JPH04304882A - Composition for animal cell culture, culturing method and production of physiologically active substance - Google Patents

Abstract

PURPOSE:To provide the subject inexpensive composition containing a polymer having phospholipid-like structure and effective for efficiently producing a physio logically active substance in an easily purifiable state by improving the substance-productivity of a cell capable of producing the physiologically active substance. CONSTITUTION:The objective composition contains a polymer having phospholipid-like structure. The polymer is preferably produced by homopolymerizing a polymerizable monomer having a hydrophilic group same as or similar to the group of phospholipid or copolymerizing the monomer with other monomer such as styrene. The polymerizable monomer having a hydrophilic group same as or similar to the hydrophilic group of phospholipid is e.g. 2- methacryloyloxyethyl phosphorylcholine.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a composition for culturing animal cells, a method for culturing animal cells, and a method for producing physiologically active substances.

0002

[Prior Art] Techniques for culturing animal cells efficiently in large quantities have been applied to the production of useful physiologically active substances such as monoclonal antibodies, but conventionally used media contain approximately 10% serum. It had been.

0003

[Problems to be Solved by the Invention] However, since serum is expensive and there are lot differences, there are problems in culturing cells in large quantities. Furthermore, since serum contains many foreign proteins, it is also inconvenient to collect and purify desired useful substances after culturing.

[0004] For this reason, various serum-free media have been developed, but these are not as satisfactory as serum-containing media in terms of cell proliferation, survival, and productivity of physiologically active substances. Furthermore, the addition of serum components such as albumin is often essential, and the disadvantages of serum-containing media, such as cost and purification, have not been completely eliminated.

[0005] In any case, the conventionally known culture medium is
Cell culture has not always been completely satisfactory in order to efficiently obtain large quantities of useful substances.

[0006]

[Means for Solving the Problems] As a result of searching for substances that increase the productivity of physiologically active substances, the present inventor has developed an animal cell culture composition containing a polymer with a phospholipid-like structure. They found that culturing increases the productivity of physiologically active substances, and as a result of further research, they completed the present invention.

Specifically, the present invention provides (1) an animal cell culture composition characterized in that it contains a polymer having a phospholipid-like structure; (2) a composition comprising a polymer having a phospholipid-like structure;
- The composition according to (1) above, which is a homopolymer of methacryloyloxyethylphosphorylcholine,

[0008]
(3) The composition according to (1) above, wherein the polymer having a phospholipid-like structure is a copolymer of 2-methacryloyloxyethylphosphorylcholine and another monomer; (4) the medium containing the polymer having a phospholipid-like structure; A method for culturing animal cells, characterized by culturing animal cells;

(5) A physiologically active substance, which is characterized by culturing animal cells that produce the physiologically active substance in a medium containing a polymer having a structure similar to phospholipids, producing and accumulating the substance in the culture, and collecting it. (6) the method described in (5) above, wherein the physiologically active substance is a monoclonal antibody;
It is related to.

The composition of the present invention comprises a basal medium and a polymer having a phospholipid-like structure. The basal medium may be any medium used for culturing animal cells.

For example, various basic media available on the market (Eagle MEM, Eagle BME, Dulbecco's modified Eagle medium, Iscove's medium, L-15 medium, McCoy's 5a medium, Medium 199, Ham's F10, Ham's F12, RP)
MI1640, etc.) or a mixed medium of these.

[0012] Furthermore, these media require factors necessary for cell proliferation (insulin, transferrin, steroid hormones, epidermal growth factor, fibroblast growth factor, ethanolamine, 2-mercaptoethanol, sodium selenite, etc.). Examples include medium added depending on the condition.

[0013] Furthermore, a medium to which serum or albumin is added in an amount normally used or a lower amount may be used.

[0014] Also, commercially available serum-free media [for example, HB-10
2 (manufactured by Hana Media), HL-1 (manufactured by Bentlett), ASF medium (manufactured by Ajinomoto Co., Ltd.), e-RDF (manufactured by Kyokuto Pharmaceutical), etc.] can also be used as the basal medium.

The polymer having a phospholipid-like structure used in the present invention refers to a hydrophilic group having the same or similar structure as a phospholipid such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, etc., such as the following formula [A]. ]~Formula [E
] Refers to polymers with hydrophilic groups such as

[0016]

[Chemical formula 1]

[0017]

[Case 2]

[0018]

[Chemical 3]

[0019]

[C4]

[0020]

[C5]

The polymer can be obtained by homopolymerization of a polymerizable monomer having a hydrophilic group identical or similar to that of a phospholipid, or by copolymerization with other monomers. Various partner monomers can be used for copolymerization, and are not particularly limited, and include, for example, polymerizable unsaturated monomers such as acrylic esters, methacrylic esters, styrene and derivatives thereof.

The amount of other monomers used is not particularly limited, but it is preferable that the proportion of the polymerizable monomer having the same or similar hydrophilic group as the phospholipid is 5 mol % or more, particularly 30 mol % or more of the total amount. It is preferable to use it in an amount of mol % or more.

[0023] The polymer can be synthesized by conventionally known methods. For example, radical polymerization using azobisisobutyronitrile or the like as an initiator can be mentioned. Conventional techniques are used to purify the polymer. For example, reprecipitation, dialysis, chromatography, etc. are applied.

An example of a polymerizable monomer having a hydrophilic group the same as or similar to a phospholipid is 2-methacryloyloxyethylphosphorylcholine having a structure of the following formula [F].

[0025]

[C6]

The average molecular weight of the polymer used in the present invention is preferably 103 to 106, particularly preferably 10
It is about 4 to 105. Further, the concentration of the polymer used in the present invention in the basal medium is preferably 10 to 100
00 μg/ml, particularly preferably 100 to 5000 μg
/ml. The polymer may be mixed in advance into the culture composition, or may be mixed in at the time of use.

Animal cells to be cultured in the culture method of the present invention are not particularly limited, and include, for example, human, mouse, rat, monkey,
Various lymphoid cells, hybridomas, normal diploid cells, established tumor cells, and other cells derived from mammals such as cows and hamsters can be cultured. Specifically, Ball-1.
Namalva, Jurkat. NS-1, P3
U1, Hela, Vero, CHO, BHK-
21. Examples include cells such as L929.

Examples of animal cells used in the method for producing physiologically active substances of the present invention include Namalva which produces interferon, Jurkat which produces interleukin-2, and various hybridomas (mouse-mouse, mouse-human, mouse-human, etc.) which produce monoclonal antibodies. (human-human, etc.), human lung cancer cells or melanoma cells that produce tissue plasminogen activators.

[0029] Also included are cells that have been genetically engineered to produce various useful substances, and virus-susceptible cells that are infected with viruses and used in the production of vaccines.

[0030] When culturing in the culturing method or manufacturing method of the present invention, containers or devices commonly used for culturing can be used. Examples include multiwell plates, petri dishes, culture flasks, spinner flasks, jar fermenters, fermenters, roller bottles, hollow fibers, microcarriers, and the like.

[0031] The culture conditions may be those normally used for culture, and good results can be obtained, for example, at 37°C in a 5% CO2 atmosphere.

[0032] To collect cells from a culture solution, in the case of floating cells, for example, the culture solution is directly collected using a centrifuge or filter. In the case of adherent cells, for example, a 0.25% trypsin-0.02% EDTA solution is added to suspend the cells, and then the cells are collected by centrifugation or filtration.

[0033] When the physiologically active substance produced by cell culture is accumulated in the culture medium, a supernatant is obtained by filtration or centrifugation, and the supernatant is collected from the supernatant. In the case of substances that accumulate within cells, cells obtained by filtration or centrifugation are subjected to homogenization, ultrasonic treatment, chemical treatment, etc. to obtain a supernatant from which products are extracted.

[0034] In order to separate and purify the physiologically active substance from the above supernatant, known methods may be appropriately combined. For example, ammonium sulfate precipitation, dialysis, ultrafiltration, electrophoresis, gel filtration, ion exchange chromatography, reversed phase chromatography, affinity chromatography, etc. are applied.

By using the composition of the present invention, physiologically active substances can be efficiently obtained in large quantities.

[0036]

[Examples] The present invention will be explained in more detail below with reference to Examples.

Example 1 Insulin 0.2U/e-RDF (manufactured by Kyokuto Pharmaceutical Co., Ltd.)
ml, transferrin 35μg/ml, ethanolamine 20μM, sodium selenite 2.5x10-8
A medium supplemented with M is used as a basal medium, and various concentrations of 2
- A medium supplemented with a homopolymer of methacryloyloxyethylphosphorylcholine, a copolymer of 2-methacryloyloxyethylphosphorylcholine and butyl methacrylate, or bovine serum albumin was dispensed into a petri dish (Falcon 1008, n=5).

[0038] Monoclonal antibody (IgG)-producing mouse-mouse hydridoma Lu243 cells were added to this at 2.5%.
Inoculate at ×106 cells/ml and use 5% CO2
After culturing in an incubator at 37°C for 3 days, the amount of antibody in the supernatant was measured by EIA using a plate coated with α-mouse Ig. The results are shown in Table 1. The numbers are shown as relative values when the result when cultured only with basal medium is set as 100.

Table 1. Effect of polymers with phospholipid-like structure on antibody production Added concentration (μg/ml)
10 100
1000 additives PMPC
120 250
700P (MPC-BMA)
120 300 1
000BSA
100 120
250

PMPC: homopolymer of 2-methacryloyloxyethylphosphorylcholine (
Average molecular weight approximately 100,000) P(MPC-BMA): 2-
Methacryloyloxyethylphosphorylcholine and
Copolymer with butyl methacrylate (average molecular weight approx. 7
10,000,2-Methacryloyloxyethylphosphorylcoli
BSA: Butyl methacrylate = 47:53) BSA: Bovine serum albumin As seen in Table 1, antibody production is significantly improved by the addition of a polymer having a structure similar to phospholipids.

Example 2 An experiment similar to Example 1 was conducted using monoclonal antibody (IgM)-producing mouse-mouse hybridoma ST439 cells instead of Lu243. The results are shown in Table 2.

Table 2 Effect of polymers with phospholipid-like structures on antibody production Addition concentration (μg/ml
) 10 100
1000 additives PMPC
170 600
1100P (MPC-BMA) 1
90 650 14
00 BSA
170 250
350

As seen in Table 2,
Regarding ST439, antibody production was also significantly improved by the addition of a polymer having a phospholipid-like structure.

[0044]

Effects of the Invention: When cells that produce physiologically active substances are cultured using the composition of the present invention, the substance productivity increases, making it possible to efficiently produce physiologically active substances, which is useful for industrial production. It's advantageous. The polymer with a phospholipid-like structure used in the present invention is cheaper than serum or albumin, and contains almost no impurities, so it is cheaper than conventional culture media and it is easier to purify the target substance. It is advantageous for industrial production.

Claims (6)

[Claims] 1. A composition for culturing animal cells, which comprises a polymer having a structure similar to that of a phospholipid. 2. The composition according to claim 1, wherein the polymer having a phospholipid-like structure is a homopolymer of 2-methacryloyloxyethylphosphorylcholine. 3. The composition according to claim 1, wherein the polymer having a phospholipid-like structure is a copolymer of 2-methacryloyloxyethylphosphorylcholine and another monomer. 4. A method for culturing animal cells, which comprises culturing the animal cells in a medium containing a polymer having a structure similar to that of a phospholipid. 5. A method of producing a physiologically active substance, which comprises culturing animal cells that produce a physiologically active substance in a medium containing a polymer having a structure similar to a phospholipid, producing and accumulating the substance in the culture, and collecting the same. Manufacturing method. 6. The production method according to claim 5, wherein the physiologically active substance is a monoclonal antibody.