JP2021166474A - Cell culture medium additive and use thereof - Google Patents
Cell culture medium additive and use thereof Download PDFInfo
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- JP2021166474A JP2021166474A JP2020070126A JP2020070126A JP2021166474A JP 2021166474 A JP2021166474 A JP 2021166474A JP 2020070126 A JP2020070126 A JP 2020070126A JP 2020070126 A JP2020070126 A JP 2020070126A JP 2021166474 A JP2021166474 A JP 2021166474A
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- JP
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- Prior art keywords
- cell culture
- meth
- culture medium
- vinyl
- cells
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- 239000000654 additive Substances 0.000 title claims abstract description 42
- 230000000996 additive effect Effects 0.000 title claims abstract description 36
- 239000006143 cell culture medium Substances 0.000 title claims abstract description 26
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- 238000012258 culturing Methods 0.000 claims abstract description 14
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- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 13
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 8
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- QYZFTMMPKCOTAN-UHFFFAOYSA-N n-[2-(2-hydroxyethylamino)ethyl]-2-[[1-[2-(2-hydroxyethylamino)ethylamino]-2-methyl-1-oxopropan-2-yl]diazenyl]-2-methylpropanamide Chemical compound OCCNCCNC(=O)C(C)(C)N=NC(C)(C)C(=O)NCCNCCO QYZFTMMPKCOTAN-UHFFFAOYSA-N 0.000 description 1
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- 125000001400 nonyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 150000001451 organic peroxides Chemical class 0.000 description 1
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- HVAMZGADVCBITI-UHFFFAOYSA-M pent-4-enoate Chemical compound [O-]C(=O)CCC=C HVAMZGADVCBITI-UHFFFAOYSA-M 0.000 description 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
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- 238000011002 quantification Methods 0.000 description 1
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- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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- 238000003756 stirring Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
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- GJBRNHKUVLOCEB-UHFFFAOYSA-N tert-butyl benzenecarboperoxoate Chemical compound CC(C)(C)OOC(=O)C1=CC=CC=C1 GJBRNHKUVLOCEB-UHFFFAOYSA-N 0.000 description 1
- 125000000383 tetramethylene group Chemical group [H]C([H])([*:1])C([H])([H])C([H])([H])C([H])([H])[*:2] 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- LDHQCZJRKDOVOX-UHFFFAOYSA-N trans-crotonic acid Natural products CC=CC(O)=O LDHQCZJRKDOVOX-UHFFFAOYSA-N 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 125000003258 trimethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])[*:1] 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- FUSUHKVFWTUUBE-UHFFFAOYSA-N vinyl methyl ketone Natural products CC(=O)C=C FUSUHKVFWTUUBE-UHFFFAOYSA-N 0.000 description 1
- NLVXSWCKKBEXTG-UHFFFAOYSA-N vinylsulfonic acid Chemical compound OS(=O)(=O)C=C NLVXSWCKKBEXTG-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
Abstract
Description
本発明は、動物細胞培養時における生理活性物質の高産生化に適した細胞用培地添加剤に関する。さらに本発明は該細胞用培地添加剤を含有する培地を用いた生理活性物質の製造方法に関する。 The present invention relates to a cell culture medium additive suitable for increasing the production of physiologically active substances during animal cell culture. Furthermore, the present invention relates to a method for producing a physiologically active substance using a medium containing the cell culture medium additive.
ｉＰＳ細胞やＥＳ細胞、間葉系幹細胞等の幹細胞を用いた再生医療では、これらの細胞を効率よく増殖させ、その後に目的組織へ分化させ移植することが想定されている。また、動物細胞を大量培養することにより、モノクローナル抗体をはじめ有用な生理活性物質(ホルモン、神経伝達物質、サイトカイン、ビタミンやミネラル、酵素、核酸など)を工業的に量産することが非常に多くなってきている。その際には増殖因子として、ウシ胎児血清（ＦＢＳ）などの動物血清が頻繁に使用される。具体的にはＲＰＭ１１６４０、イーグルＭＥＭ、Ｈａｍ‘ｓ Ｆ１２などの合成基礎培地に１０〜２０％程度のウシ胎児血清（ＦＢＳ）などの動物血清を添加した培養液が一般に用いられる。 In regenerative medicine using stem cells such as iPS cells, ES cells, and mesenchymal stem cells, it is assumed that these cells are efficiently proliferated and then differentiated into target tissues and transplanted. In addition, by culturing animal cells in large quantities, it is extremely common to industrially mass-produce useful physiologically active substances (hormones, neurotransmitters, cytokines, vitamins and minerals, enzymes, nucleic acids, etc.) such as monoclonal antibodies. It's coming. In that case, animal serum such as fetal bovine serum (FBS) is frequently used as a growth factor. Specifically, a culture medium obtained by adding about 10 to 20% of animal serum such as fetal bovine serum (FBS) to a synthetic basal medium such as RPM11640, Eagle MEM, or Ham's F12 is generally used.
しかしながら、培地にウシ胎児血清（ＦＢＳ）などの血清を加えることは培地コストの上昇を招き、また、血清由来のタンパク質を含む培養液から目的とする生理活性物質を単離することが困難になるなどの欠点がある。また、多くの場合、血清にはロット間に品質のばらつきがみられる。そこで血清の使用に先立ってロットチェックを十分に行い、使用可能な血清を選択する必要があるが、この血清の選択には多くの労力を要する。 However, the addition of serum such as fetal bovine serum (FBS) to the medium leads to an increase in the cost of the medium, and it becomes difficult to isolate the desired bioactive substance from the culture medium containing the protein derived from the serum. There are drawbacks such as. Also, in many cases, serum quality varies from lot to lot. Therefore, it is necessary to perform a sufficient lot check prior to the use of serum and select a usable serum, but selection of this serum requires a lot of labor.
そこで上記課題を解決すべく、細胞培養の効率化、低コスト化が可能となる培地添加剤の検討が行われてきた。
例えば、特許文献１には、グリシルグリシンを培地に添加することで、動物細胞に高濃度に有用物質を生産させることができることが開示されている。
また、特許文献２には、リン脂質類似構造を有するポリマーを含有する培地で細胞培養した場合に、生理活性物質を効率的に生産できることが開示されている。
また、特許文献３には、疎水性Ｄ−アミノ酸を含有する培地が、細胞増殖を促進させ、有用物質の生産量を増大させることが開示されている。
Therefore, in order to solve the above problems, studies have been conducted on medium additives that can improve the efficiency and cost of cell culture.
For example, Patent Document 1 discloses that by adding glycylglycine to a medium, animal cells can produce a useful substance at a high concentration.
Further, Patent Document 2 discloses that a physiologically active substance can be efficiently produced when cells are cultured in a medium containing a polymer having a phospholipid-like structure.
Further, Patent Document 3 discloses that a medium containing a hydrophobic D-amino acid promotes cell proliferation and increases the production of useful substances.
しかし、従来の無血清培地の多くは、抗体産生細胞の増殖性、生存性および抗体生産性の面で血清含有培地に比べて十分なものとは言えない。 However, many of the conventional serum-free media are not sufficient as compared with serum-containing media in terms of proliferation, viability and antibody productivity of antibody-producing cells.
上記事情に鑑み、本発明の目的は、コストや手間の増加を伴わずに、抗体の生産性を高めるための、抗体産生細胞用培地添加剤、該添加剤を用いた抗体産生細胞の培養方法、細胞用培地及び抗体の製造方法を提供することにある。 In view of the above circumstances, an object of the present invention is an antibody-producing cell medium additive, and a method for culturing antibody-producing cells using the additive, in order to increase antibody productivity without increasing cost and labor. , A method for producing a cell culture medium and an antibody.
すなわち、本発明は以下の〔１〕〜〔６〕に関する。
〔１〕下記一般式（１）〜（５）で表される少なくともいずれかのモノマー由来の構造単位を有するビニル系ポリマー（Ａ）を含むことを特徴とする細胞用培地添加剤。
一般式（１）
一般式（２）
一般式（３）
一般式（４）
一般式（５）
[1] A cell culture medium additive containing a vinyl polymer (A) having a structural unit derived from at least one of the monomers represented by the following general formulas (1) to (5).
General formula (1)
General formula (2)
General formula (3)
General formula (4)
General formula (5)
［一般式（１）〜（５）中、
Ｒ１およびＲ４は、それぞれ独立して水素原子またはメチル基、
Ｒ２およびＲ３は、それぞれ独立して水素原子、炭素数１〜６のアルキル基、炭素数１〜６のヒドロキシアルキル基またはジアルキルアミノプロピル基
Ｒ５およびＲ６は、それぞれ独立して、炭素数１〜３のアルカンジイル基、
Ｒ７は、炭素数１〜５のアルカンジイル基、
Ｒ８〜Ｒ１０は、それぞれ独立して水素原子またはメチル基、
Ｒ１１〜Ｒ１５のうち４つは、それぞれ独立して、水素原子または炭素数１〜６のアルキル基を表し、Ｒ１１〜Ｒ１５のうち１つは、ビニル基を表す。］
[In general formulas (1) to (5),
R 1 and R 4 are independent hydrogen atoms or methyl groups, respectively.
R 2 and R 3 are independent hydrogen atoms, alkyl groups having 1 to 6 carbon atoms, hydroxyalkyl groups having 1 to 6 carbon atoms or dialkylaminopropyl groups R 5 and R 6 are independent carbon atoms, respectively. Alkanediyl groups of numbers 1 to 3,
R 7 is an alkanediyl group having 1 to 5 carbon atoms.
R 8 to R 10 are independent hydrogen atoms or methyl groups, respectively.
Four of R 11 to R 15 independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and one of R 11 to R 15 represents a vinyl group. ]
〔２〕ビニル系ポリマー（Ａ）が、炭素数１〜１８のアルキル基を有するモノマー（ａ）由来の構造単位を有することを特徴とする上記細胞用培地添加剤。 [2] The above-mentioned cell culture medium additive, wherein the vinyl-based polymer (A) has a structural unit derived from the monomer (a) having an alkyl group having 1 to 18 carbon atoms.
〔３〕ビニル系ポリマー（Ａ）１００質量％中、前記一般式（１）〜（５）で表される少なくともいずれかのモノマー由来の構造を５質量％以上含むことを特徴とする上記細胞用培地添加剤。 [3] For the above-mentioned cells, which contains 5% by mass or more of a structure derived from at least one of the monomers represented by the general formulas (1) to (5) in 100% by mass of the vinyl-based polymer (A). Medium additive.
〔４〕上記細胞用培地添加剤を含む、細胞用培地。 [4] A cell culture medium containing the above-mentioned cell culture medium additive.
〔５〕ビニル系ポリマー（Ａ）の培地中の濃度が０．００１〜０．５質量％である上記細胞用培地。 [5] The cell medium having a concentration of the vinyl polymer (A) in the medium of 0.001 to 0.5% by mass.
〔６〕上記細胞用培地中で細胞を培養することを特徴とする生理活性物質の製造方法。 [6] A method for producing a physiologically active substance, which comprises culturing cells in the above-mentioned cell medium.
本発明の細胞用培地添加物は、該添加物を細胞用培地に添加し、得られた培地を用いて、生理活性物質の産生可能な細胞を培養した際に、生理活性物質の生産量を増大させることが可能である。特に細胞活性やＤＮＡ量あたりの生理活性物質の産生性を高めることができる。また、本発明の細胞用培地添加剤を添加することにより、所望の生理活性物質の産生性を促進することができるため、製造コストや手間を少なくすることができる点でも有用である。例えば、抗体の場合では抗体医薬品等のバイオ医薬品の製造に関して、大量供給に大きく貢献することができる。さらに、本発明の細胞用培地添加物は水溶性であるため、培養液に溶解させることができ、取り扱いが容易である。 The cell medium additive of the present invention determines the amount of the physiologically active substance produced when the additive is added to the cell medium and the obtained medium is used to culture cells capable of producing the physiologically active substance. It can be increased. In particular, cell activity and productivity of physiologically active substances per amount of DNA can be enhanced. Further, by adding the cell culture medium additive of the present invention, the productivity of a desired physiologically active substance can be promoted, which is also useful in that the production cost and labor can be reduced. For example, in the case of an antibody, it can greatly contribute to mass supply in the production of biopharmaceuticals such as antibody drugs. Furthermore, since the cell culture medium additive of the present invention is water-soluble, it can be dissolved in a culture solution and is easy to handle.
＜ビニル系ポリマー（Ａ）＞
本発明の細胞用培地添加剤は、ビニル系ポリマー（Ａ）を含むものである。
<Vinyl polymer (A)>
The cell culture medium additive of the present invention contains a vinyl-based polymer (A).
本発明のビニル系ポリマー（Ａ）は、下記一般式（１）〜（５）で表される少なくともいずれかのモノマー由来の構造を有していればよい。
一般式（１）
一般式（４）
一般式（５）
General formula (1)
General formula (4)
General formula (5)
［一般式（１）〜（５）中、
Ｒ１およびＲ４は、それぞれ独立して水素原子またはメチル基、
Ｒ２およびＲ３は、それぞれ独立して水素原子、炭素数１〜６のアルキル基、炭素数１〜６のヒドロキシアルキル基またはジアルキルアミノプロピル基
Ｒ５およびＲ６は、それぞれ独立して、炭素数１〜３のアルカンジイル基、
Ｒ７は、炭素数１〜５のアルカンジイル基、
Ｒ８〜Ｒ１０は、それぞれ独立して水素原子またはメチル基、
Ｒ１１〜Ｒ１５のうち４つは、それぞれ独立して、水素原子または炭素数１〜６のアルキル基を表し、Ｒ１１〜Ｒ１５のうち１つは、ビニル基を表す。］
[In general formulas (1) to (5),
R 1 and R 4 are independent hydrogen atoms or methyl groups, respectively.
R 2 and R 3 are independent hydrogen atoms, alkyl groups having 1 to 6 carbon atoms, hydroxyalkyl groups having 1 to 6 carbon atoms or dialkylaminopropyl groups R 5 and R 6 are independent carbon atoms, respectively. Alkanediyl groups of numbers 1 to 3,
R 7 is an alkanediyl group having 1 to 5 carbon atoms.
R 8 to R 10 are independent hydrogen atoms or methyl groups, respectively.
Four of R 11 to R 15 independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and one of R 11 to R 15 represents a vinyl group. ]
本発明のビニル系ポリマー（Ａ）は、従来のポリマーに対し、下記一般式（１）〜（５）で表される少なくともいずれかのモノマー由来の構造を有しているため、シアル酸由来の負電荷を有する細胞との静電相互作用が制御され、細胞の周囲の環境を適切に制御することができ、例えば抗体産生細胞の抗体産生性や株化細胞の増殖性、生存性を向上することができる。また、本発明のビニル系ポリマー（Ａ）は、生物学的安全性の効果を最大限に発揮できるように、分子量、共重合モノマーの種類および共重合モノマーの導入量をその使用目的に応じて最適化することが可能である。 Since the vinyl-based polymer (A) of the present invention has a structure derived from at least one of the monomers represented by the following general formulas (1) to (5) with respect to the conventional polymer, it is derived from sialic acid. Electrostatic interaction with negatively charged cells can be controlled, and the environment around the cells can be appropriately controlled, for example, improving the antibody productivity of antibody-producing cells, the proliferative potential of established cells, and the viability. be able to. In addition, the vinyl-based polymer (A) of the present invention has a molecular weight, a type of copolymerized monomer, and an amount of the copolymerized monomer introduced according to the purpose of use so as to maximize the effect of biological safety. It is possible to optimize.
ビニル系ポリマー（Ａ）は、全モノマーの合計１００質量％中、一般式（１）〜（５）で表されるモノマーを５質量％以上含むことが好ましく、２０質量％以上含むことがより好ましく、５０質量％以上含むことが更に好ましく５５質量％以上であることが更に好ましい。上記範囲とすることで、培養液に溶解させることができ、取り扱いが容易となる。 The vinyl-based polymer (A) preferably contains 5% by mass or more of the monomers represented by the general formulas (1) to (5), and more preferably 20% by mass or more, based on 100% by mass of all the monomers. , 50% by mass or more is more preferable, and 55% by mass or more is further preferable. Within the above range, it can be dissolved in the culture solution and can be easily handled.
ビニル系ポリマー（Ａ）は、前記一般式（１）〜（５）で表されるモノマーの単独重合体であってもよく、共重合体であってもよい。共重合体の場合、ブロック共重合体、ランダム共重合体、交互共重合体のいずれであってもよい。
また、一般式（１）〜（５）のうち一般式（１）または一般式（２）で示されるモノマーが好ましい。
The vinyl-based polymer (A) may be a homopolymer or a copolymer of the monomers represented by the general formulas (1) to (5). In the case of a copolymer, it may be a block copolymer, a random copolymer, or an alternating copolymer.
Further, among the general formulas (1) to (5), the monomer represented by the general formula (1) or the general formula (2) is preferable.
また、ビニル系ポリマー（Ａ）は、公知の方法により合成できる。例えば、溶液重合、会場重合、乳化重合、分散（沈殿）重合などが好ましいが、各モノマーを所定の溶媒に溶解した溶液に重合開始剤を加えてラジカル重合する溶液重合がより好ましい。 Further, the vinyl polymer (A) can be synthesized by a known method. For example, solution polymerization, venue polymerization, emulsion polymerization, dispersion (precipitation) polymerization and the like are preferable, but solution polymerization in which a polymerization initiator is added to a solution in which each monomer is dissolved in a predetermined solvent and radical polymerization is more preferable.
一般式（１）で示されるモノマーについて説明する。
一般式（１）
The monomer represented by the general formula (1) will be described.
General formula (1)
一般式（１）中、Ｒ２、Ｒ３で示されるアルキル基の炭素数は好ましくは１〜３である。また、アルキル基は直鎖上でも分岐鎖状でも良く、好適な具体例としては、メチル基、エチル基、ｎ−プロピル基、イソプロピル基が挙げられる。 In the general formula (1), the number of carbon atoms of the alkyl group represented by R 2 and R 3 is preferably 1 to 3. Further, the alkyl group may be linear or branched, and suitable specific examples thereof include a methyl group, an ethyl group, an n-propyl group and an isopropyl group.
また、Ｒ２、Ｒ３で示されるヒドロキシアルキル基の炭素数は、好ましくは１〜４であり、より好ましくは１〜２である。ヒドロキシアルキル基に含まれるアルキル基は直鎖状でも分岐鎖状でもよく、ヒドロキシアルキル基の好適な具体例としては、ヒドロキシメチル基、ヒドロキシエチル基、ヒドロキシプロピル基、ヒドロキシイソプロピル基が挙げられる。なお、ヒドロキシイソプロピル基におけるヒドロキシ基の置換位置は任意である。 The hydroxyalkyl group represented by R 2 and R 3 has preferably 1 to 4 carbon atoms, more preferably 1 to 2 carbon atoms. The alkyl group contained in the hydroxyalkyl group may be linear or branched, and preferred specific examples of the hydroxyalkyl group include a hydroxymethyl group, a hydroxyethyl group, a hydroxypropyl group and a hydroxyisopropyl group. The position of substitution of the hydroxy group in the hydroxyisopropyl group is arbitrary.
また、Ｒ２、Ｒ３で示されるジアルキルアミノプロピル基としては、ジメチルアミノプロピル基が挙げられる。 Further, examples of the dialkylaminopropyl group represented by R 2 and R 3 include a dimethylaminopropyl group.
Ｒ２、Ｒ３としては、水素原子、炭素数１〜６のアルキル基または炭素数１〜６のヒドロキシアルキル基が好ましい。 As R 2 and R 3 , a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a hydroxyalkyl group having 1 to 6 carbon atoms is preferable.
一般式（１）の構造を有するモノマーとしては、例えば、
ジメチル（メタ）アクリルアミド、ジエチル（メタ）アクリルアミド、Ｎ−イソプロピル（メタ）アクリルアミド、Ｎ−（ヒドロキシメチル）（メタ）アクリルアミド、Ｎ−（２−ヒドロキシエチル）（メタ）アクリルアミド、Ｎ，Ｎ−ジメチルアミノプロピル（メタ）アクリルアミド等が挙げられる。
Examples of the monomer having the structure of the general formula (1) include, for example.
Dimethyl (meth) acrylamide, diethyl (meth) acrylamide, N-isopropyl (meth) acrylamide, N- (hydroxymethyl) (meth) acrylamide, N- (2-hydroxyethyl) (meth) acrylamide, N, N-dimethylamino Examples thereof include propyl (meth) acrylamide.
一般式（２）で示されるモノマーについて説明する。
一般式（２）
The monomer represented by the general formula (2) will be described.
General formula (2)
式（２）中、Ｒ５、Ｒ６で示されるアルカンジイル基の炭素数は、好ましくは１〜２である。
また、上記アルカンジイル基は直鎖状でも分岐鎖状でもよいが、直鎖状が好ましい。好適な具体例としては、メタン−１，１−ジイル基、エタン−１，２−ジイル基が挙げられる。
In the formula (2), the number of carbon atoms of the alkanediyl group represented by R 5 and R 6 is preferably 1 to 2.
The alkanediyl group may be linear or branched, but linear is preferable. Suitable specific examples include a methane-1,1-diyl group and an ethane-1,2-diyl group.
一般式（２）の構造を有するモノマーとしては、例えば、４−（メタ）アクリロイルモルホリン等が挙げられる。 Examples of the monomer having the structure of the general formula (2) include 4- (meth) acryloyl morpholine and the like.
一般式（３）で示されるモノマーについて説明する。
一般式（３）
The monomer represented by the general formula (3) will be described.
General formula (3)
一般式（３）中、Ｒ７で示されるアルカンジイル基の炭素数は、好ましくは３〜５である。
また、上記アルカンジイル基は直鎖状でも分岐鎖状でもよいが、直鎖状が好ましい。好適な具体例としては、プロパン−１，３−ジイル基、ブタン−１，４−ジイル基、ペンタン−１，５−ジイル基が挙げられる。
In the general formula (3), the number of carbon atoms of the alkanediyl group represented by R 7 is preferably 3 to 5.
The alkanediyl group may be linear or branched, but linear is preferable. Suitable specific examples include a propane-1,3-diyl group, a butane-1,4-diyl group, and a pentane-1,5-diyl group.
一般式（３）の構造を有するモノマーとしては、例えば、１−ビニル−２−ピロリドン、Ｎ−ビニル−ε−カプロラクタム等が挙げられる。 Examples of the monomer having the structure of the general formula (3) include 1-vinyl-2-pyrrolidone, N-vinyl-ε-caprolactam and the like.
一般式（４）で示されるモノマーについて説明する。
一般式（４）
The monomer represented by the general formula (4) will be described.
General formula (4)
一般式（４）の構造を有するモノマーとしては、例えば、
１−ビニルイミダゾール、２−メチル−１−ビニルイミダゾール、４−メチル−１−ビニルイミダゾール、５−メチル−１−ビニルイミダゾール等が挙げられる。
Examples of the monomer having the structure of the general formula (4) include, for example.
Examples thereof include 1-vinylimidazole, 2-methyl-1-vinylimidazole, 4-methyl-1-vinylimidazole, 5-methyl-1-vinylimidazole and the like.
一般式（５）で示されるモノマーについて説明する。
一般式（５）
The monomer represented by the general formula (5) will be described.
General formula (5)
一般式（５）の構造を有するモノマーとしては、例えば、
２−ビニルピリジン、３−ビニルピリジン、４−ビニルピリジン、２−メチル−３−ビニルピリジン、２−メチル−４−ビニルピリジン、３−メチル−４−ビニルピリジン、２−メチル−５−ビニルピリジン、３−メチル−５−ビニルピリジン、４−メチル−５−ビニルピリジン、２−ラウリル−４−ビニルピリジン、２−ラウリル−５−ビニルピリジン、２−（ｔ−ブチル）−４−ビニルピリジン、２−（ｔ−ブチル）−５−ビニルピリジン等が挙げられる。
Examples of the monomer having the structure of the general formula (5) include, for example.
2-Vinylpyridine, 3-vinylpyridine, 4-vinylpyridine, 2-methyl-3-vinylpyridine, 2-methyl-4-vinylpyridine, 3-methyl-4-vinylpyridine, 2-methyl-5-vinylpyridine , 3-Methyl-5-vinylpyridine, 4-methyl-5-vinylpyridine, 2-lauryl-4-vinylpyridine, 2-lauryl-5-vinylpyridine, 2- (t-butyl) -4-vinylpyridine, Examples thereof include 2- (t-butyl) -5-vinylpyridine.
＜炭素数１〜１８のアルキル基を有するモノマー（ａ）＞
ビニル系ポリマー（Ａ）を得る際に、一般式（１）〜（５）に記載したモノマーの他に、炭素数１〜１８のアルキル基を有するモノマー（ａ）を用いることができる。炭素数１〜１８のアルキル基を有するモノマー（ａ）に基づく構造の導入により、極性やＴｇが適切に制御され、溶媒溶解性等を制御することができる。
<Monomer (a) having an alkyl group having 1 to 18 carbon atoms>
When obtaining the vinyl polymer (A), in addition to the monomers described in the general formulas (1) to (5), the monomer (a) having an alkyl group having 1 to 18 carbon atoms can be used. By introducing a structure based on the monomer (a) having an alkyl group having 1 to 18 carbon atoms, the polarity and Tg can be appropriately controlled, and the solvent solubility and the like can be controlled.
モノマー（ａ）としては、特に限定はされないが、例えば、
メチル(メタ)アクリレート、エチル(メタ)アクリレート、プロピル(メタ)アクリレート、ブチル（メタ）アクリレート、ペンチル（メタ）アクリレート、ヘプチル（メタ）アクリレート、２−エチルヘキシル（メタ）アクリレート、オクチル（メタ）アクリレート、ノニル（メタ）アクリレート、デシル（メタ）アクリレート、ドデシル（メタ）アクリレート、ステアリル（メタ）アクリレート、セチル（メタ）アクリレート、シクロヘキシル（メタ）アクリレートなどのアルキル（メタ）アクリレートなどが挙げられる。
The monomer (a) is not particularly limited, but for example,
Methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, butyl (meth) acrylate, pentyl (meth) acrylate, heptyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, octyl (meth) acrylate, Examples thereof include alkyl (meth) acrylates such as nonyl (meth) acrylate, decyl (meth) acrylate, dodecyl (meth) acrylate, stearyl (meth) acrylate, cetyl (meth) acrylate, and cyclohexyl (meth) acrylate.
＜（ａ）以外のモノマー（ｂ）＞
さらに、ビニル系ポリマーを得る際に、前記モノマー（ａ）以外のモノマー（ｂ）を用いてもよい。（ａ）以外のモノマー（ｂ）は、一般式（１）〜（５）記載のモノマーやモノマー（ａ）と共重合可能であり、モノマー（ａ）以外の、１分子中に１つのエチレン性不飽和基を有するモノマーであることが好ましい。
<Monomer (b) other than (a)>
Further, when obtaining a vinyl polymer, a monomer (b) other than the monomer (a) may be used. The monomer (b) other than the monomer (a) can be copolymerized with the monomers and the monomers (a) represented by the general formulas (1) to (5), and has one ethylenic property in one molecule other than the monomer (a). It is preferably a monomer having an unsaturated group.
モノマー（ｂ）としては、特に限定はされないが、
フェニル（メタ）アクリレート、ベンジル（メタ）アクリレート、フェノキシエチル（メタ）アクリレート等の芳香族エステル（メタ）アクリレート；
パーフルオロメチルメチル（メタ）アクリレート、パーフルオロエチルメチル（メタ）アクリレート、２−パーフルオロブチルエチル（メタ）アクリレート、２−パーフルオロヘキシルエチル（メタ）アクリレート、２−パーフルオロオクチルエチル（メタ）アクリレート、２−パーフルオロイソノニルエチル（メタ）アクリレート、２−パーフルオロノニルエチル（メタ）アクリレート、２−パーフルオロデシルエチル（メタ）アクリレート、パーフルオロプロピルプロピル（メタ）アクリレート、パーフルオロオクチルプロピル（メタ）アクリレート、パーフルオロオクチルアミル（メタ）アクリレート、パーフルオロオクチルウンデシル（メタ）アクリレートなどの炭素数１〜２０のパーフルオロアルキル基を有するパーフルオロアルキル基含有エチレン性不飽和モノマーなどの（メタ）アクリレート系モノマー；
２−ヒドロキシエチル（メタ）アクリレート、２−ヒドロキシプロピル（メタ）アクリレート、４−ヒドロキシブチル（メタ）アクリレート、２−（メタ）アクリロイロキシエチル−２−ヒドロキシエチルフタル酸、グリセロールモノ（メタ）アクリレート、４−ヒドロキシビニルベンゼン、１−エチニル−１−シクロヘキサノール、アリルアルコールなどの水酸基を有するモノマー；
マレイン酸、フマル酸、イタコン酸、シトラコン酸、又は、これらのアルキル若しくはアルケニルモノエステル、フタル酸β−（メタ）アクリロキシエチルモノエステル、イソフタル酸β−（メタ）アクリロキシエチルモノエステル、テレフタル酸β−（メタ）アクリロキシエチルモノエステル、コハク酸β−（メタ）アクリロキシエチルモノエステル、アクリル酸、メタクリル酸、クロトン酸、けい皮酸などのカルボン酸基、若しくはその無水物を有するモノマー；
ビニルスルホン酸、スチレンスルホン酸などのスルホン酸基を有するモノマー；
（２−ヒドロキシエチル）メタクリレートアシッドホスフェートなどのリン酸基を有するモノマー；
ポリエチレングリコール（メタ）アクリレート、メトキシポリエチレングリコール（メタ）アクリレート、エトキシポリエチレングリコール（メタ）アクリレート、プロポキシポリエチレングリコール（メタ）アクリレート、ｎ−ブトキシポリエチレングリコール（メタ）アクリレート、ｎ−ペンタキシポリエチレングリコール（メタ）アクリレート、フェノキシポリエチレングリコール（メタ）アクリレート、ポリプロピレングリコール（メタ）アクリレート、メトキシポリプロピレングリコール（メタ）アクリレート、エトキシポリプロピレングリコール（メタ）アクリレート、プロポキシポリプロピレングリコール（メタ）アクリレート、ｎ−ブトキシポリプロピレングリコール（メタ）アクリレート、ｎ−ペンタキシポリプロピレングリコール（メタ）アクリレート、フェノキシポリプロピレングリコール（メタ）アクリレート、ポリテトラメチレングリコール（メタ）アクリレート、メトキシポリテトラメチレングリコール（メタ）アクリレート、フェノキシテトラエチレングリコール（メタ）アクリレート、ヘキサエチレングリコール（メタ）アクリレート、メトキシヘキサエチレングリコール（メタ）アクリレートなどのポリエーテル鎖を有するモノマー；
ラクトン変性（メタ）アクリレートなどのポリエステル鎖を有するエチレン性不飽和化合物などの側鎖に高分子構造を有する（メタ）アクリレート系モノマー；
スチレン、α−メチルスチレン、２−メチルスチレン、クロロスチレン、アリルベンゼン、エチニルベンゼン等の芳香族ビニルモノマー；
（メタ）アクリロニトリルなどのニトリル基含有エチレン性不飽和モノマー；
酢酸ビニル、酪酸ビニル、プロピオン酸ビニル、ヘキサン酸ビニル、カプリル酸ビニル、ラウリル酸ビニル、パルミチン酸ビニル、ステアリン酸ビニルなどの脂肪酸ビニル系化合物；
ブチルビニルエーテル、エチルビニルエーテルなどのビニルエーテル系エチレン性不飽和モノマー；
酢酸アリル、シアン化アリルなどのアリルモノマー；
シアン化ビニル、ビニルシクロヘキサン、ビニルメチルケトンなどのビニルモノマー；
アセチレン、エチニルトルエンなどのエチニルモノマーパーフルオロブチルエチレン、パーフルオロヘキシルエチレン、パーフルオロオクチルエチレン、パーフルオロデシルエチレンなどのパーフルオロアルキル、アルキレン類などのパーフルオロアルキル基含有エチレン性不飽和化合物等の、（メタ）アクリレートではないエチレン性不飽和結合を有するモノマーなどが挙げられる。
モノマー（ｂ）としては、カルボン酸基、若しくはその無水物を有するモノマー、
スルホン酸基を有するモノマー、リン酸基を有するモノマー等のアニオン性モノマーを実質的に含まないことが好ましい。
The monomer (b) is not particularly limited, but is not particularly limited.
Aromatic ester (meth) acrylates such as phenyl (meth) acrylate, benzyl (meth) acrylate, and phenoxyethyl (meth) acrylate;
Perfluoromethylmethyl (meth) acrylate, perfluoroethylmethyl (meth) acrylate, 2-perfluorobutylethyl (meth) acrylate, 2-perfluorohexylethyl (meth) acrylate, 2-perfluorooctylethyl (meth) acrylate , 2-Perfluoroisononyl ethyl (meth) acrylate, 2-perfluorononyl ethyl (meth) acrylate, 2-perfluorodecylethyl (meth) acrylate, perfluoropropylpropyl (meth) acrylate, perfluorooctylpropyl (meth) ) Perfluoroalkyl group-containing ethylenically unsaturated monomers having 1 to 20 carbon atoms such as acrylates, perfluorooctyl amyl (meth) acrylates, and perfluorooctyl undecyl (meth) acrylates (meth). Acrylate-based monomer;
2-Hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, 4-hydroxybutyl (meth) acrylate, 2- (meth) acryloyloxyethyl-2-hydroxyethylphthalic acid, glycerol mono (meth) acrylate , 4-Hydroxyvinylbenzene, 1-ethynyl-1-cyclohexanol, allyl alcohol, and other monomers having hydroxyl groups;
Maleic acid, fumaric acid, itaconic acid, citraconic acid, or alkyl or alkenyl monoesters of these, phthalic acid β- (meth) acryloxyethyl monoester, isophthalic acid β- (meth) acryloxyethyl monoester, terephthalic acid Monomer having a carboxylic acid group such as β- (meth) acryloxyethyl monoester, succinic acid β- (meth) acryloxyethyl monoester, acrylic acid, methacrylic acid, crotonic acid, carcinic acid, or an anhydride thereof;
Monomer having a sulfonic acid group such as vinyl sulfonic acid and styrene sulfonic acid;
Monomer having a phosphate group such as (2-hydroxyethyl) methacrylate acid phosphate;
Polyethylene glycol (meth) acrylate, methoxypolyethylene glycol (meth) acrylate, ethoxypolyethylene glycol (meth) acrylate, propoxypolyethylene glycol (meth) acrylate, n-butoxypolyethylene glycol (meth) acrylate, n-pentoxypolyethylene glycol (meth) Acrylate, phenoxypolyethylene glycol (meth) acrylate, polypropylene glycol (meth) acrylate, methoxypolypropylene glycol (meth) acrylate, ethoxypolypropylene glycol (meth) acrylate, propoxypolyethylene glycol (meth) acrylate, n-butoxypolyethylene glycol (meth) acrylate , N-pentoxypolypropylene glycol (meth) acrylate, phenoxypolyethylene glycol (meth) acrylate, polytetramethylene glycol (meth) acrylate, methoxypolytetramethylene glycol (meth) acrylate, phenoxytetraethylene glycol (meth) acrylate, hexaethylene Monomer having a polyether chain such as glycol (meth) acrylate and methoxyhexaethylene glycol (meth) acrylate;
A (meth) acrylate-based monomer having a polymer structure in the side chain of an ethylenically unsaturated compound having a polyester chain such as a lactone-modified (meth) acrylate;
Aromatic vinyl monomers such as styrene, α-methylstyrene, 2-methylstyrene, chlorostyrene, allylbenzene, ethynylbenzene;
Nitrile group-containing ethylenically unsaturated monomers such as (meth) acrylonitrile;
Fatty acid vinyl compounds such as vinyl acetate, vinyl butyrate, vinyl propionate, vinyl hexanoate, vinyl caprylate, vinyl laurate, vinyl palmitate, vinyl stearate;
Vinyl ether-based ethylenically unsaturated monomers such as butyl vinyl ether and ethyl vinyl ether;
Allyl monomers such as allyl acetate and allyl cyanide;
Vinyl monomers such as vinyl cyanide, vinyl cyclohexane, vinyl methyl ketone;
Ethynyl monomers such as acetylene and ethynyltoluene Perfluorobutylethylene, perfluorohexylethylene, perfluorooctylethylene, perfluoroalkyl such as perfluorodecylethylene, perfluoroalkyl group-containing ethylenically unsaturated compounds such as alkylenes, etc. Examples thereof include monomers having an ethylenically unsaturated bond that is not a (meth) acrylate.
As the monomer (b), a monomer having a carboxylic acid group or an anhydride thereof,
It is preferable that the anionic monomer such as a monomer having a sulfonic acid group and a monomer having a phosphoric acid group is substantially not contained.
＜重合開始剤＞
本発明では、重合開始剤としてラジカル重合開始剤を使用することが好ましい。ラジカル重合を開始する能力を有するものであれば使用でき、公知の油溶性重合開始剤や水溶性重合開始剤を用いることができる。
<Polymerization initiator>
In the present invention, it is preferable to use a radical polymerization initiator as the polymerization initiator. Any material having the ability to initiate radical polymerization can be used, and known oil-soluble polymerization initiators and water-soluble polymerization initiators can be used.
油溶性重合開始剤としては、例えば、ベンゾイルパーオキサイド、ｔｅｒｔ−ブチルパーオキシベンゾエート、ｔｅｒｔ−ブチルハイドロパーオキサイド、ｔｅｒｔ−ブチルパーオキシ（２−エチルヘキサノエート）、ｔｅｒｔ−ブチルパーオキシ−３，５，５−トリメチルヘキサノエート、ジ−ｔｅｒｔ−ブチルパーオキサイドなどの有機過酸化物；
２，２’−アゾビスイソブチロニトリル、２，２’−アゾビス−２，４−ジメチルバレロニトリル、２，２’−アゾビス（４−メトキシ−２，４−ジメチルバレロニトリル）、１，１’−アゾビス−シクロヘキサン−１−カルボニトリルなどのアゾビス化合物などが挙げられる。
これらは１種類または２種類以上を混合して使用することができる。
Examples of the oil-soluble polymerization initiator include benzoyl peroxide, tert-butylperoxybenzoate, tert-butylhydroperoxide, tert-butylperoxy (2-ethylhexanoate), and tert-butylperoxy-3. Organic peroxides such as 5,5-trimethylhexanoate, di-tert-butyl peroxide;
2,2'-azobisisobutyronitrile, 2,2'-azobis-2,4-dimethylvaleronitrile, 2,2'-azobis (4-methoxy-2,4-dimethylvaleronitrile), 1,1 Examples thereof include azobis compounds such as'-azobis-cyclohexane-1-carbonitrile.
These can be used alone or in admixture of two or more.
水溶性重合開始剤としては、例えば、過硫酸アンモニウム、過硫酸カリウム、過酸化水素、２，２’−アゾビス（２−メチルプロピオンアミジン）ジハイドロクロライド、２，２’−アゾビス［Ｎ−（２−カルボキシエチル）−２−メチルプロピオンアミジン］・四水和物、４，４’−アゾビス（４−シアノ吉草酸）、２，２’−アゾビス［２−メチル−Ｎ−（２−ヒドロキシエチル）プロピオンアミド］、２，２’−アゾビス［２−（２−イミダゾリン−２−イル）プロパン］・二塩酸塩、２，２’ −アゾビス［２−（２−イミダゾリン−２−イル）プロパン］二硫酸塩・二水和物、２，２’−アゾビス［２−（２−イミダゾリン−２−イル）プロパン］など、従来既知のものを好適に使用することができる。 Examples of the water-soluble polymerization initiator include ammonium persulfate, potassium persulfate, hydrogen peroxide, 2,2'-azobis (2-methylpropionamidine) dihydrochloride, and 2,2'-azobis [N- (2- (2-). Carboxyethyl) -2-methylpropion amidine] -tetrahydrate, 4,4'-azobis (4-cyanovaleric acid), 2,2'-azobis [2-methyl-N- (2-hydroxyethyl) propion Amid], 2,2'-azobis [2- (2-imidazolin-2-yl) propane] -dihydrochloride, 2,2'-azobis [2- (2-imidazolin-2-yl) propane] disulfate Conventionally known substances such as salt / dihydrate and 2,2'-azobis [2- (2-imidazolin-2-yl) propane] can be preferably used.
これら重合開始剤は、モノマー１００質量部に対して、０．１〜１０質量部の量を用いることが好ましい。 It is preferable to use 0.1 to 10 parts by mass of these polymerization initiators with respect to 100 parts by mass of the monomer.
＜質量平均分子量（Ｍｗ）＞
ビニル系ポリマー（Ａ）の質量平均分子量は、好ましくは２，０００〜１０，０００，０００であり、より好ましくは５，０００以上であり、さらに好ましくは１０，０００以上であり、特に好ましくは２０，０００以上である。より好ましくは、５００，０００以下であり、特に好ましくは、２００，０００以下である。分子量が上記範囲であることにより、増粘作用を低下させゲル化等の不具合を防ぐことができる。またピペット操作などの取り扱いが容易になる。
<Mass average molecular weight (Mw)>
The mass average molecular weight of the vinyl polymer (A) is preferably 2,000 to 1,000,000, more preferably 5,000 or more, still more preferably 10,000 or more, and particularly preferably 20. It is over 000. More preferably, it is 500,000 or less, and particularly preferably 200,000 or less. When the molecular weight is in the above range, the thickening action can be reduced and problems such as gelation can be prevented. In addition, handling such as pipette operation becomes easy.
（質量平均分子量（Ｍｗ）の測定方法）
ビニル系ポリマー（Ａ）の質量平均分子量は、ゲルパーミエーションクロマトグラフィー（ＧＰＣ）によって標準プルラン換算で計測した値を採用した。測定装置及び測定条件としては下記条件１を用いた。その他の事項については、ＪＩＳＫ７２５２−１〜４：２００８を参照した。なお、難溶の高分子化合物については下記条件の下、溶解可能な濃度で測定した。また、条件１での分子量測定が困難な場合は、下記条件２によることを基本とした。
(Measurement method of mass average molecular weight (Mw))
For the mass average molecular weight of the vinyl polymer (A), a value measured by gel permeation chromatography (GPC) in terms of standard pullulan was adopted. The following condition 1 was used as the measuring device and the measuring conditions. For other matters, JIS K7252-1 to 4: 2008 was referred to. The poorly soluble polymer compound was measured at a soluble concentration under the following conditions. When it is difficult to measure the molecular weight under condition 1, the condition 2 below is basically used.
（条件１）
カラム：ＯＨｐａｋ ＳＢ−Ｇ、
ＯＨｐａｋ ＳＢ−８０６Ｍ ＨＱを３本及び、
ＯＨｐａｋ ＳＢ−８０２．５ ＨＱを連結したもの。
キャリア：１／１５ ｍｏｌ／Ｌ ｐＨ７．０ リン酸緩衝液
（りん酸緩衝剤粉末１／１５ ｍｏｌ／Ｌ ｐＨ７．０（富士フイルム和光純薬(株)製）をイオン交換水１Ｌに溶解させたもの）
キャリア流量：１．０ｍＬ／ｍｉｎ
試料濃度：０．５質量％
検出器：ＲＩ（屈折率）検出器
注入量：０．１ｍＬ
(Condition 1)
Column: OHpak SB-G,
Three OHpak SB-806M HQs and
OHpak SB-802.5 HQ is connected.
Carrier: 1/15 mol / L pH 7.0 Phosphate buffer (phosphate buffer powder 1/15 mol / L pH 7.0 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 1 L of ion-exchanged water. thing)
Carrier flow rate: 1.0 mL / min
Sample concentration: 0.5% by mass
Detector: RI (refractive index) detector Injection volume: 0.1 mL
（条件２）
カラム：ＴＯＳＯＨＴＳＫｇｅｌＳｕｐｅｒＡＷ４０００、
ＴＯＳＯＨＴＳＫｇｅｌＳｕｐｅｒＡＷ３０００ 及び
ＴＯＳＯＨＴＳＫｇｅｌＳｕｐｅｒＡＷ２５００を連結したもの。
キャリア：Ｎ，Ｎ−ジメチルホルムアミド（１Ｌ）、トリエチルアミン（３．０４ｇ）、ＬｉＢｒ（０．８７ｇ）の混合液
測定温度：４０℃
キャリア流量：０．６ｍＬ／ｍｉｎ
(Condition 2)
Column: TOSOHTSKgelSuperAW4000,
A combination of TOSOHTSKgelSuperAW3000 and TOSOHTSKgelSuperAW2500.
Carrier: Mixture of N, N-dimethylformamide (1L), triethylamine (3.04g), LiBr (0.87g) Measurement temperature: 40 ° C.
Carrier flow rate: 0.6 mL / min
＜細胞用培地＞
ビニル系ポリマー（Ａ）を添加する細胞用培地としては、従来公知の細胞用培地を使用することができる、例えば、市販されている各種培地（αＭＥＭ、ＭＥＭ、ＤＭＥＭ、ＩＭＤＥＭ、ＲＰＭＩ１６４０、ＤＭＥＭ／Ｆ１２等）や、これらの組み合わせが挙げられる。
細胞用培地添加剤であるビニル系ポリマー（Ａ）の培地中の濃度は０．００１〜０．５質量％が好ましく、０．０１〜０．５質量％がより好ましく、０．０５〜０．５質量％がさらに好ましい。
<Medium for cells>
As the cell medium to which the vinyl polymer (A) is added, a conventionally known cell medium can be used, for example, various commercially available media (αMEM, MEM, DMEM, IMDEM, RPMI1640, DMEM / F12). Etc.) and combinations of these.
The concentration of the vinyl polymer (A), which is a medium additive for cells, in the medium is preferably 0.001 to 0.5% by mass, more preferably 0.01 to 0.5% by mass, and 0.05 to 0. 5% by mass is more preferable.
細胞用培地には、必要に応じて、各種増殖因子（上皮成長因子やインスリン様成長因子、神経成長因子、肝細胞増殖因子、血管内皮増殖因子、塩基性繊維芽細胞増殖因子、トランスフェリン、ステロイドホルモン、２−メルカプトエタノール等）や各種動物血清（ウシ胎児血清（ＦＢＳ）やウシ血清等）、血清代替物などを添加するのが好ましい。 Various growth factors (epithelial growth factor, insulin-like growth factor, nerve growth factor, hepatocellular growth factor, vascular endothelial growth factor, basic fibroblast growth factor, transferrin, steroid hormone) are added to the cell medium as needed. , 2-Mercaptoethanol, etc.), various animal serums (fetal bovine serum (FBS), bovine serum, etc.), serum substitutes, etc. are preferably added.
＜生理活性物質の製造方法＞
本発明の細胞用培地中で細胞を培養することで、生理活性物質を高効率で生産することができる。
本実施形態に係る生理活性物質の製造方法では、前述のビニル系ポリマー（Ａ）を含む細胞用培地添加剤を含む細胞用培地を用いて、細胞を培養することを含む。
<Manufacturing method of bioactive substances>
By culturing cells in the cell medium of the present invention, a physiologically active substance can be produced with high efficiency.
The method for producing a physiologically active substance according to the present embodiment includes culturing cells using a cell culture medium containing the above-mentioned cell culture medium additive containing the vinyl polymer (A).
本実施形態に係る生理活性物質とは、例えば、抗体タンパク質（以下抗体という）やアルブミンなどである。ここで産生される抗体は特に限定されず、例えば、マウスモノクローナル抗体、ヒト化モノクローナル抗体またはヒトモノクローナル抗体である。また、免疫グロブリンのクラスは特に限定されないが、例えば、ＩｇＧ（例えば、ＩｇＧ１、ＩｇＧ２など）である。 The physiologically active substance according to the present embodiment is, for example, an antibody protein (hereinafter referred to as an antibody), albumin, or the like. The antibody produced here is not particularly limited, and is, for example, a mouse monoclonal antibody, a humanized monoclonal antibody, or a human monoclonal antibody. The class of immunoglobulin is not particularly limited, but is, for example, IgG (for example, IgG1, IgG2, etc.).
本発明の培養方法で培養される動物細胞は特に限定されず、細胞については生理活性物質の生産に使用可能な細胞であれば特に限定されず、特に抗体生産可能な細胞であれば、例えば、ＣＨＯ細胞、ＢＨＫ細胞、ＨｅｐＧ２細胞、ｒｏｄｅｎｔｍｙｅｌｏｍａ細胞、（ＳＰ２／Ｏ細胞、ＮＳＯ細胞等のマウス骨髄腫細胞など）、ハイブリドーマ、昆虫細胞、および、それらの細胞に外来遺伝子を導入した形質転換細胞が挙げられるが、例えばＣＨＯ細胞、ＳＰ２/Ｏ細胞またはＮＳＯ細胞等を細胞融合することによって得られるハイブリドーマ等を抗体産生細胞として採用することができる。 The animal cells cultured by the culture method of the present invention are not particularly limited, and the cells are not particularly limited as long as they can be used for the production of physiologically active substances, and particularly any cells capable of producing antibodies, for example. CHO cells, BHK cells, HepG2 cells, rodentmyeloma cells, mouse myeloma cells such as SP2 / O cells and NSO cells, hybridomas, insect cells, and transformed cells in which foreign genes have been introduced into these cells. However, for example, a hybridoma obtained by fusing CHO cells, SP2 / O cells, NSO cells, or the like can be adopted as an antibody-producing cell.
本発明の培養方法または製造方法において、培養を行う際、通常培養に用いられている容器または装置を使用することができる。例えば、マルチウェルプレート、シャーレ、培養フラスコ、スピナーフラスコ、ジャーファーメンター、ファーメンター、ローラーボトル、ホローファイバー、マイクロキャリアーなどが挙げられる。 In the culturing method or production method of the present invention, when culturing, the container or device usually used for culturing can be used. Examples include multi-well plates, petri dishes, culture flasks, spinner flasks, jar fermenters, fermenters, roller bottles, hollow fibers, microcarriers and the like.
本実施形態における培養条件は、通常の動物細胞の培養条件でよく、例えば、５体積％ＣＯ２雰囲気下で、温度３７℃である条件とすることができる。 The culture conditions in the present embodiment may be normal animal cell culture conditions, for example, a condition in which the temperature is 37 ° C. under a 5% by volume CO 2 atmosphere.
培養液から細胞を採取するには、浮遊細胞の場合は、例えば、培養液を直接遠心分離機やろ過機にかけて集める。接着細胞の場合は、例えば０．２５％トリプシン−０．０２％ＥＤＴＡ液を添加して細胞を浮遊させた後、遠心分離やろ過により集める。 To collect cells from the culture medium, in the case of suspended cells, for example, the culture medium is directly collected by centrifuging or filtering. In the case of adherent cells, for example, 0.25% trypsin-0.02% EDTA solution is added to suspend the cells, and then the cells are collected by centrifugation or filtration.
細胞培養によって生産される生理活性物質、特に抗体は、その物質が培養液中に蓄積される場合、ろ過または遠心分離により上澄み液を得、これから採取される。また、細胞内に蓄積される物質の場合には、ろ過または遠心分離により得た細胞をホモジナイズ、超音波処理、化学薬品処理等を施し、生産物を抽出した上澄み液を得る。 Physiologically active substances produced by cell culture, especially antibodies, are collected from the supernatant obtained by filtration or centrifugation when the substance accumulates in the culture medium. In the case of substances accumulated in cells, the cells obtained by filtration or centrifugation are subjected to homogenization, sonication, chemical treatment, etc. to obtain a supernatant from which the product has been extracted.
上記上澄みから抗体を分離、精製するには、公知の方法を適宜組み合わせて行う。例えば、硫安沈殿、透析、限外ろ過、電気泳動、ゲルろ過、イオン交換クロマトグラフィ、逆相クロマトグラフィ、アフィニティクロマトグラフィなどが好ましい。 In order to separate and purify the antibody from the above supernatant, a known method is appropriately combined. For example, ammonium sulfate precipitation, dialysis, ultrafiltration, electrophoresis, gel filtration, ion exchange chromatography, reverse phase chromatography, affinity chromatography and the like are preferable.
以下に、実施例により本発明をさらに具体的に説明するが、以下の実施例は本発明の権利範囲を何ら制限するものではない。尚、実施例及び比較例における「部」及び「％」は、「質量部」及び「質量％」を表し、ｍｏｌは物質量を表し、ｍｏｌ％は全単量体中の物質量の割合を表す。 Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples do not limit the scope of rights of the present invention at all. In addition, "part" and "%" in Examples and Comparative Examples represent "parts by mass" and "mass%", mol represents the amount of substance, and mol% represents the ratio of the amount of substance in all the monomers. show.
＜抗体産生細胞用培地添加剤の製造＞
［実施例１］
（細胞用培地添加剤１）
温度計、撹拌機、窒素導入管、還流冷却器、滴下管を備えた反応容器に、窒素気流下、溶媒として水１５０質量部を仕込み、撹拌下８０℃で３０分加熱した。滴下管にモノマーとしてＮ，Ｎ−ジメチルアクリルアミドを１００質量部、重合開始剤として２，２′−アゾビス（２−アミジノプロパン）二塩酸塩（富士フイルム和光純薬社製、Ｖ５０）を０．５質量部、溶媒として水を１０質量部仕込み、２時間かけて滴下した。滴下終了後６時間加熱を継続した。その後、室温に冷却し反応を停止した。その後、真空ポンプで溶剤および未反応のモノマーを除去し、ビニル系ポリマー（Ａ−１）を得た。
得られたビニル系ポリマー（Ａ−１）の質量平均分子量は４５０００であった。
次に、２５℃のリン酸緩衝生理食塩水（以下、ＰＢＳという）９９ｇ中に、得られたビニル系ポリマー（Ａ−１）を１ｇ入れて撹拌して溶解させた後、２５℃で２４時間放置して、ＰＢＳで１質量％に希釈された細胞用培地添加剤１を得た。
その結果、ビニル系ポリマー（Ａ−１）の分離、析出ともに見られず、完全に溶解可能であり、ＰＢＳに対して可溶であることが示された。
<Manufacturing of medium additives for antibody-producing cells>
[Example 1]
(Cell medium additive 1)
A reaction vessel equipped with a thermometer, a stirrer, a nitrogen introduction tube, a reflux condenser, and a dropping tube was charged with 150 parts by mass of water as a solvent under a nitrogen stream, and heated at 80 ° C. for 30 minutes under stirring. In the dropping tube, 100 parts by mass of N, N-dimethylacrylamide as a monomer and 0.5 of 2,2'-azobis (2-amidinopropane) dihydrochloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., V50) as a polymerization initiator. 10 parts by mass and 10 parts by mass of water were charged as a solvent, and the mixture was added dropwise over 2 hours. Heating was continued for 6 hours after the completion of the dropping. Then, it was cooled to room temperature and the reaction was stopped. Then, the solvent and the unreacted monomer were removed by a vacuum pump to obtain a vinyl polymer (A-1).
The mass average molecular weight of the obtained vinyl polymer (A-1) was 45,000.
Next, 1 g of the obtained vinyl-based polymer (A-1) was added to 99 g of phosphate buffered saline (hereinafter referred to as PBS) at 25 ° C., stirred and dissolved, and then dissolved at 25 ° C. for 24 hours. The mixture was left to obtain 1 mass% diluted cell medium additive 1.
As a result, neither separation nor precipitation of the vinyl polymer (A-1) was observed, and it was shown that it was completely soluble and soluble in PBS.
［実施例２〜１８、比較例１、２］
（細胞用培地添加剤２〜２０）
表１に示す組成に変更した以外は、細胞用培地添加剤１と同様にして、ビニル系ポリマー（Ａ−２〜１８）及び、ビニル系ポリマー（Ａ−１９、２０）を得た後、ＰＢＳに溶解させて、ＰＢＳで１質量％に希釈された細胞用培地添加剤２〜２０を得た。
[Examples 2 to 18, Comparative Examples 1 and 2]
(Cell medium additives 2 to 20)
PVC-based polymers (A-2 to 18) and vinyl-based polymers (A-19, 20) were obtained in the same manner as in Cell Medium Additive 1, except that the compositions were changed to those shown in Table 1. To give cells medium additives 2-20 diluted with PBS to 1% by mass.
表１中の略称を以下に示す。
ＤＭＡＡ：Ｎ，Ｎ−ジメチルアクリルアミド
ＨＥＡＡ： Ｎ−ヒドロキシエチルアクリルアミド
ＤＭＡＰＡＡ：Ｎ，Ｎ−ジメチルアミノプロピルアクリルアミド
ＡＣＭＯ：アクリロイルモルホリン
ＮＶＰ：N-ビニル-2-ピロリドン
ＶＩ：1-ビニルイミダゾール
ＶＰ：4-ビニルピリジン
ＢＡ：ｎ−ブチルアクリレート
２ＥＨＡ：２−エチルヘキシルアクリレート
ＩＳＴＡ：イソステアリルアクリレート
Ｓｔ：スチレン
ＨＥＡ：２−ヒドロキシエチルアクリレート
ＡＭＥ−４００：メトキシポリエチレングリコールアクリレート
The abbreviations in Table 1 are shown below.
DMAA: N, N-dimethylacrylamide HEAA: N-hydroxyethylacrylamide DMAPAA: N, N-dimethylaminopropyl acrylamide ACMO: Acryloylmorpholin NVP: N-vinyl-2-pyrrolidone VI: 1-vinylimidazole VP: 4-vinylpyridine BA: n-butyl acrylate 2EHA: 2-ethylhexyl acrylate ISTA: isostearyl acrylate St: styrene HEA: 2-hydroxyethyl acrylate AME-400: methoxypolyethylene glycol acrylate
＜抗体産生細胞用培地添加剤の評価＞
得られた細胞用培地添加剤について、以下の評価を実施した。結果を表２に示す。
<Evaluation of medium additives for antibody-producing cells>
The following evaluations were carried out on the obtained cell culture medium additives. The results are shown in Table 2.
＜抗体生産性＞
培地はＤｙｎａｍｉｓ Ｍｅｄｉｕｍ（Ｇｉｂｃｏ製）を用い、Ｌ−Ｇｌｕｔａｍｉｎを１質量％になるように添加した。以後、これを基礎培地と呼ぶ。これを２セット用意し、得られた細胞用培地添加剤をビニル系ポリマー（Ａ）の濃度が０．０５質量％又は０．５質量％になるようにそれぞれ添加した。ビニル系ポリマー（Ａ）の濃度が異なる２種の培地にそれぞれＩｇＧ遺伝子を導入しＩｇＧ抗体を分泌産生するＣＨＯ細胞株（ＡＴＣＣ製ＣＲＬ−１２４５５）を加え、ＣＨＯ細胞株の濃度が１，０００，０００ｃｅｌｌｓ／ｍｌとなる細胞懸濁液を作製した。得られた細胞懸濁液２０ｍＬを、１２５ｍＬの三角フラスコに播種し、３７℃にて培養した。なお、培養中、栄養源が枯渇する前に３〜４日に一度、上澄み液１５ｍＬを回収し、新たな基礎培地１５ｍＬと交換し、これを５回繰り返した。培地交換を５回繰り返した後の三角フラスコの上澄み液中の抗体量を、Ｂｅｔｈｙｌ Ｌａｂｏｒａｔｏｒｉｅｓ，Ｉｎｃ製のＨｕｍａｎ ＩｇＧ ＥＬＩＳＡ Ｑｕａｎｔｉｔａｔｉｏｎ Ｓｅｔを用いて測定した。抗体産生細胞用培地添加剤を加えないで培養した場合の成績を１とした場合の相対値で判定した。
ａ：１．２以上（非常に良好）
ｂ：１以上〜１．２未満（良好）
ｃ：１未満（不良）
<Antibody productivity>
The medium was Dynamis Medium (manufactured by Gibco), and L-Glutamine was added in an amount of 1% by mass. Hereinafter, this is referred to as a basal medium. Two sets of these were prepared, and the obtained cell medium additive was added so that the concentration of the vinyl polymer (A) was 0.05% by mass or 0.5% by mass, respectively. A CHO cell line (CRL-12455 manufactured by ATCC), which secretes and produces IgG antibody by introducing an IgG gene into each of two media having different concentrations of vinyl polymer (A), was added, and the concentration of the CHO cell line was 1,000. A cell suspension at 000 cells / ml was prepared. 20 mL of the obtained cell suspension was seeded in a 125 mL Erlenmeyer flask and cultured at 37 ° C. During culturing, 15 mL of the supernatant was collected once every 3 to 4 days before the nutrient source was depleted, replaced with a new basal medium of 15 mL, and this was repeated 5 times. The amount of antibody in the supernatant of the Erlenmeyer flask after repeating the medium exchange 5 times was measured using Human IgG ELISA Quantitation Set manufactured by Bethyl Laboratories, Inc. The result was determined by a relative value when the result when cultured without adding the medium additive for antibody-producing cells was 1.
a: 1.2 or more (very good)
b: 1 or more and less than 1.2 (good)
c: Less than 1 (defective)
＜アルブミン産生性＞
培地にはＯｐｉｔｉ−ＭＥＭ（Ｒｅｄｕｃｅｄ Ｓｅｒｕｍ Ｍｅｄｉｕｍ、ＧｌｕｔａＭＡＸ Ｓｕｐｐｌｅｍｅｎｔ）を使用した。Ｏｐｉｔｉ−ＭＥＭに細胞用培地添加剤をビニル系ポリマー（Ａ）の濃度が０．０５質量％又は０．５質量％になるようにそれぞれ添加し、攪拌をすることで培地組成物を調製した。ヒト肝がん細胞株ＨｅｐＧ２細胞を、２５０，０００細胞／ｍＬとなるように上記のビニル系ポリマー（Ａ）を含む細胞用培地添加剤を添加した培地組成物に播種した後、９６ウェルＵ底マイクロプレートのウェルに１ウェルあたり２００μＬになるように分注した。その後、本プレートをＣＯ２インキュベーター（３７℃、５％ＣＯ２）内にて静置状態で３日間培養した。３日間培養後の肝細胞を含む培養液を回収し、遠心分離にすることで培地上清を回収した。培地中のアルブミン量を、Ｂｅｔｈｙｌ Ｌａｂｏｒａｔｏｒｉｅｓ，Ｉｎｃ製Ａｌｂｕｍｉｎ ＥＬＩＳＡ Ｑｕａｎｔｉｔａｔｉｏｎ Ｓｅｔを用いて測定した。また、沈降した細胞群のＤＮＡ量も定量し、単位ＤＮＡ量あたりの抗体生産量として算出した。ビニル系ポリマー（Ａ）を含む細胞用培地添加剤を加えないで培養した場合の成績を１とした場合の相対値で判定した。評価結果を表２に示す。
ａ：１．２以上（非常に良好）
ｂ：１以上〜１．２未満（良好）
ｃ：１未満（不良）
<Albumin productivity>
Opti-MEM (Reduced Serum Medium, GlutaMAX Supplement) was used as the medium. A cell medium additive was added to Opiti-MEM so that the concentration of the vinyl polymer (A) was 0.05% by mass or 0.5% by mass, respectively, and the mixture was stirred to prepare a medium composition. Human hepatoma cell line HepG2 cells were seeded in a medium composition containing the above-mentioned vinyl-based polymer (A) -containing cell medium additive so as to have a concentration of 250,000 cells / mL, and then a 96-well U-bottom was added. The cells were dispensed into the wells of the microplate so as to be 200 μL per well. Then, this plate was cultured in a CO 2 incubator (37 ° C., 5% CO 2 ) in a stationary state for 3 days. After culturing for 3 days, the culture solution containing hepatocytes was collected, and the culture medium supernatant was collected by centrifugation. The amount of albumin in the medium was measured using Bethyl Laboratories, Inc. Albumin ELISA Quantification Set. In addition, the amount of DNA in the precipitated cell group was also quantified and calculated as the amount of antibody produced per unit amount of DNA. The result was determined by a relative value when the result when the cells were cultured without adding the cell culture medium additive containing the vinyl polymer (A) was 1. The evaluation results are shown in Table 2.
a: 1.2 or more (very good)
b: 1 or more and less than 1.2 (good)
c: Less than 1 (defective)
＜細胞のＡＴＰアッセイ＞
Ｏｐｉｔｉ−ＭＥＭに細胞用培地添加剤をビニル系ポリマー（Ａ）の濃度が０．０５質量％又は０．５質量％になるようにそれぞれ添加し、攪拌をすることで培地組成物を調製した。マウス線維芽細胞用細胞株（ＮＩＨ／３Ｔ３細胞）を、１００，０００細胞／ｍＬとなるように上記のビニル系ポリマー（Ａ）を含む細胞用培地添加剤を添加した培地組成物に播種した後、９６ウェルＵ底マイクロプレート（住友ベークライト製Ｐｒｉｍｅｓｕｒｆａｃｅ）のウェルに１ウェルあたり１００μＬになるように分注した。その後、本プレートをＣＯ２インキュベーター（３７℃、５％ＣＯ２）内にて静置状態で５日間培養した。
その後、５日目まで培養した細胞のＡＴＰ（アデノシン−５’−三リン酸）アッセイを行い、生細胞の割合を評価した。具体的には、培養後のウェルに１００μＬのＡＴＰ試薬（『塊』のＡＴＰ測定試薬：東洋ビーネット社製）を添加、５回ピペッティングし、５分間室温で静置した後、１００μＬの試薬・細胞溶解液を別プレートに分取し１分間撹拌した。これをＭｉｔｈｒａｓＬＢ９４０（Ｂｅｒｔｈｏｌｄ社製）を用いて発光量を測定した。
生細胞の割合＝（培養５日後の発光量）／（細胞用培地添加剤を加えずに５日間培養
した際の発光量）×１００
ａ：１２０％以上（非常に良好）
ｂ：１００％以上１２０％未満（良好）
ｃ：１００％未満（不良）
<Cell ATP assay>
A cell medium additive was added to Opiti-MEM so that the concentration of the vinyl polymer (A) was 0.05% by mass or 0.5% by mass, respectively, and the mixture was stirred to prepare a medium composition. After seeding a mouse fibroblast cell line (NIH / 3T3 cells) into a medium composition containing the above-mentioned vinyl-based polymer (A) -containing cell medium additive so as to have a concentration of 100,000 cells / mL. , 96-well U-bottom microplate (Prime-surface manufactured by Sumitomo Bakelite) was dispensed to 100 μL per well. Then, this plate was cultured in a CO 2 incubator (37 ° C., 5% CO 2 ) in a static state for 5 days.
Then, ATP (adenosine-5'-triphosphate) assay of the cells cultured up to the 5th day was performed to evaluate the proportion of living cells. Specifically, 100 μL of ATP reagent (“lump” ATP measurement reagent: manufactured by Toyo Beanet Co., Ltd.) is added to the well after culturing, pipetting 5 times, allowing to stand at room temperature for 5 minutes, and then 100 μL of reagent. -The cytolytic solution was separated into a separate plate and stirred for 1 minute. The amount of light emitted from this was measured using MisrasLB940 (manufactured by Berthold).
Percentage of living cells = (amount of luminescence after 5 days of culturing) / (amount of luminescence when cultivated for 5 days without adding a cell medium additive) × 100
a: 120% or more (very good)
b: 100% or more and less than 120% (good)
c: Less than 100% (defective)
表２に示すように、一般式（１）〜（５）で表される少なくともいずれかの構造を有するビニル系ポリマー（Ａ）を含む、本発明の細胞用培地添加剤を培地に添加することで、優れた抗体生産性、アルブミン産生性を示した。また、細胞のＡＴＰ活性も向上し、生細胞数が増えていることが示された。
これは、ビニル系ポリマー（Ａ）が一般式（１）〜（５）で表される少なくともいずれかの構造を有することで、シアル酸由来の負電荷を有する細胞との静電相互作用が制御され、抗体産生細胞の周囲の環境を適切に制御したため抗体産生細胞の抗体産生性、および株化細胞の増殖性、生存性の面で有効に働いたためであると考えられる。
As shown in Table 2, the cell medium additive of the present invention containing the vinyl polymer (A) having at least one of the structures represented by the general formulas (1) to (5) is added to the medium. It showed excellent antibody productivity and albumin productivity. It was also shown that the ATP activity of cells was improved and the number of living cells was increased.
This is because the vinyl-based polymer (A) has at least one of the structures represented by the general formulas (1) to (5), so that electrostatic interaction with cells having a negative charge derived from sialic acid is controlled. It is considered that this is because the environment around the antibody-producing cells was appropriately controlled, and thus the antibody-producing properties of the antibody-producing cells and the proliferative and viable properties of the established cells worked effectively.
それに対して、一般式（１）〜（５）で表される少なくともいずれかの構造を有しないビニル系ポリマーを使用した比較例１、２は、抗体生産性、アルブミン産生性、細胞のＡＴＰ活性も向上しなかった。これは、一般式（１）〜（５）で表される少なくともいずれかの構造を有していないことにより、細胞の周囲の環境を適切に制御できなかったためであると考えられる。 On the other hand, Comparative Examples 1 and 2 using a vinyl polymer not having at least one of the structures represented by the general formulas (1) to (5) show antibody productivity, albumin productivity, and cellular ATP activity. Did not improve. It is considered that this is because the environment around the cell could not be appropriately controlled because it did not have at least one of the structures represented by the general formulas (1) to (5).
Claims (6)
一般式（１）
一般式（４）
一般式（５）
［一般式（１）〜（５）中、
Ｒ１およびＲ４は、それぞれ独立して水素原子またはメチル基、
Ｒ２およびＲ３は、それぞれ独立して水素原子、炭素数１〜６のアルキル基、炭素数１〜６のヒドロキシアルキル基またはジアルキルアミノプロピル基
Ｒ５およびＲ６は、それぞれ独立して、炭素数１〜３のアルカンジイル基、
Ｒ７は、炭素数１〜５のアルカンジイル基、
Ｒ８〜Ｒ１０は、それぞれ独立して水素原子またはメチル基、
Ｒ１１〜Ｒ１５のうち４つは、それぞれ独立して、水素原子または炭素数１〜６のアルキル基を表し、Ｒ１１〜Ｒ１５のうち１つは、ビニル基を表す。］ A cell culture medium additive containing a vinyl polymer (A) having a structural unit derived from at least one of the monomers represented by the following general formulas (1) to (5).
General formula (1)
General formula (4)
General formula (5)
[In general formulas (1) to (5),
R 1 and R 4 are independent hydrogen atoms or methyl groups, respectively.
R 2 and R 3 are independent hydrogen atoms, alkyl groups having 1 to 6 carbon atoms, hydroxyalkyl groups having 1 to 6 carbon atoms or dialkylaminopropyl groups R 5 and R 6 are independent carbon atoms, respectively. Alkanediyl groups of numbers 1 to 3,
R 7 is an alkanediyl group having 1 to 5 carbon atoms.
R 8 to R 10 are independent hydrogen atoms or methyl groups, respectively.
Four of R 11 to R 15 independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and one of R 11 to R 15 represents a vinyl group. ]
A method for producing a physiologically active substance, which comprises culturing cells in the cell culture medium according to claim 4 or 5.
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