice spleen of generation sheep red blood cell (SRBC) is thin
Born of the same parents' fusion, the hybridoma of formation can generate antibody can be carried out schizogamy again, and the antibody generated can only identify one kind
Antigenic determinant, referred to as monoclonal antibody（Abbreviation monoclonal antibody）.Monoclonal antibody is mainly used in the prevention of disease, diagnoses and control at present
It treats, in the purifying of biological substance and the research of protein structure function.The market of therapeutic monoclonal antibody has in new drug development to be increased year by year
Long trend, the sales volume of monoclonal antibody in 1997 are 5,000,000,000 U.S. dollars, hereafter have big growth every year, until 2003 up to 50,000,000,000
U.S. dollar, it can be seen that monoclonal antibody technology is the nova risen in field of biotechnology.
The compound growth rate of 2000-2010, global monoclonal antibody drug market are up to 32%.2011, monoclonal was anti-
Body drug continues global drug market of taking the lead in race, and the market scale of global biopharmaceutical products crosses hundred million dollars, accounts for biopharmaceutical products
25%.There is data to show, accounting of the biopharmaceutical products a few years from now on world market will obviously rise.
There are mainly two types of the methods for largely preparing monoclonal antibody that laboratory is widely used both at home and abroad at present：First is that in vitro culture
Method, second is that working system in animal body.
Hybridoma cell line is not stringent wall dependent cells, therefore can both carry out monolayer cell culture, and can
To carry out suspension culture.The monolayer method of hybridoma is the most common means in each laboratory, i.e., by hybridoma
Cell is added in culture bottle, with the culture medium culture containing calf serum, hybridoma concentration 1.0 × 107A/ware, is then received
Collect culture supernatant, wherein monoclonal antibody content 0.3mg/ ware.
Since the monoclonal antibody amount of above method preparation is extremely limited, it is not suitable for the large-scale production of monoclonal antibody undoubtedly.To
Monoclonal antibody is largely prepared in vitro, must just carry out a large amount of High Density Cultivations of hybridoma, unit volume inner cell quantity is more,
The hybridoma time-to-live is longer, and the concentration of monoclonal antibody is higher, and yield is bigger.
In the Maitland culture of hybridoma, if the small-scale culture that suspends, mostly uses rolling bottle to be cultivated, passes through
Stirring makes cell in suspended state；And the culture that suspends on a large scale, then it mostly uses the bioreactor of fermentation type to be cultivated, trains
Feeding mode can be divided into pure batch experiments and stream plus formula culture.Since the decline of pure batch experiments Cell viability is fast, lead to antibody production
It is not high, therefore apply less.Stream plus formula culture, since the time-to-live of cell can be extended by flow feeding culture medium, significantly
Improve antibody production.But the high-quality but price of existing supplemented medium foreign vendor is high, increases production cost；State
Interior producer's feed-batch culture based formulas is unstable, there are difference between batch, antibody-secreting amount is caused to decline after use, being unable to meet production needs
It asks.
Summary of the invention
That the purpose of the present invention is to provide a kind of differences between batches is small, quality is consistent for Hybridoma Cell Culture
Supplemented medium, the present invention also provides the preparation methods of the supplemented medium.
To achieve the above object, the present invention can take following technical proposals：
Supplemented medium of the present invention for Hybridoma Cell Culture, is wrapped in the water for injection of 1000ml unit volume
Containing following components：Glucose 10g, glutamine 3g, ox blood albumin 60g, valine 52mg, 54 mg of isoleucine, benzene
35 mg of alanine, 59 mg of leucine, 9 mg of tryptophan, 53 mg of threonine, 17 mg of methionine, 91 mg of lysine.
Preparation method of the present invention for the supplemented medium of Hybridoma Cell Culture includes the following steps：
800ml water for injection is heated to 30 ~ 35 DEG C by the first step in beaker, and by valine, isoleucine, phenylalanine is bright
Propylhomoserin, tryptophan, threonine, methionine, lysine are added in beaker successively, stirring while adding, are allowed to sufficiently dissolve；
Second step, after completely dissolution to raw materials all in the first step, then by glucose, glutamine, ox blood albumin successively add
Enter into beaker, it is stirring while adding, it is allowed to sufficiently dissolve；
Third step, the raw material to add in second step are settled to 1000ml after completely dissolution, by solution, filter through sterilizing filter
Afterwards, supplemented medium finished product is obtained.
The filtering accuracy of the sterilizing filter is 0.2 μm.
The advantage of the invention is that raw material proportioning is scientific, preparation method is simple.It is white using glucose, glutamine, ox blood
The formula of albumen and Amino acid profile supplemented medium, wherein carbon source needed for wherein glucose mainly provides cell production, paddy
Glutamine can not only be used for the important composition that carbon source is also nucleosides synthesis, and amino acid is the required raw material of protein synthesis.The present invention
The supplemented medium of preparation overcomes difference between batch existing for existing supplemented medium, has reached stationary flow and has added culture process, stablizes
Improve the purpose of antibody production.When using stream plus formula cultural method, as supplemented medium, after inoculated and cultured 72 hours, warp
The antibody production of purifying is crossed not less than 300mg/L, and process stabilizing, antibody production are stablized.
Specific embodiment
More detailed explanation is done to the present invention below by specific embodiment.
Supplemented medium of the present invention for Hybridoma Cell Culture, in the water for injection of 1000ml unit volume
In include following components：Glucose（It analyzes pure）10g, glutamine（Biochemical reagents）3g, ox blood albumin（Biochemical reagents）
60g, valine（Biochemical reagents）52mg, isoleucine（Biochemical reagents）54 mg, phenylalanine（Biochemical reagents）35 mg, it is bright
Propylhomoserin（Biochemical reagents）59 mg, tryptophan（Biochemical reagents）9 mg, threonine（Biochemical reagents）53 mg, methionine（Biochemistry examination
Agent）17 mg, lysine（Biochemical reagents）91 mg.
When prepared by supplemented medium of the invention, preparation condition and equipment：Ambient pressure is an atmospheric pressure, environment temperature
For room temperature.
The following raw materials of precise according to the following ratio：Glucose 10g, glutamine 3g, ox blood albumin 60g, valine
52mg, 54 mg of isoleucine, 5 mg of phenylalanine-3,4-quinone, 59 mg of leucine, 9 mg of tryptophan, 53 mg of threonine, methionine 17
Mg, 91 mg of lysine；Water for injection 1000ml is spare.
Preparation method is as follows：
800ml water for injection is poured into beaker be heated to 30 ~ 35 DEG C first by the first step, by valine, isoleucine, phenylpropyl alcohol
Propylhomoserin, leucine, tryptophan, threonine, methionine, lysine are added in beaker successively, stirring while adding, and raw material all adds
Continue stir about 10min after entering, is allowed to sufficiently dissolve, liquid is bright, free from admixture；
Second step, after completely dissolution to raw materials all in the first step, then by glucose, glutamine, ox blood albumin successively add
Enter into beaker, stirring while adding, raw material continues stir about 10min after being all added, and is allowed to sufficiently dissolve, liquid is bright, nothing
Impurity；
Third step, the raw material to add in second step are settled to 1000ml after completely dissolution, by solution, through 0.2 μm of degerming
After filter filtering, supplemented medium finished product is obtained, 4 DEG C, be sealed.
In use, the 5% of addition volume of culture, addition in every 24 hours once, after Cell viability is down to 20% or less, stop every time
It only adds, and collects culture solution.
Three kinds of supplemented mediums are made by oneself using import, the domestic, embodiment of the present invention separately below, and stream is carried out on shaking table and is added
Formula culture contrast verification.It spends 48 hours and cultivates, carry out 1000rpm, 10min centrifugation and collect supernatant, use affinity chromatography column purification
Antibody, and antibody content is detected using A280, it the results are shown in Table 1.
Table 1
Conclusion：It can be seen that from the result of three of the above feed-batch culture culture compared to negative control, antibody production is all mentioned
It rises.The homemade yield of the embodiment of the present invention is higher than domestic 64.5%, is lower than import about 7.3%.Import is optimal in terms of yield, but from
The comprehensive aspect such as cost, yield, technological progress is considered, and the homemade supplemented medium of the present invention is with greater advantage.
Claims (3)
1. a kind of supplemented medium for Hybridoma Cell Culture, it is characterised in that：In the injection of 1000ml unit volume
It include following components in water：Glucose 10g, glutamine 3g, ox blood albumin 60g, valine 52mg, isoleucine 54
Mg, 5 mg of phenylalanine-3,4-quinone, 59 mg of leucine, 9 mg of tryptophan, 53 mg of threonine, 17 mg of methionine, 91 mg of lysine.
2. the preparation method described in claim 1 for the supplemented medium of Hybridoma Cell Culture, it is characterised in that：Including under
State step：
800ml water for injection is heated to 30 ~ 35 DEG C by the first step in beaker, and by valine, isoleucine, phenylalanine is bright
Propylhomoserin, tryptophan, threonine, methionine, lysine are added in beaker successively, stirring while adding, are allowed to sufficiently dissolve；
Second step, after completely dissolution to raw materials all in the first step, then by glucose, glutamine, ox blood albumin successively add
Enter into beaker, it is stirring while adding, it is allowed to sufficiently dissolve；
Third step, the raw material to add in second step are settled to 1000ml after completely dissolution, by solution, filter through sterilizing filter
Afterwards, supplemented medium finished product is obtained.
3. the preparation method for the supplemented medium of Hybridoma Cell Culture according to claim 2, it is characterised in that：Institute
The filtering accuracy for stating sterilizing filter is 0.2 μm.
Priority Applications (1)
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| CN201810818028.4A CN108823173A (en) | 2018-07-24 | 2018-07-24 | Supplemented medium and preparation method thereof for Hybridoma Cell Culture |
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| CN201810818028.4A CN108823173A (en) | 2018-07-24 | 2018-07-24 | Supplemented medium and preparation method thereof for Hybridoma Cell Culture |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6048728A (en) * | 1988-09-23 | 2000-04-11 | Chiron Corporation | Cell culture medium for enhanced cell growth, culture longevity, and product expression |
| CN1311257A (en) * | 2000-03-02 | 2001-09-05 | 上海复旦张江生物医药有限公司 | Double specific monoclonal anti-body, its preparing method and thromobolysin made therefrom |
| CN1772883A (en) * | 2005-10-10 | 2006-05-17 | 华东理工大学 | Animal cell feeding culture process |
| CN101535469A (en) * | 2006-11-08 | 2009-09-16 | 惠氏公司 | Rationally designed media for cell culture |
| CN104812891A (en) * | 2012-11-14 | 2015-07-29 | 默克专利有限公司 | Cell culture media |
-
2018
- 2018-07-24 CN CN201810818028.4A patent/CN108823173A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6048728A (en) * | 1988-09-23 | 2000-04-11 | Chiron Corporation | Cell culture medium for enhanced cell growth, culture longevity, and product expression |
| CN1311257A (en) * | 2000-03-02 | 2001-09-05 | 上海复旦张江生物医药有限公司 | Double specific monoclonal anti-body, its preparing method and thromobolysin made therefrom |
| CN1772883A (en) * | 2005-10-10 | 2006-05-17 | 华东理工大学 | Animal cell feeding culture process |
| CN101535469A (en) * | 2006-11-08 | 2009-09-16 | 惠氏公司 | Rationally designed media for cell culture |
| CN104812891A (en) * | 2012-11-14 | 2015-07-29 | 默克专利有限公司 | Cell culture media |
Non-Patent Citations (4)
| Title |
|---|
| WEICHANG ZHOU ET AL.: ""Alteration of mammalian cell metabolism by dynamic nutrient feeding"", 《CYTOTECHNOLOGY》 * |
| WEICHANG ZHOU ET AL.: ""Fed-Batch Culture of Recombinant NS0 Myeloma Cells with High Monoclonal Antibody Production"", 《BIOTECHNOLOGY AND BIOENGINEERING》 * |
| 文道源 等: "《羊膜建库及在眼科临床的应用》", 31 December 2006, 江西高校出版社 * |
| 骆海燕 等: ""动物细胞流加培养工艺研究进展"", 《中国畜牧兽医》 * |
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Application publication date: 20181116 |