CN114645010A

CN114645010A - Low-protein serum-free cell culture medium for producing monoclonal antibody and application thereof

Info

Publication number: CN114645010A
Application number: CN202011498943.3A
Authority: CN
Inventors: 於瑞敏; 陈玲; 吴倍熠
Original assignee: Nanjing Getein Biomedical Co ltd
Current assignee: Nanjing Getein Biomedical Co ltd
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2022-06-21

Abstract

The invention discloses a low-protein serum-free cell culture medium for producing monoclonal antibodies, which comprises a basic physiological buffering agent, bovine serum albumin, amino acid, vitamins, inorganic salt and lipid; the bovine serum albumin content is 10.0-20.0 mg/L; the inorganic salts are as follows: 5-15 mg/L of ferric citrate, 40-60 mg/L of sodium pyruvate, 1-5 mg/L of calcium chloride, 1-5 mg/L of magnesium sulfate, 4-10 mg/L of potassium chloride, 80-100 mg/L of sodium chloride, 100-200 mg/L of sodium dihydrogen phosphate and 4-10 mg/L of zinc sulfate. The highest cell density and antibody yield of the hybridoma cells cultured by the culture medium reach or even exceed the effect of a commercial culture medium, the production cost is greatly reduced, and the performance of the monoclonal antibody is stable.

Description

Low-protein serum-free cell culture medium for producing monoclonal antibody and application thereof

Technical Field

The invention relates to cell culture in the technical field of biology, in particular to a low-protein serum-free cell culture medium for producing monoclonal antibodies and application thereof.

Background

The cell culture is realized by simulating the growth environment in a cell body, and the in vitro culture technology of animal cells is widely applied to the research fields of physiological, biochemical and pharmacological activities of cells and tissues and the industrial production field of biological products such as antibodies, hormones, vaccines and the like. Wherein the cell culture medium is a vital part of the in vitro cell culture technology. Although the traditional serum-containing culture medium contains a large amount of components necessary for cell growth and proliferation such as various growth factors, plasma proteins, polypeptides, hormones and the like, the serum has the problems of large batch difference, potential pollution risk, undefined components, unfavorable separation and purification of target products, easy infection by viruses and mycoplasma and the like, and the serum-free culture medium has relatively definite components and simple preparation process, and can be widely applied in the field of modern biotechnology.

Serum-free medium is relatively clear in composition, consistent in quality and low in protein content, so that the stability of cell product production is improved, the cell product is easy to purify, and more importantly, different cells can be continuously cultured in high density in an environment which is most beneficial to growth or expression of a target product by optimizing the serum-free medium composition on the basis.

At present, commercial serum-free culture media mainly are imported and expensive, and few domestic serum-free culture medium manufacturers exist, and the prior literature also discloses a serum-free culture medium which is suitable for rapid growth of hybridoma cells and high in monoclonal antibody expression, for example, patent 200510030198.9 discloses a serum-free culture medium which is suitable for large-scale culture of various animal cells, but the serum-free culture medium contains 5g/L of insulin, 10g/L of transferrin and 100g/L of albumin, the protein content of the serum-free culture medium is too high to be beneficial to antibody purification, and patent 201811394132.1 discloses a low-protein serum-free cell culture medium which contains bovine serum albumin, transferrin and insulin, and although the protein content is very low, the serum-free culture medium still contains insulin and transferrin which have high cost and poor stability.

Disclosure of Invention

The invention aims to provide a low-protein serum-free cell culture medium for producing monoclonal antibodies, only one protein component of bovine serum albumin is added, separation and purification of a useful product are facilitated, and the quality of the monoclonal antibodies is improved; and simultaneously, the highest cell density and the antibody yield of the culture medium reach or even exceed the effects of a commercial culture medium.

The technical scheme adopted by the invention is as follows:

a low-protein serum-free cell culture medium for producing monoclonal antibodies, said medium comprising basic physiological buffer, bovine serum albumin, amino acids, vitamins, inorganic salts, lipids, trace elements, lipids and hormones;

the bovine serum albumin content is 10.0-20.0 mg/L;

the inorganic salts are as follows: 5-15 mg/L of ferric citrate, 40-60 mg/L of sodium pyruvate, 1-5 mg/L of calcium chloride, 1-5 mg/L of magnesium sulfate, 4-10 mg/L of potassium chloride, 80-100 mg/L of sodium chloride, 100-200 mg/L of sodium dihydrogen phosphate and 4-10 mg/L of zinc sulfate.

Preferably, the culture medium further comprises trace elements

Preferably, the culture medium further comprises a buffer.

Preferably, the culture medium further comprises an anti-shear force protective agent.

Specifically, the serum-free culture medium takes DMEM/F12(v/v, 0.8-1.2:0.8-1.2) as a basic culture medium, and the following components are added:

bovine serum albumin Bovine serum albumin 10.0-20.0 mg/L

Arginine L-Arginine 36-58 mg/L

Cystine L-cysteine 8-12 mg/L

Histidine L-Histidine 9-15 mg/L

Isoleucine L-Isoleucine 13-23 mg/L

Leucine L-Leucine 13-23 mg/L

Lysine L-Lysine 14-22 mg/L

Methionine L-Methionine 4-7 mg/L

Phenylalanine L-phenylalkanine 9-15 mg/L

Threonine L-Threonine 18-22 mg/L

Tryptophan L-Tryptophan 3-5 mg/L

Tyrosine L-Tyrosine 9-16 mg/L

Valine L-Valine 18-22 mg/L

Glycine Glycine 4-7 mg/L

Alanine L-Alanine 5-9 mg/L

Asparagine L-Asparaginine 8-12 mg/L

Aspartic acid L-Aspartic acid 8-12 mg/L

Glutamic Acid L-Glutamic Acid 8-12 mg/L

Proline L-Proline 5-9 mg/L

Serine L-Serine 5-9 mg/L

Choline chloride 0.7-1.8 mg/L

Calcium pantothenate D-Calcium pantothenate 0.7-1.8 mg/L

Folic acid 0.7-1.8 mg/L

Nicotinamide 0.7-1.8 mg/L

Pyridoxal hydrochloride 0.7-1.8 mg/L

Riboflavin 0.7-1.8 mg/L

Thiamine hydrochloride 0.7-1.8 mg/L

Inositol 0.7-1.8 mg/L

5-15 mg/L Ferric citrate ferrite citrate

Sodium pyruvate Sodium pyruvate 40-60 mg/L

Calcium chloride 1-5 mg/L

Magnesium sulfate 1-5 mg/L

Potassium chloride Potassside chloride 4-10 mg/L

Sodium chloride 80-100 mg/L

Sodium dihydrogen phosphate Sodium phosphate monobasic-H20100-200 mg/L

Zinc sulfate zincsulfate 4-10 mg/L

Arachidonic acid Arachidonic acid 10-50 mug/L

Cholesterol 1500-

Linoleic acid is 50-250 mu g/L

Linolenic acid Linolenic acid 50-250 mu g/L

Oleic acid 50-250 mu g/L

Palmitic acid palmitate 50-250 mu g/L

Stearic acid Stearic acid 50-250 mu g/L

Ethanolamine 1500-

Selenious acid 3-8 mug/L

Sodium bicarbonate Sodium bicarbonate 1.0-1.8 g/L

Hepes 3.2-4.5 mg/L

Pluronic F-68 700-2000 mg/L

The invention also provides the application of the culture medium in the suspension culture of the hybridoma cells for producing the monoclonal antibody.

The invention has the following beneficial effects:

1. compared with the existing scheme, the invention only adds the protein component bovine serum albumin, and simultaneously adds 8 inorganic salts, so that the zinc sulfate component of the inorganic salts is increased, the insulin is replaced, the cell glycometabolism is promoted, and the highest cell density and the antibody yield of the hybridoma cultured by the culture medium reach the effect of a commercial culture medium and even exceed the effect of the commercial culture medium.

2. The invention only contains bovine serum albumin, greatly reduces the production cost, is beneficial to the separation and purification of products and ensures the quality of the monoclonal antibody.

Drawings

In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious to those skilled in the art that other drawings can be obtained according to the drawings without any inventive exercise.

FIG. 1 is a medium serum-free medium culture hybridoma cell growth curve.

FIG. 2 is a graph showing the cell growth curves of the Hybridoma cells cultured in GIBCO Hybridoma-SFM medium.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the specific embodiments of the present invention and the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The technical solutions provided by the embodiments of the present invention are described in detail below with reference to the accompanying drawings.

Examples Low protein serum free Medium preparation and cell culture

1. Preparation of Low-protein serum-free culture Medium

The following were added to a basal medium of DMEM/F12(v/v, 1: 1):

bovine serum albumin 18.0 mg/L, arginine 48 mg/L, cystine 10 mg/L, histidine 12 mg/L, isoleucine 19 mg/L, leucine 18 mg/L, lysine 18 mg/L, methionine 7 mg/L, phenylalanine 11 mg/L, threonine 19 mg/L, tryptophan 4 mg/L, tyrosine 14 mg/L, valine 20 mg/L, glycine 5 mg/L, alanine 7 mg/L, asparagine 10 mg/L, aspartic acid 10 mg/L, glutamic acid 10 mg/L, proline 6 mg/L, serine 6 mg/L, choline chloride 1.2 mg/L, lysine 10 mg/L, arginine 12 mg/L, arginine 19 mg/L, threonine 19 mg/L, tryptophan 4 mg/L, tyrosine 14 mg/L, valine 20 mg/L, glycine 5 mg/L, alanine 7 mg/L, asparagine 10 mg/L, aspartic acid 10 mg/L, glutamic acid 10 mg/L, proline 6 mg/L, serine 6 mg/L, choline chloride 1.2 mg/L, and the like, 1.2 mg/L calcium pantothenate, 1.2 mg/L folic acid, 1.2 mg/L nicotinamide, 1.2 mg/L pyridoxal hydrochloride, 1.2 mg/L riboflavin, 1.2 mg/L thiamine, 1.2 mg/L inositol, 12 mg/L ferric citrate, 50mg/L sodium pyruvate, 4 mg/L calcium chloride, 3 mg/L magnesium sulfate, 7 mg/L potassium chloride, 100 mg/L sodium chloride, 120 mg/L sodium dihydrogen phosphate, 7 mg/L zinc sulfate, 30 μ g/L arachidonic acid, 2000 μ g/L cholesterol, 150 μ g/L linoleic acid, 150 μ g/L linolenic acid, 150 μ g/L oleic acid, 150 μ g/L palmitoleic acid, 150 μ g/L stearic acid, 2500 mu g/L ethanolamine, 6 mu g/L selenious acid, 1.5 g/L sodium bicarbonate, 3.8 mg/L Hepes and 78 mg/L Pluronic F-681300 mg/L. The medium was prepared according to a conventional method and then sterilized by filtration through a 0.22 μm microporous filter.

Cell culture

Frozen anti-glycosylated hemoglobin monoclonal antibody hybridoma cell strain 1D7 cells are taken out from a liquid nitrogen tank, quickly placed in a water bath at 37 ℃, centrifuged to remove supernatant after complete dissolution, lightly blown and beaten by a small amount of the culture medium to ensure that the cells are uniformly mixed, then transferred into a square bottle, and then placed in a CO2 incubator at 37 ℃ and 5% CO2 humidity saturation for subculture.

The above 1D7 cells were expanded and transferred into 4 spinner flasks and inoculated into a 15L bioreactor together with growth to the log phase of the cells at an initial culture density of about 100 x 104cells/ml for a total culture duration of 9 days.

Comparative example

The culturing process was the same as in the above example except that the culture used was GIBCO Hybridoma-SFM medium.

As shown in FIGS. 1 and 2, the serum-free medium of the present invention was mixed with GIBCO Hybridoma-SF when used for Hybridoma cell cultureM medium, the viable cell density reaches 2000 x 10 respectively at 216h⁴cells/ml and 1000X 10⁴cells/ml, and the antibody expression level is 1800mg/L and 750mg/L respectively, so that the culture effect of the protein serum-free culture medium is obviously superior to that of a GIBCO Hybridoma-SFM culture medium, and meanwhile, the serum-free culture medium is relatively low in cost, clear in chemical components and extremely low in protein content.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A low-protein serum-free cell culture medium for producing monoclonal antibodies, wherein the medium comprises a basic physiological buffer, bovine serum albumin, amino acids, vitamins, inorganic salts, and lipids;

the bovine serum albumin content is 10.0-20.0 mg/L;

2. The culture medium of claim 1, further comprising a trace element.

3. The culture medium of claim 1, further comprising a buffer.

4. The culture medium of claim 1, further comprising an anti-shear force protectant.

5. The culture medium according to any one of claims 1 to 4 for use in suspension culture of hybridoma cells for monoclonal antibody production.