CN118325843A

CN118325843A - Low-protein serum-free culture medium suitable for hybridoma cell suspension culture and monoclonal antibody production and application thereof

Info

Publication number: CN118325843A
Application number: CN202310041867.0A
Authority: CN
Inventors: 孙益军; 杨文骏; 黄莺; 张峰
Original assignee: Jiangsu Keygen Biotech Corp ltd
Current assignee: Jiangsu Keygen Biotech Corp ltd
Priority date: 2023-01-12
Filing date: 2023-01-12
Publication date: 2024-07-12

Abstract

The invention discloses a low-protein serum-free culture medium suitable for hybridoma cell suspension culture and monoclonal antibody production and application thereof. The culture medium is based on DMEM/F12 (v/v, 1:1), and bovine serum albumin, transferrin and 3 protein components of insulin, 19 amino acids, 8 vitamins, 8 lipids, trace elements, a protective agent, a buffer solution and other non-protein substances are added. The culture medium has the characteristics of low protein content and basically definite chemical composition, not only can meet the requirement of hybridoma cell suspension culture, but also can effectively avoid the problem that the product purification is not facilitated due to the use of serum; the culture medium also has the characteristic of strong universality, can obviously reduce the cost of cell culture solution, and is suitable for the mass production and industrial application of hybridoma cells.

Description

Low-protein serum-free culture medium suitable for hybridoma cell suspension culture and monoclonal antibody production and application thereof

Technical Field

The invention relates to a cell culture and culture medium research and development technology in the field of biotechnology, in particular to a serum-free culture medium suitable for hybridoma cell suspension culture and monoclonal antibody production and application thereof.

Background

Cell culture is to realize in vitro cell culture by simulating in vivo cell growth environment, and animal cell in vitro culture technology is widely applied in the fields of research on physiological, biochemical and pharmacological activities of cells and tissues and the industrial production fields of biological products such as antibodies, hormones, vaccines and the like. Wherein the cell culture medium is a vital part of the cell in vitro culture technology. The traditional serum-containing culture medium contains a large amount of components necessary for cell growth proliferation such as various growth factors, plasma proteins, polypeptides, hormones and the like, but the serum has the problems of large batch-to-batch difference, potential pollution risk, undefined components, adverse separation and purification of target products, easy infection by viruses and mycoplasma and the like, and the serum-free culture medium has the advantages of simple preparation process, consistent quality and low protein content due to relatively defined components, is favorable for improving the production stability of cell products and facilitating the purification of the cell products, and can be widely applied to the field of modern biotechnology.

There are also many reports on components of serum component substitutes that promote cell growth. Albumin is a major additive factor for many serum-free media, and has the effects of regulating osmotic pressure, protecting cells from mechanical damage, and the like. Li Ping et al (Li Ping, jiang Lin, huang Saiyang, etc.), study of serum-free culture of chinese hamster ovary engineering cells [ J ] microbiological immunological progress 2004, 32 (4): 46-50) experiments showed that albumin also had a remarkable effect of stabilizing cell growth and increasing the expression level of cell strain products. The serum-free medium for general suspension culture contains bovine serum albumin. Hu Xuemei et al (Hu Xuemei, zhang Yuanxing, amino acids acting on chinese hamster ovary cells in serum-free medium [ J ] university of eastern China university of technology 1996, 22 (6): 283-285) examined the influence of 15 amino acids and their concentrations on cell culture in CHO cell serum-free culture by orthogonal assay, and the results showed that the added arginine, leucine, proline and methionine had a remarkable promoting effect on cell growth, whereas serine and tryptophan at high concentrations had a remarkable inhibiting effect on cell growth. Insulin promotes synthesis of RNA, protein and fatty acid, inhibits apoptosis, and is an important cell survival factor. Jan et al (Jan L,Haggstrorm L.Specific growth rate as a parameter fortracing growth-limiting substances in animal cell cultures〔J〕.Journal of Biotechnology 1995,42：163～165) believe that rapid depletion of insulin in batch culture is the primary cause of a decrease in the specific growth rate of cells. Selenium has obvious effect in the growth process of mammalian cells, and trace element selenium participates in the action process of glutathione peroxidase and superoxide dismutase to eliminate the damage of oxidase and oxygen free radical to cells. Xue Qingshan (Xue Qingshan, in vitro culture principles and techniques [ M ] Beijing science and technology Press, 2001, 162-164) states that the addition of selenious acid in an appropriate amount can promote cell growth, but if the concentration is too high, it can exert toxic effects on cells.

The commercial serum-free culture medium is mainly imported, the price is high, and the formula is kept secret, so that the serum-free culture medium which can support the rapid growth of hybridoma cells and the high expression of monoclonal antibodies is independently researched and developed, and related process optimization and amplification are carried out to meet the requirement of expressing the monoclonal antibodies in a large quantity.

Disclosure of Invention

The invention aims to provide a low-protein serum-free culture medium suitable for hybridoma cell suspension culture and monoclonal antibody production so as to meet the requirements of hybridoma cell suspension culture and monoclonal antibody production. The hybridoma cell is an infinite passage cell capable of secreting a single unique antibody, and the secreted monoclonal antibody has wide application in vaccine production, in-vitro diagnosis and biological medicine. The culture medium can enable the hybridoma cells to grow normally under the suspension condition and express the monoclonal antibody.

To achieve the above object, the present invention provides a serum-free medium suitable for hybridoma cell suspension culture and monoclonal antibody production, the serum-free medium comprising a basal medium and an additive factor, wherein:

The basic culture medium is DMEM and F12, and the volume ratio is 1:1;

the additive factors comprise protein components, amino acids, microorganisms, lipids, trace elements, protective agents and buffer solutions;

The protein component is bovine serum albumin, transferrin and insulin.

Wherein the dosage of the protein components is respectively as follows: bovine serum albumin (Bovine serum albumin) 10.0-20.0mg/L, transferrin (Transferrin) 2.5-15.5mg/L, insulin (Transferrin) 5.0-15.0mg/L.

The components and the amounts of the amino acid are as follows: arginine (L-Arginine) 20-60mg/L, cystine (L-Cystine) 5-15mg/L, histidine (L-HISTIDINE) 5-20mg/L, isoleucine (L-Isoleucine) 10-25mg/L, leucine (L-Leucine) 10-25mg/L, lysine (L-Lysine) 10-25mg/L, methionine (L-Methionine) 10-20mg/L, phenylalanine (L-PHENYLALANINE) 10-25mg/L, threonine (L-Threonine) 10-25mg/L, tryptophan (L-Tryptophan) 2-20mg/L, tyrosine (L-Tyrosine) 5-20mg/L, valine (L-Valine) 10-30mg/L, glycine (Glycine) 2-10mg/L, alanine (L-Alanine) 2-10mg/L, asparagine (L-ASPARAGINE) 2-20mg/L, aspartic acid (L-ASPARTICACID) 2-20mg/L, glutamic acid (L-28) 2-20mg/L, proline (L-34) 2-30 mg/L, and Proline (L-34) 2-76 mg/L.

The components and the dosage of the vitamins are as follows: choline chloride (Choline chloride) 0.5-2.0mg/L, calcium pantothenate (D-Calcium pantothenate) 0.5-2.0mg/L, folic acid (Folic acid) 0.5-2.0mg/L, niacinamide (Nicotinamida) 0.5-2.0mg/L, pyridoxal hydrochloride (Pyridoxal hydrochloride) 0.5-2.0mg/L, riboflavin (Riboflavin) 0.5-2.0mg/L, thiamine (Thiamine hydrochloride) 0.5-2.0mg/L, inositol (Inositol) 0.5-2.0mg/L.

The lipid components and the dosage are as follows: 10-50 mug/L of arachidonic acid (Arachidonic acid), 1000-3000 mug/L of Cholesterol (Cholesterol), 30-300 mug/L of Linoleic acid (Linoleic acid), 30-300 mug/L of linolenic acid (Linolenicacid), 30-300 mug/L of oleic acid (Oleic acid), 30-300 mug/L of palmitoleic acid (PALMITIC ACID), 30-300 mug/L of stearic acid (STEARIC ACID), 1000-4000 mug/L of ethanolamine (Ethanolamine).

The microelements are sodium selenite (Selenous acid), and the dosage is 2-20 mug/L.

The buffer solution is sodium bicarbonate (Sodium bicarbonate) and Hepes, the dosage of the sodium bicarbonate is 1-3g/L, and the dosage range of the Hepes is 2-40mg/L.

The shear force resistant protective agent is Pluronic F-68, and the dosage is 500-1000mg/L.

Serum-free medium was prepared by conventional methods.

The invention also provides application of the serum-free culture medium in hybridoma cell suspension culture and monoclonal antibody production.

The invention adopts three protein substances of bovine serum albumin, transferrin and insulin with low content and definite sources to replace substances such as growth factors in the traditional serum culture medium, develops a serum-free culture medium suitable for hybridoma cell suspension culture and monoclonal antibody production, can meet the culture requirement of hybridoma cells, effectively avoid a plurality of adverse factors caused by using serum, reduce the production cost and lead the expression quantity of monoclonal antibodies to be equivalent to that of commercial serum-free culture.

The serum-free culture medium suitable for hybridoma cell suspension culture and monoclonal antibody production has the positive effects that:

(1) The hybridoma cells can be well supported, and the highest cell density and the antibody yield of the hybridoma cells reach the commercial culture medium effect of the GIBCO brand;

(2) The components are clear, and only three protein components are contained, so that the separation and purification of the product are utilized, and the quality of the product is improved;

(3) The hybridoma cells are supported to be stably subcultured, and can be directly cultured without adaptation;

(4) The components are clear, the preparation is convenient, and the preparation method is suitable for mass production and application;

(5) The use cost of the serum-free culture medium is reduced, and the serum-free culture medium of Gibco brand hybrid-SFM is known through inquiry, and the sales price of the official net is 977 yuan/500 mL; the domestic standard product, the official network shows that the selling price interval is 300-400 yuan/500 mL; the initial accounting cost of the product is less than 250 yuan/500 mL, and the cost can be further reduced after the large-scale preparation is realized.

Drawings

The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.

FIG. 1AE-1 cell resuscitates to the cellular State in T75 flask

FIG. 2 shows a graph of growth of hybridoma cell AE-1 strain in serum-free medium of the invention;

FIG. 3 recovery of Hybridoma-2 cells to cellular status in 125mL shake flask

FIG. 4 shows a graph of growth of Hybridoma cell lines in serum-free medium according to the invention and domestic homogeneous serum-free medium.

Detailed Description

Example 1 serum-free medium preparation and cell culture.

(1) Preparation of the culture Medium

In a basal medium based on DMEM/F12 (v/v, 1:1), the following substances were added to the basal medium:

Bovine serum albumin 10.0mg/L, transferrin 6mg/L, insulin 5mg/L, arginine 116mg/L, cystine 22mg/L, histidine 35mg/L, isoleucine 42mg/L, leucine 42mg/L, lysine 65mg/L, methionine 120mg/L, phenylalanine 23mg/L, threonine 40mg/L, tryptophan 8mg/L, tyrosine 26mg/L, valine 40mg/L, glycine 6.5mg/L, alanine 7.9mg/L, asparagine 12.2mg/L, aspartic acid 12.3mg/L, glutamic acid 13.7mg/L, proline 10.5mg/L, serine 8mg/L, glutamine 700mg/L, choline chloride 1mg/L, calcium pantothenate 1mg/L, folic acid 1mg/L, niacinamide 1mg/L, pyridoxal hydrochloride 1mg/L, riboflavin 0.1mg/L, thiamine 1mg/L, inositol 2mg/L, ferric ammonium citrate 8mg/L, sodium chloride 100mg/L, sodium oleate 130 μg/L, sodium linoleate 130 μg/L, sodium bicarbonate 130 μg/L, 50 μg/g of oleic acid, and 130 μg/L of linolenic acid. The medium was prepared according to the conventional method and then sterilized by filtration through a 0.22 μm microporous filter.

(2) Cell culture

The hybridoma cell line AE-1 (purchased from the cell bank of the department of chinese sciences, AE-1 is the cell name in a T75 square bottle, cell density at recovery is 0.59×10 ⁶ cells/mL, the activity is 92% as shown in fig. 1), and then placed in a CO ₂ incubator saturated with 5% CO ₂ humidity for subculture at 37 ℃.

(3) Comparison with GIBCO brand serum-free Medium Hybridoma-SFM

The cells were subjected to expansion culture to prepare a cell suspension, inoculated into 125 shake flasks at an initial culture density of about 0.2X10 ⁶ cells/ml, and then subjected to suspension culture in a saturated incubator with 5% CO ₂ humidity at 37 ℃.

As shown in FIG. 2, the yellow line graph shows the growth curve of the Hybridoma cells cultured in the serum-free medium according to the present invention, and the blue line graph shows the growth curve of the Hybridoma cells cultured in the GIBCO Hybridoma-SFM medium. The maximum growth density of AE-1 cells in the serum-free medium of the present invention was significantly higher than that of the control SFM medium.

(4) Monoclonal antibody expression level detection

Collecting culture supernatant from the cell culture to day 4 (Batch culture), purifying protein sample with protein A filler, and performing concentration analysis on the purified sample with microfluidic ultraviolet spectrophotometer. According to the calculation, the expression level of the monoclonal antibody in the culture supernatant was 101.5. Mu.g/ml.

Example 2 cell resuscitative test and comparison test with domestic commercial media.

(1) Preparation of the culture Medium

The following were added to the DMEM/F12 (v/v, 1:1) based medium:

Bovine serum albumin 15.0mg/L, transferrin 8mg/L, insulin 8mg/L, arginine 125mg/L, cystine 28mg/L, histidine 45mg/L, isoleucine 62mg/L, leucine 52mg/L, lysine 75mg/L, methionine 150mg/L, phenylalanine 30mg/L, threonine 50mg/L, tryptophan 10mg/L, tyrosine 40mg/L, valine 40mg/L, glycine 13mg/L, alanine 16mg/L, asparagine 18mg/L, aspartic acid 19mg/L, glutamic acid 25mg/L, proline 20mg/L, serine 12mg/L, glutamine 900mg/L, choline chloride 1mg/L, calcium pantothenate 1mg/L, folic acid 1mg/L, niacinamide 1mg/L, pyridoxal hydrochloride 1mg/L, riboflavin 0.1mg/L, thiamine 1mg/L, inositol 2mg/L, ferric ammonium citrate 8mg/L, sodium pyruvate 100mg/L, sodium chloride 85mg/L, tetramine oleic acid 85mg/L, oleic acid 130 μg/L, oleic acid and 130 μg/L. The medium was prepared according to the conventional method and then sterilized by filtration through a 0.22 μm microporous filter.

(2) Cell resuscitation test

The Hybridoma cell line Hybridoma-2 cells (the Hybridoma-2 cells were prepared in the laboratory, the mouse myeloma cells Sp2/0 and the immunized mouse B lymphocytes were prepared by electrofusion techniques, and the obtained Hybridoma cells were selected) were resuscitated using the above serum-free medium into a 125mL shake flask in a culture volume of 30mL, and then cultured in a shake flask at 100rpm and 37℃under 5% CO ₂ humidity.

Cell growth was observed after resuscitating cells: on the day of resuscitation, the cell density was 0.42×10 ⁶ cells/ml and the viability was 96.5%, as shown in fig. 3; after 24h of culture, the cell density is 0.74×10 ⁶ cells/ml, and the activity rate is 97.3%; after 48h of culture, the cell density is 2.05×10 ⁶ cells/ml, and the activity rate is 98.1%; after 72h of incubation, the cell density was 3.62×10 ⁶ cells/mL and the viability was 96.6%. The result shows that the serum-free culture medium can be suitable for the recovery operation of hybridoma cells.

(3) Comparing and testing with similar serum-free culture medium products of different domestic brands

The Hybridoma-2 cells are subjected to expansion culture to prepare cell suspension, then the culture medium (LPM) and other two similar serum-free culture medium products of different brands in China are respectively used, the first comparison product is HybriSFM-P1B serum-free culture medium of the company of Om Pu Mai, the second comparison product is Hyber-B100 serum-free culture medium of the company of double-skill, the initial culture density is about 0.3 x10 ⁶ cells/mL, the culture volume is 30mL, and then the culture is placed in a saturated culture box with the humidity of 37 ℃ and 5% CO ₂ for suspension culture.

As shown in FIG. 4, the orange-yellow line is the growth curve of the hybridoma cells cultured by the serum-free culture medium, the red line is the growth curve of the hybridoma cells of the first domestic similar comparison product, and the green line is the growth curve of the hybridoma cells of the second domestic similar comparison product. The viable cell density and the cell viability of the hybrid oma-2 cells cultured in the serum-free medium for 96 hours are higher than those of the domestic similar comparison medium.

The culture medium has the characteristics of low protein content and basically definite chemical composition, not only can meet the requirement of hybridoma cell suspension culture, but also can effectively avoid the problem that the product purification is not facilitated due to the use of serum; the culture medium also has the characteristic of strong universality, can obviously reduce the cost of cell culture solution, and is suitable for the mass production and industrial application of hybridoma cells.

The invention provides a thought and a method of a low-protein serum-free culture medium suitable for hybridoma cell suspension culture and monoclonal antibody production, and the method and the way for realizing the technical scheme are numerous, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by those skilled in the art without departing from the principle of the invention, and the improvements and the modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.

Claims

1. A serum-free medium suitable for hybridoma cell suspension culture and monoclonal antibody production, wherein the serum-free medium comprises a basal medium and an additive factor, wherein:

The basic culture medium is DMEM and F12, and the volume ratio is 1:1;

The protein component is bovine serum albumin, transferrin and insulin.

2. Serum-free medium according to claim 1, characterized in that the protein components are used in amounts of: bovine serum albumin 10.0-20.0 mg/L, transferrin 2.5-15.5 mg/L, insulin 5.0-15.0 mg/L.

3. The serum-free medium of claim 1, wherein the amino acids are present in the following composition and amounts: arginine 20-60 mg/L, cystine 5-15 mg/L, histidine 5-20 mg/L, isoleucine 10-25 mg/L, leucine 10-25 mg/L, lysine 10-25 mg/L, methionine 10-20 mg/L, phenylalanine 10-25 mg/L, threonine 10-25 mg/L, tryptophan 2-20 mg/L, tyrosine 5-20 mg/L, valine 10-30 mg/L, glycine 2-10 mg/L, alanine 2-10 mg/L, asparagine 2-20 mg/L, aspartic acid 2-20 mg/L, glutamic acid 2-20 mg/L, proline 2-20 mg/L and serine 2-20 mg/L.

4. The serum-free medium of claim 1, wherein the vitamins are present in the following amounts and components: 0.5 to 2.0 mg/L of choline chloride, 0.5 to 2.0 mg/L of calcium pantothenate, 0.5 to 2.0 mg/L of folic acid, 0.5 to 2.0 mg/L of niacinamide, 0.5 to 2.0 mg/L of pyridoxal hydrochloride, 0.5 to 2.0 mg/L of riboflavin, 0.5 to 2.0 mg/L of thiamine and 0.5 to 2.0 mg/L of inositol.

5. The serum-free medium of claim 1, wherein the lipid component and amount are: 10-50 mug/L of arachidonic acid, 1000-3000 mug/L of cholesterol, 30-300 mug/L of linoleic acid, 30-300 mug/L of linolenic acid, 30-300 mug/L of oleic acid, 30-300 mug/L of palmitoleic acid, 30-300 mug/L of stearic acid and 1000-4000 mug/L of ethanolamine.

6. The serum-free medium according to claim 1, wherein the trace element is sodium selenite in an amount of 2-20 μg/L.

7. The serum-free medium of claim 1, wherein the buffer is sodium bicarbonate and Hepes, the amount of sodium bicarbonate is 1-3 g/L, and the amount of Hepes is 2-40 mg/L.

8. The serum-free medium according to claim 1, wherein the shear resistant protective agent is Pluronic F-68 in an amount of 500-1000 mg/L.

9. Use of the serum-free medium according to any one of claims 1-8 for hybridoma cell suspension culture and monoclonal antibody production.