CN116731956A - Culture medium of mammalian cells, culture method thereof and preparation method of antibody - Google Patents

Culture medium of mammalian cells, culture method thereof and preparation method of antibody Download PDF

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CN116731956A
CN116731956A CN202310853168.6A CN202310853168A CN116731956A CN 116731956 A CN116731956 A CN 116731956A CN 202310853168 A CN202310853168 A CN 202310853168A CN 116731956 A CN116731956 A CN 116731956A
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medium
cell
feed medium
culture
sodium
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李娅娅
鲍勇
田晓平
赵巧辉
李桂林
付光宇
吴学炜
杨增利
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Zhengzhou Immuno Biotech Co Ltd
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Zhengzhou Immuno Biotech Co Ltd
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Abstract

The invention relates to the technical field of cell culture, in particular to a mammalian cell culture method and a preparation method of an antibody. The invention adopts a specific feed supplement adding method in the high-density inoculation process, so that the cell density and the antibody expression level are finally and effectively improved, and compared with the traditional process, the cell density is improved by 20 percent, and the antibody expression level is improved by more than 30 percent.

Description

Culture medium of mammalian cells, culture method thereof and preparation method of antibody
Technical Field
The invention relates to the technical field of cell culture, in particular to a culture medium of mammalian cells, a culture method thereof and a preparation method of antibodies.
Background
The commercial application of recombinant antibodies is becoming more and more widespread, and it is extremely important to reduce the production cost of antibodies for their commercial application. Among factors affecting the production cost of antibodies, the expression level is one of the most important factors, and the improvement of the productivity of cell lines can significantly reduce the cost. The method for improving the expression quantity mainly comprises high-expression clone screening, cell strain transformation, carrier transformation, culture medium optimization, culture process optimization and the like, wherein the transformation period of the cell strain and the carrier is generally more than 6 months, and the improvement of the expression quantity through the culture medium and the culture process optimization with shorter and more flexible period is important.
The development of cell culture process has been carried out for many years, from low-density inoculation culture to high-density inoculation culture, and from batch culture mode and feeding flow to continuous flow culture mode, especially the domestic biopharmaceutical industry is in the rising stage, and there is still much room for improvement in the research of the culture process.
High density inoculation culture means cultivation at a higher density (generally higher than 5X 10) 5 cells/ml), and then carrying out fed-batch culture, the method can lead the cells to start a culture period in the most metabolizing state and reach the highest cell density in the shortest time, thereby shortening the culture period and improving the yield of target proteins. However, this process is not universal, and some cells undergo rapid apoptosis after high-density inoculation reaches the highest cell density, and it is difficult to maintain high density for protein production, which in turn leads to a significant reduction in final yield. How to promote cell proliferation to reach the platform as soon as possible after high-density inoculation and how to maintain cells on the platform for longer protein expression are key to developing the process. In addition, the current high-density inoculation is realized by centrifuging and liquid-changing after N-1 seed large-volume culture or N-1 perfusion culture, the two modes can obviously improve the production cost during commercial production, and the realization difficulty of the centrifuging or perfusion process is high.
Disclosure of Invention
In view of this, the present invention provides a culture medium for mammalian cells, a method for culturing the same, and a method for producing antibodies. The culture medium and the culture method can effectively improve the cell density and the antibody yield, and compared with the traditional process, the cell density is improved by 20 percent, and the antibody expression quantity is improved by more than 30 percent.
In order to achieve the above object, the present invention provides the following technical solutions:
a feed medium A for high-density cell culture, which comprises water and the following components:
1410-141100 mg/L of amino acid, 100-1000mg/L of magnesium sulfate, 100-1000mg/L of calcium chloride, 1000-5000mg/L of potassium chloride, 1000-5000mg/L of choline chloride, 1000-5000mg/L of inositol, 1000-5000mg/L of sodium pyruvate, 1000-5000mg/L, poloxamer 188 1000-5000mg/L of sodium dihydrogen phosphate, 50-100g/L of glucose, 10-100mg/L of riboflavin and vitamin B12
10-100mg/L, 1-10mg/L glutathione, 1-10mg/L folic acid, 100-500mg/L ferric citrate, 1-10mg/L biotin, 1-10mg/L lipoic acid, 1-10mg/L thiamine hydrochloride, 1-10mg/L nicotinamide, 1-10mg/L ethanolamine hydrochloride, 1-10mg/L pyridoxine hydrochloride, 1-10 mg/L4-aminobenzoic acid, 1-10mg/L sodium linoleate, 1-10mg/L, D-calcium pantothenate, 1-10mg/L putrescine hydrochloride, 1-10mg/L sodium selenite, 1-10mg/L linolenic acid, 0.1-10mg/L cupric sulfate pentahydrate, 1-10mg/L cobalt chloride, 1-10mg/L manganese chloride tetrahydrate, 1-10mg/L nickel chloride, 1-10mg/L sodium metasilicate, 1-10mg/L ammonium metavanadate and 1-10mg/L ammonium molybdate.
In some embodiments, the feed medium A comprises feed medium A1 and feed medium A2, wherein,
the feed medium A1 consists of water and the following components:
l-methionine 1286mg/L, L-phenylalanine 4652mg/L, L-asparagine 10800mg/L,
L-proline 4091mg/L, L-lysine 5363mg/L, L-leucine 8600mg/L,
9200mg/L, L-serine 6600mg/L, L-threonine 7448mg/L, L-valine 6030mg/L, L-isoleucine 6664mg/L, L-glutamic acid 5851mg/L,
L-arginine 7292mg/L, L-hydroxyproline 200mg/L, L-histidine 2379mg/L,
Glycine 150mg/L, magnesium sulfate 20mg/L, calcium chloride 232mg/L,
650mg/L potassium chloride, 700mg/L choline chloride, 2000mg/L inositol,
1600mg/L, poloxamer 188 1000mg/L sodium pyruvate, 1200mg/L sodium dihydrogen phosphate,
D-glucose 80000mg/L, riboflavin 10mg/L, vitamin B12 mg/L,
50mg/L, L-reduced glutathione 3mg/L, folic acid 8mg/L,
Biotin 2mg/L, (+ -.) -alpha-lipoic acid 1100mg/L, thiamine hydrochloride 1mg/L,
2mg/L of ethanolamine hydrochloride, 1mg/L of pyridoxine hydrochloride, 1mg/L of 4-aminobenzoic acid,
Linoleic acid sodium salt 3mg/L, D-pantothenic acid hemicalcium salt 2mg/L, putrescine hydrochloride 0.1mg/L,
0.4mg/L nicotinamide, 0.5mg/L sodium selenite, 0.0001mg/L cobalt chloride hexahydrate,
Manganese chloride tetrahydrate 0.00001mg/L, nickel chloride hexahydrate 0.00006mg/L, sodium metasilicate 0.013mg/L, ammonium metavanadate 0.05mg/L, ammonium molybdate tetrahydrate 0.00001mg/L and copper sulfate pentahydrate 0.1mg/L;
the feed medium A2 consists of water and the following components:
l-methionine 1286mg/L, L-phenylalanine 4652mg/L, L-asparagine 8800mg/L, L-proline 4091mg/L, L-lysine 5363mg/L, L-leucine 7600mg/L,
9200mg/L, L-serine 6600mg/L, L-threonine 7448mg/L, L-valine 6030mg/L, L-isoleucine 6664mg/L, L-glutamic acid 5851mg/L,
L-arginine 7292mg/L, L-hydroxyproline 200mg/L, L-histidine 2379mg/L,
Glycine 150mg/L, magnesium sulfate 20mg/L, calcium chloride 232mg/L,
650mg/L potassium chloride, 700mg/L choline chloride, 2000mg/L inositol,
1600mg/L, poloxamer 188 1000mg/L sodium pyruvate, 1200mg/L, D-glucose 80000mg/L sodium dihydrogen phosphate, 10mg/L riboflavin, 12 mg/L vitamin B,
50mg/L, L-reduced glutathione 3mg/L, folic acid 8mg/L,
Biotin 2mg/L, (+ -.) -alpha-lipoic acid 1100mg/L, thiamine hydrochloride 1mg/L,
2mg/L of ethanolamine hydrochloride, 1mg/L of pyridoxine hydrochloride, 1mg/L of 4-aminobenzoic acid,
Linoleic acid sodium salt 3mg/L, D-pantothenic acid hemicalcium salt 2mg/L, putrescine hydrochloride 0.1mg/L,
0.4mg/L nicotinamide, 0.5mg/L sodium selenite, 0.0001mg/L cobalt chloride hexahydrate,
Manganese chloride tetrahydrate 0.00001mg/L, nickel chloride hexahydrate 0.00006mg/L, sodium metasilicate 0.013mg/L, ammonium metavanadate 0.05mg/L, ammonium molybdate tetrahydrate 0.00001mg/L and copper sulfate pentahydrate 0.1mg/L.
The invention also provides a culture medium combination for high-density cell culture, which comprises a Dynamis culture medium, a feed medium A and a feed medium B as described above; the feed medium B is a Cell boost7B medium.
Research results show that the feed medium A provided by the invention has reasonable composition, and is used for high-density culture of cells, thereby being beneficial to improving cell density and antibody yield. The feed medium A of the invention is purposefully improved and optimized according to the problems in cell culture, and the components and the concentration are properly matched. Early experiments show that the replacement, addition, deletion or concentration adjustment of each component in the feed medium can bring adverse effects to the culture effect. The culture medium provided by the invention can be matched with the culture method provided by the invention, so that the cell density and the antibody yield can be effectively improved, and compared with the traditional process, the cell density is improved by 20%, and the antibody expression quantity is improved by more than 30%.
The invention also provides the application of the feed medium or the culture medium combination in high-density culture of mammalian cells or in preparation of antibodies by culturing the mammalian cells.
In some embodiments, the mammalian cell is a CHO cell secreting an antibody. Wherein, the antibody can be monoclonal antibody or polyclonal antibody, specific types of the monoclonal antibody and the polyclonal antibody are not particularly required, and a person skilled in the art can construct CHO cells secreting specific antibodies according to a conventional method in the art according to actual needs for culturing.
The invention also provides a high-density cell culture method, which utilizes the culture medium combination to culture cells.
Specifically, the culture method of the invention comprises the following steps:
a: resuscitating the cells, and then inoculating the cells to Dynamis for culture to obtain seed liquid;
b: and (3) carrying out amplification culture on the seed solution, and adding the feed medium A and the feed medium B on the 2 nd to 14 th days of the amplification culture.
In some embodiments, the medium used for resuscitation is CD Forti medium, and the density after resuscitation is 5X 10 5 The method for resuscitation comprises the following steps of: resuscitating the culture system at a rotation speed of 100rpm and CO 2 The cells were incubated in a shaker at a concentration of 8% (volume percent) at 37.0deg.C for 3 days.
In some embodiments, the addition feed medium comprises:
feed medium a1 2% and Cell boost7b medium 0.2% were added on day 2;
feed medium a1 2% and Cell boost7b medium 0.2% were added on day 3;
feed medium a1 3% and Cell boost7b medium 0.3% were added on day 4;
feed medium a2 3% and Cell boost7b medium 0.3% were added on day 5;
feed medium a2 3% and Cell boost7b medium 0.3% were added on day 6;
feed medium a2 4% and Cell boost7b medium 0.4% were added on day 8;
feed medium a2 3% and Cell boost7b medium 0.3% were added on day 10;
feed medium a2 2% and Cell boost7b medium 0.2% were added on day 12;
feed medium a2 2% and Cell boost7b medium 0.2% were added on day 14.
In some embodiments, the feed medium a added in step b1 is the feed medium A1 mentioned above, the feed medium a added in step b2 is the feed medium A2 mentioned above, the feed medium A2 added in step b2 has an asparagine concentration reduced by 2000mg/L and a leucine concentration reduced by 1000mg/L as compared to the feed medium A1 added in step b1, i.e. the asparagine concentration in the feed medium A1 is 10800mg/L and the leucine concentration is 8600mg/L; in the feed medium A2, the concentration of asparagine was 8800mg/L and the concentration of leucine was 7600mg/L.
In some embodiments, in steps b1 and b2, feed medium a is added in a volume percentage of the scaled-up culture system. In some embodiments, the cell density after primary culture is 80-120X 10 5 cells/ml, the cell inoculum size of the amplification culture is 40-50×10 5 cells/ml。
In the method for high-density cell culture provided by the invention, when the cell activity rate is reduced to 70%, the amplification culture is ended.
Compared with the prior art, the invention has the following beneficial effects:
(1) The high-density inoculation is combined with different feeding strategies, and the process can ensure that higher protein yield can be obtained in the same production period, and the yield is improved by more than 30 percent;
(2) The inoculation method can improve the cell density by more than 20%;
(3) The different culture stages are added with different feed supplements, so that the proliferation of early cells can be promoted, the maintenance time of later cells can be prolonged, and the cells can be more suitable for a high-density inoculation process, thereby improving the yield.
(4) High-density inoculation is realized without centrifugation and perfusion liquid exchange, so that the method is convenient to amplify to a production scale, and is simple to operate and low in cost.
Detailed Description
The invention provides a culture medium of mammalian cells, a culture method thereof and a preparation method of antibodies. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The chinese hamster ovary cell line CHO in the examples of the invention was derived from Thermo and expressed IgG monoclonal antibodies against IgM.
The basic culture medium used in the invention is Dynamis TM Culture medium, feed medium a included A1 and A2, feed medium B was Cell boost7B (purchased from Hyclone); the medium used to resuscitate the cells was CD Forti (purchased from Gibco).
All biochemical reagents used in the present invention were purchased from Sigma.
The feed medium A1 comprises the following components:
l-methionine 1286mg/L, L-phenylalanine 4652mg/L, L-asparagine 10800mg/L,
L-proline 4091mg/L, L-lysine 5363mg/L, L-leucine 8600mg/L,
9200mg/L, L-serine 6600mg/L, L-threonine 7448 mg/L-aspartic acid,
L-valine 6030mg/L, L-isoleucine 6664mg/L, L-glutamic acid 5851mg/L,
L-arginine 7292mg/L, L-hydroxyproline 200mg/L, L-histidine 2379mg/L,
Glycine 150mg/L, magnesium sulfate 20mg/L, calcium chloride 232mg/L,
650mg/L potassium chloride, 700mg/L choline chloride, 2000mg/L inositol,
1600mg/L, poloxamer 188 1000mg/L sodium pyruvate, 1200mg/L sodium dihydrogen phosphate,
D-glucose 80000mg/L, riboflavin 10mg/L, vitamin B12 mg/L,
50mg/L, L-reduced glutathione 3mg/L, folic acid 8mg/L,
Biotin 2mg/L, (+ -.) -alpha-lipoic acid 1100mg/L, thiamine hydrochloride 1mg/L,
2mg/L of ethanolamine hydrochloride, 1mg/L of pyridoxine hydrochloride, 1mg/L of 4-aminobenzoic acid,
Linoleic acid sodium salt 3mg/L, D-pantothenic acid hemicalcium salt 2mg/L, putrescine hydrochloride 0.1mg/L,
0.4mg/L nicotinamide, 0.5mg/L sodium selenite, 0.0001mg/L cobalt chloride hexahydrate,
Manganese chloride tetrahydrate 0.00001mg/L, nickel chloride hexahydrate 0.00006mg/L, sodium metasilicate 0.013mg/L,
Ammonium metavanadate 0.05mg/L, ammonium molybdate tetrahydrate 0.00001mg/L and cupric sulfate pentahydrate 0.1mg/L. The feed medium A2 comprises the following components:
l-methionine 1286mg/L, L-phenylalanine 4652mg/L, L-asparagine 8800mg/L,
L-proline 4091mg/L, L-lysine 5363mg/L, L-leucine 7600mg/L,
9200mg/L, L-serine 6600mg/L, L-threonine 7448 mg/L-aspartic acid,
L-valine 6030mg/L, L-isoleucine 6664mg/L, L-glutamic acid 5851mg/L,
L-arginine 7292mg/L, L-hydroxyproline 200mg/L, L-histidine 2379mg/L,
Glycine 150mg/L, magnesium sulfate 20mg/L, calcium chloride 232mg/L,
650mg/L potassium chloride, 700mg/L choline chloride, 2000mg/L inositol,
1600mg/L, poloxamer 188 1000mg/L sodium pyruvate, 1200mg/L sodium dihydrogen phosphate,
D-glucose 80000mg/L, riboflavin 10mg/L, vitamin B12 mg/L,
50mg/L, L-reduced glutathione 3mg/L, folic acid 8mg/L,
Biotin 2mg/L, (+ -.) -alpha-lipoic acid 1100mg/L, thiamine hydrochloride 1mg/L,
2mg/L of ethanolamine hydrochloride, 1mg/L of pyridoxine hydrochloride, 1mg/L of 4-aminobenzoic acid,
Linoleic acid sodium salt 3mg/L, D-pantothenic acid hemicalcium salt 2mg/L, putrescine hydrochloride 0.1mg/L,
0.4mg/L nicotinamide, 0.5mg/L sodium selenite, 0.0001mg/L cobalt chloride hexahydrate,
Manganese chloride tetrahydrate 0.00001mg/L, nickel chloride hexahydrate 0.00006mg/L, sodium metasilicate 0.013mg/L,
Ammonium metavanadate 0.05mg/L, ammonium molybdate tetrahydrate 0.00001mg/L and cupric sulfate pentahydrate 0.1mg/L.
The invention is further illustrated by the following examples:
example 1
CHO monoclonal antibodies expressing mab a (CHO from Thermo, constructed by ambam biology) were taken from a cell bank and rapidly thawed by rapid shaking in a 37 ℃ water bath for 3 minutes or less. CD Forti CHO was used for resuscitation TM Culture medium (containing 200nM MTX), recovery density 5X 10 5 cell/mL, placed at a speed of 100rpm, CO 2 Culturing in shaking table at 37.0deg.C for 3 days until the density reaches 25X10 5 At the time of cells/mL, passage expansion was started, and the inoculation density was 5X 10 5 cells/mL。
Feed medium A2 and feed medium A1 were prepared, all media were sterilized by filtration and stored at 4 ℃.
The low-density group, the control high-density group and the high-density group of the invention are arranged, wherein the control high-density group is divided into control groups 1-3 according to the different culture modes (without changing liquid and centrifuging), and the specific culture method comprises the following steps (see table 1):
low density inoculation: CHO cells resuscitated and expanded in CD Forti (containing 200nM MTX) were centrifuged at 5X 10 5 Inoculating cells/mL into Dynamis culture medium, and placing at 100rpm and CO 2 Culturing in shaking table at 37.0deg.C at concentration of 8% (volume) to reach cell density of 20X10 5 At a cell/mL ratio of 5X 10 5 cells/mL were inoculated into fresh Dynamis medium. And (4) starting to add a feeding material on the 4 th day, wherein the feeding material procedure is as follows:
day 4: feed medium a1 5%, cell boost7b medium 0.25%;
day 6: feed medium a1 4%, cell boost7b medium 0.4%;
day 8: feed medium a1 7%, cell boost7b medium 0.7%;
day 10: feed medium a1 4%, cell boost7b medium 0.4%;
day 12: feed medium a1 3%, cell boost7b medium 0.3%;
day 14: feed medium a1 3%, cell boost7b medium 0.3%.
Sugar was fed to 6g/L daily. When the cell viability was reduced to 70%, the culture was terminated and the supernatant was collected by centrifugation.
High-density inoculation:
control group 1:
CHO cells resuscitated and expanded in CD Forti (containing 200nM MTX) were centrifuged at 5X 10 5 Inoculating cells/mL into dynamics, and placing at 100rpm and CO 2 Culturing in shaking table at 37.0deg.C at concentration of 8% (volume) to obtain cell density of 100deg.C 10 5 Fresh Dynamis was added without centrifugation (i.e., without changing the liquid) at cells/mL TM The culture medium is grown to a density of 40×10 5 The feed is added from the cell/mL day 2, and the feed procedure is as follows:
feed medium a1 2% and Cell boost7b medium 0.2% were added on day 2;
feed medium a1 2% and Cell boost7b medium 0.2% were added on day 3;
feed medium a1 3% and Cell boost7b medium 0.3% were added on day 4;
feed medium a1 3% and Cell boost7b medium 0.3% were added on day 5;
feed medium a1 3% and Cell boost7b medium 0.3% were added on day 6;
feed medium a1 4% and Cell boost7b medium 0.4% were added on day 8;
feed medium a1 3% and Cell boost7b medium 0.3% were added on day 10;
feed medium a1 2% and Cell boost7b medium 0.2% were added on day 12;
feed medium a1 2% and Cell boost7b medium 0.2% were added on day 14.
Sugar was fed to 6g/L daily. When the cell viability was reduced to 70%, the culture was terminated and the supernatant was collected by centrifugation.
Control group 2:
CHO cells resuscitated and expanded in CD Forti (containing 200nM MTX) were centrifuged at 5X 10 5 Inoculating cells/mL into dynamics, and placing at 100rpm and CO 2 Culturing in shaking table at 37.0deg.C at concentration of 8% (volume) to obtain cell density of 100deg.C 10 5 At cells/mL, after centrifugation to remove the old medium, fresh Dynamics medium was added at 40X 10 5 Inoculating cells/mL, and starting to add feed on the 2 nd day, wherein the feed procedure is as follows:
feed medium a1 2% and Cell boost7b medium 0.2% were added on day 2;
feed medium a1 2% and Cell boost7b medium 0.2% were added on day 3;
feed medium a1 3% and Cell boost7b medium 0.3% were added on day 4;
feed medium a1 3% and Cell boost7b medium 0.3% were added on day 5;
feed medium a1 3% and Cell boost7b medium 0.3% were added on day 6;
feed medium a1 4% and Cell boost7b medium 0.4% were added on day 8;
feed medium a1 3% and Cell boost7b medium 0.3% were added on day 10;
feed medium a1 2% and Cell boost7b medium 0.2% were added on day 12;
feed medium a1 2% and Cell boost7b medium 0.2% were added on day 14.
Control group 3:
CHO cells resuscitated and expanded in CD Forti (containing 200nM MTX) were centrifuged at 5X 10 5 Inoculating cells/mL into dynamics, and placing at 100rpm and CO 2 Culturing in shaking table at 37.0deg.C at concentration of 8% (by volume), and sealing cellsThe degree reaches 100 multiplied by 10 5 At cells/mL, after centrifugation to remove the old medium, fresh Dynamis medium was added to 100X 10 5 Inoculating cells/mL, and starting to add feed on the 2 nd day, wherein the feed procedure is as follows:
feed medium a1 2% and Cell boost7b medium 0.2% were added on day 2;
feed medium a1 2% and Cell boost7b medium 0.2% were added on day 3;
feed medium a1 3% and Cell boost7b medium 0.3% were added on day 4;
feed medium a1 3% and Cell boost7b medium 0.3% were added on day 5;
feed medium a1 3% and Cell boost7b medium 0.3% were added on day 6;
feed medium a1 4% and Cell boost7b medium 0.4% were added on day 8;
feed medium a1 3% and Cell boost7b medium 0.3% were added on day 10;
feed medium a1 2% and Cell boost7b medium 0.2% were added on day 12;
feed medium a1 2% and Cell boost7b medium 0.2% were added on day 14.
Experimental group
CHO cells resuscitated and expanded in CD Forti (containing 200nM MTX) were centrifuged at 5X 10 5 Inoculating cells/mL into dynamics, and placing at 100rpm and CO 2 Culturing in shaking table at 37.0deg.C at concentration of 8% (volume) to obtain cell density of 100deg.C 10 5 Without centrifugation (i.e., without changing the liquid) at cells/mL, fresh Dynamis medium was added to a density of 40X 10 5 The cells/mL was fed starting on day 2 with the following feed procedure:
day 2: feed medium a1 2%, cell boost7b medium 0.2%;
day 3: feed medium a1 2%, cell boost7b medium 0.2%;
day 4: feed medium a1 3%, cell boost7b medium 0.3%;
day 5: feed medium a2 3%, cell boost7b medium 0.3%;
day 6: feed medium a2 3%, cell boost7b medium 0.3%;
day 8: feed medium a2 4%, cell boost7b medium 0.4%;
day 10: feed medium a2 3%, cell boost7b medium 0.3%;
day 12: feed medium a2 2%, cell boost7b medium 0.2%;
day 14: feed medium a2 2%, cell boost7b medium 0.2%;
day 16: feed medium a2 2%, cell boost7b medium 0.2%.
Sugar was fed to 6g/L daily. When the cell viability was reduced to 70%, the culture was terminated and the supernatant was collected by centrifugation.
The antibody expression level was quantitatively measured by HPLC, and the results are shown in Table 1.
TABLE 1 antibody expression level
The result shows that the invention realizes high-density inoculation under the conditions of not replacing the old culture medium and not using centrifugation and perfusion, and the cell density can be effectively improved by more than 20%; in the high-density culture process, the maintenance time of cells can be prolonged by adding proper feed, and the antibody expression quantity of a relatively low-density inoculation group can be improved by 35 percent.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (9)

1. A feed medium A for high-density cell culture, which is characterized by comprising water and the following components:
1410-141100 mg/L of amino acid, 100-1000mg/L of magnesium sulfate, 100-1000mg/L of calcium chloride, 1000-5000mg/L of potassium chloride, 1000-5000mg/L of choline chloride, 1000-5000mg/L of inositol, 1000-5000mg/L of sodium pyruvate, 1000-5000mg/L of sodium dihydrogen phosphate, 50-100g/L of glucose, 10-100mg/L of riboflavin, 1210-100mg/L of vitamin B, 1-10mg/L of glutathione, 1-10mg/L of folic acid, 100-500mg/L of ferric citrate, 1-10mg/L of biotin, 1-10mg/L of lipoic acid, 1-10mg/L of thiamine hydrochloride, 1-10mg/L of nicotinamide, 1-10mg/L of ethanolamine hydrochloride, 1-10mg/L of pyridoxine hydrochloride, 1-10mg/L of 4-aminobenzoic acid, 1-10mg/L, D-10 mg/L of sodium linoleate, 1-10mg/L of selenium amine hydrochloride, 1-10mg/L of sodium linoleate, 1-10mg/L of sodium metasilicate, 1-10mg/L of ammonium chloride, 1-10mg/L of sodium metasilicate chloride, 1-10mg/L of ammonium molybdate, 1-10mg/L of sodium metasilicate chloride, 1-10mg/L of aqueous solution, 1-10mg/L of sodium metasilicate chloride, 1-10mg/L of sodium metasilicate, 1-10mg/L of sodium chloride, 1-10mg/L of sodium metasilicate, 1-sodium chloride, 1-10mg/L, 10mg/L of sodium sulfate, and 10mg/L.
2. Feed medium A according to claim 1, characterized in that,
comprises a feed medium A1 and a feed medium A2, wherein,
the feed medium A1 consists of water and the following components:
l-methionine 1286mg/L, L-phenylalanine 4652mg/L, L-asparagine 10800mg/L,
L-proline 4091mg/L, L-lysine 5363mg/L, L-leucine 8600mg/L,
9200mg/L, L-serine 6600mg/L, L-threonine 7448 mg/L-aspartic acid,
L-valine 6030mg/L, L-isoleucine 6664mg/L, L-glutamic acid 5851mg/L,
L-arginine 7292mg/L, L-hydroxyproline 200mg/L, L-histidine 2379mg/L,
Glycine 150mg/L, magnesium sulfate 20mg/L, calcium chloride 232mg/L,
650mg/L potassium chloride, 700mg/L choline chloride, 2000mg/L inositol,
1600mg/L, poloxamer 188 1000mg/L sodium pyruvate, 1200mg/L sodium dihydrogen phosphate,
D-glucose 80000mg/L, riboflavin 10mg/L, vitamin B12 mg/L,
50mg/L, L-reduced glutathione 3mg/L, folic acid 8mg/L,
Biotin 2mg/L, (+ -.) -alpha-lipoic acid 1100mg/L, thiamine hydrochloride 1mg/L,
2mg/L of ethanolamine hydrochloride, 1mg/L of pyridoxine hydrochloride, 1mg/L of 4-aminobenzoic acid,
Linoleic acid sodium salt 3mg/L, D-pantothenic acid hemicalcium salt 2mg/L, putrescine hydrochloride 0.1mg/L,
0.4mg/L nicotinamide, 0.5mg/L sodium selenite, 0.0001mg/L cobalt chloride hexahydrate,
Manganese chloride tetrahydrate 0.00001mg/L, nickel chloride hexahydrate 0.00006mg/L, sodium metasilicate 0.013mg/L,
Ammonium metavanadate 0.05mg/L, ammonium molybdate tetrahydrate 0.00001mg/L and cupric sulfate pentahydrate 0.1mg/L;
the feed medium A2 consists of water and the following components:
l-methionine 1286mg/L, L-phenylalanine 4652mg/L, L-asparagine 8800mg/L,
L-proline 4091mg/L, L-lysine 5363mg/L, L-leucine 7600mg/L,
9200mg/L, L-serine 6600mg/L, L-threonine 7448 mg/L-aspartic acid,
L-valine 6030mg/L, L-isoleucine 6664mg/L, L-glutamic acid 5851mg/L,
L-arginine 7292mg/L, L-hydroxyproline 200mg/L, L-histidine 2379mg/L,
Glycine 150mg/L, magnesium sulfate 20mg/L, calcium chloride 232mg/L,
650mg/L potassium chloride, 700mg/L choline chloride, 2000mg/L inositol,
1600mg/L, poloxamer 188 1000mg/L sodium pyruvate, 1200mg/L sodium dihydrogen phosphate,
D-glucose 80000mg/L, riboflavin 10mg/L, vitamin B12 mg/L,
50mg/L, L-reduced glutathione 3mg/L, folic acid 8mg/L,
Biotin 2mg/L, (+ -.) -alpha-lipoic acid 1100mg/L, thiamine hydrochloride 1mg/L,
2mg/L of ethanolamine hydrochloride, 1mg/L of pyridoxine hydrochloride, 1mg/L of 4-aminobenzoic acid,
Linoleic acid sodium salt 3mg/L, D-pantothenic acid hemicalcium salt 2mg/L, putrescine hydrochloride 0.1mg/L,
0.4mg/L nicotinamide, 0.5mg/L sodium selenite, 0.0001mg/L cobalt chloride hexahydrate,
Manganese chloride tetrahydrate 0.00001mg/L, nickel chloride hexahydrate 0.00006mg/L, sodium metasilicate 0.013mg/L,
Ammonium metavanadate 0.05mg/L, ammonium molybdate tetrahydrate 0.00001mg/L and cupric sulfate pentahydrate 0.1mg/L.
3. A culture medium combination for high-density cell culture is characterized by comprising dynamos TM A culture medium, a feed medium a and a feed medium B according to claims 1-2;
the feed medium B is Cellboost 7B medium.
4. Use of the feed medium of any one of claims 1-2, or of the medium combination of claim 3, for high-density culture of mammalian cells, or for production of antibodies in culture of mammalian cells.
5. The use according to claim 4, wherein the mammalian cells are CHO cells secreting antibodies.
6. A method of high density cell culture wherein cells are cultured using the medium combination of claim 3.
7. The method of claim 6, wherein the culturing comprises the steps of:
a: resuscitating the cells, and then inoculating the cells to a Dynamis basic culture medium for culture to obtain seed liquid;
b: and (3) carrying out amplification culture on the seed solution, and adding the feed medium A and the feed medium B on the 2 nd to 14 th days of the amplification culture.
8. The method of claim 7, wherein the adding feed medium comprises:
feed medium a1 2% and Cell boost7b medium 0.2% were added on day 2;
feed medium a1 2% and Cell boost7b medium 0.2% were added on day 3;
feed medium a1 3% and Cell boost7b medium 0.3% were added on day 4;
feed medium a2 3% and Cell boost7b medium 0.3% were added on day 5;
feed medium a2 3% and Cell boost7b medium 0.3% were added on day 6;
feed medium a2 4% and Cell boost7b medium 0.4% were added on day 8;
feed medium a2 3% and Cell boost7b medium 0.3% were added on day 10;
feed medium a2 2% and Cell boost7b medium 0.2% were added on day 12;
feed medium a2 2% and Cell boost7b medium 0.2% were added on day 14.
9. The method according to claim 8, wherein the cell density after the culturing in step a is 80X 10 5 cells/ml-120×10 5 cells/ml; the cell inoculum size of the amplification culture in step b was 40X 10 5 cells/ml-50×10 5 cells/ml。
CN202310853168.6A 2023-07-12 2023-07-12 Culture medium of mammalian cells, culture method thereof and preparation method of antibody Pending CN116731956A (en)

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