JP6634202B2 - Carrier, method for producing carrier, and method for purifying target substance - Google Patents
Carrier, method for producing carrier, and method for purifying target substance Download PDFInfo
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- JP6634202B2 JP6634202B2 JP2014168929A JP2014168929A JP6634202B2 JP 6634202 B2 JP6634202 B2 JP 6634202B2 JP 2014168929 A JP2014168929 A JP 2014168929A JP 2014168929 A JP2014168929 A JP 2014168929A JP 6634202 B2 JP6634202 B2 JP 6634202B2
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- 239000003446 ligand Substances 0.000 claims description 42
- 239000002245 particle Substances 0.000 claims description 33
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- 238000010494 dissociation reaction Methods 0.000 claims description 14
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- 239000002184 metal Substances 0.000 description 1
- 229910044991 metal oxide Inorganic materials 0.000 description 1
- 150000004706 metal oxides Chemical class 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical compound OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- UCUUFSAXZMGPGH-UHFFFAOYSA-N penta-1,4-dien-3-one Chemical compound C=CC(=O)C=C UCUUFSAXZMGPGH-UHFFFAOYSA-N 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920000058 polyacrylate Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000003505 polymerization initiator Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000003361 porogen Substances 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000012475 sodium chloride buffer Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- FTCLAXOKVVLHEG-UHFFFAOYSA-N sodium;3-sulfanylpropane-1-sulfonic acid Chemical group [Na].OS(=O)(=O)CCCS FTCLAXOKVVLHEG-UHFFFAOYSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 125000000213 sulfino group Chemical group [H]OS(*)=O 0.000 description 1
- 150000003460 sulfonic acids Chemical class 0.000 description 1
- DHCDFWKWKRSZHF-UHFFFAOYSA-N sulfurothioic S-acid Chemical compound OS(O)(=O)=S DHCDFWKWKRSZHF-UHFFFAOYSA-N 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000010557 suspension polymerization reaction Methods 0.000 description 1
- 229960003080 taurine Drugs 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 1
- 229960000575 trastuzumab Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Peptides Or Proteins (AREA)
Description
本発明は、担体、担体の製造方法、及び標的物の精製方法に関する。より詳細には、優れた防汚性を有する担体、担体の製造方法、及び標的物の精製方法に関する。 The present invention relates to a carrier, a method for producing the carrier, and a method for purifying a target. More specifically, the present invention relates to a carrier having excellent antifouling properties, a method for producing a carrier, and a method for purifying a target.
タンパク質等の標的物を、分離精製、定量又は検出する方法として、標的物に対して親和性又は反応性のある化合物（リガンド）を不溶性支持体上に固定した担体を用いる手法が知られている。
この担体には、標的物への結合能を備えることの他に、不純物が非特異吸着しにくいこと等が要求される。非特異吸着は、標的物の検出感度や定量性、精製した標的物を生体内に応用する場合の安全性に大きく影響を与えるため、これを低減することは重要である。
As a method for separating, purifying, quantifying, or detecting a target such as a protein, a method using a carrier in which a compound (ligand) having affinity or reactivity with the target is immobilized on an insoluble support is known. .
The carrier is required not only to have a binding ability to a target substance but also to be difficult for non-specific adsorption of impurities. Since non-specific adsorption greatly affects the detection sensitivity and quantification of the target substance and the safety when the purified target substance is applied to a living body, it is important to reduce the non-specific adsorption.
一方、担体には、天然高分子又は合成高分子で構成される有機系支持体を用いたもの、多孔性シリカゲル粒子等の無機系支持体を用いたものがある。天然高分子で構成される支持体を用いた担体として、多孔性アガロース粒子や多孔性デキストラン粒子に各種官能基を導入したもの等が知られている（特許文献１等）。斯様な天然高分子系の担体は非特異吸着しにくいものではあるが、物理的強度が小さいため耐圧性に欠け、高圧下での分析操作を行うことが困難という欠点がある。
他方、合成高分子で構成される支持体を用いた担体として、ポリアクリルアミドゲル、ポリアクリレートゲル、ポリスチレン、エチレン−無水マレイン酸共重合物等から構成される粒子が開発されているが（特許文献２）、斯様な合成高分子系の担体は、非特異吸着が起こり易いという欠点がある。また、無機系支持体を用いた担体も同様に、非特異吸着が起こり易い欠点がある。このように、支持体の種類や組成等によっては、非特異吸着が起こり易くなる、すなわち担体の防汚性が不十分になるという問題がある。
On the other hand, the carrier includes one using an organic support composed of a natural polymer or a synthetic polymer, and one using an inorganic support such as porous silica gel particles. As a carrier using a support composed of a natural polymer, those obtained by introducing various functional groups into porous agarose particles or porous dextran particles are known (Patent Document 1 and the like). Although such a natural polymer-based carrier is difficult to non-specifically adsorb, it has a drawback that it has a low physical strength and thus lacks pressure resistance, making it difficult to perform an analysis operation under high pressure.
On the other hand, as a carrier using a support composed of a synthetic polymer, particles composed of polyacrylamide gel, polyacrylate gel, polystyrene, ethylene-maleic anhydride copolymer, etc. have been developed (Patent Documents) 2) Such a synthetic polymer-based carrier has a disadvantage that non-specific adsorption is apt to occur. Similarly, a carrier using an inorganic support also has a disadvantage that non-specific adsorption easily occurs. As described above, depending on the type and composition of the support, there is a problem that non-specific adsorption is likely to occur, that is, the antifouling property of the carrier becomes insufficient.
本発明が解決しようとする課題は、標的物の動的結合容量が高く、且つ非特異吸着が起こり易い支持体を用いた場合であったとしても優れた防汚性を有する担体を提供することにある。 The problem to be solved by the present invention is to provide a carrier having a high dynamic binding capacity for a target substance and having excellent antifouling properties even when using a support which is likely to cause non-specific adsorption. It is in.
そこで本発明者らが鋭意検討した結果、酸性基を含む基とリガンドをそれぞれ支持体表面上に結合させた担体が、標的物の動的結合容量が高く、且つ非特異吸着が起こり易い支持体を用いた場合であったとしても優れた防汚性を有することを見出し、本発明を完成した。 Therefore, as a result of intensive studies by the present inventors, a support in which a group containing an acidic group and a ligand are bonded on the support surface, respectively, has a high dynamic binding capacity of a target substance, and the support is susceptible to non-specific adsorption. The present invention was found to have an excellent antifouling property even when the compound was used, and the present invention was completed.
すなわち、本発明は、以下の〔１〕〜〔４〕を提供するものである。 That is, the present invention provides the following [1] to [4].
〔１〕支持体と、酸性基を含む基と、当該酸性基を含む基を介さずに前記支持体表面上に結合したリガンドとを有し、前記酸性基を含む基が、前記支持体表面上に結合していることを特徴とする、担体。 [1] having a support, a group containing an acidic group, and a ligand bound on the surface of the support without going through the group containing the acidic group, wherein the group containing the acidic group is formed on the surface of the support A carrier, characterized in that it is bonded on top.
〔２〕上記〔１〕の担体がカラム容器に充填されているクロマトグラフィーカラム。 [2] A chromatography column in which the carrier of the above [1] is packed in a column container.
〔３〕上記〔１〕の担体を用いることを特徴とする、試料中の標的物を精製する方法。 [3] A method for purifying a target in a sample, which comprises using the carrier according to [1].
〔４〕以下の工程１及び２を含むことを特徴とする、担体の製造方法。
（工程１）リガンドと結合可能な反応性基を表面に有する支持体の前記反応性基の一部に、リガンドを結合する工程
（工程２）残りの反応性基に、当該反応性基と反応可能な官能基及び酸性基を有する化合物を反応させる工程
[4] A method for producing a carrier, comprising the following steps 1 and 2.
(Step 1) A step of binding a ligand to a part of the reactive group of the support having a reactive group capable of binding to the ligand on the surface. (Step 2) Reacting the remaining reactive group with the reactive group. Reacting a compound having a possible functional group and an acidic group
本発明の担体は、標的物の動的結合容量が高く、且つ非特異吸着が起こり易い支持体を用いた場合であったとしても、防汚性に優れる。したがって、本発明の担体は、支持体の組成等に依存することなく、優れた防汚性を示し、極めて高い分離選択性を有する。 The carrier of the present invention is excellent in antifouling property even when a support having a high dynamic binding capacity for a target substance and non-specific adsorption is likely to be used. Therefore, the carrier of the present invention shows excellent antifouling property and has extremely high separation selectivity without depending on the composition of the support.
〔担体〕
本発明の担体は、支持体と、酸性基を含む基と、当該酸性基を含む基を介さずに前記支持体表面上に結合したリガンドとを有し、前記酸性基を含む基が、前記支持体表面上に結合していることを特徴とする。
(Carrier)
The carrier of the present invention has a support, a group containing an acidic group, and a ligand bonded on the surface of the support without going through the group containing the acidic group, wherein the group containing the acidic group is It is characterized in that it is bound on the surface of the support.
＜支持体＞
支持体としては、合成高分子系支持体、天然高分子系支持体等の有機系支持体；無機系支持体；これらを組み合わせた有機−有機複合系支持体や有機−無機複合系支持体等が挙げられる。
合成高分子系支持体としては、例えば、ポリビニルアルコール類、ポリ（メタ）アクリレート類、ポリ（メタ）アクリルアミド類、ポリスチレン類、エチレン−無水マレイン酸共重合物等で構成されるもの挙げられる。天然高分子系支持体としては、例えば、セルロース（結晶性セルロース等）、アガロース、デキストラン等の多糖類で構成されるものが挙げられる。無機系支持体としては、ガラスビーズ、シリカゲル、金属、金属酸化物等で構成されるものが挙げられる。
これらの中でも、支持体の耐圧性の観点から、合成高分子系支持体が好ましく、ポリビニルアルコール類、ポリ（メタ）アクリレート類及びポリ（メタ）アクリルアミド類から選ばれる合成高分子で構成される支持体がより好ましい。
<Support>
As the support, an organic support such as a synthetic polymer support or a natural polymer support; an inorganic support; an organic-organic composite support or an organic-inorganic composite support obtained by combining these supports; Is mentioned.
Examples of the synthetic polymer-based support include those composed of polyvinyl alcohols, poly (meth) acrylates, poly (meth) acrylamides, polystyrenes, ethylene-maleic anhydride copolymers, and the like. Examples of the natural polymer-based support include those composed of polysaccharides such as cellulose (eg, crystalline cellulose), agarose, and dextran. Examples of the inorganic support include those composed of glass beads, silica gel, metal, metal oxide, and the like.
Among these, a synthetic polymer support is preferable from the viewpoint of the pressure resistance of the support, and a support composed of a synthetic polymer selected from polyvinyl alcohols, poly (meth) acrylates, and poly (meth) acrylamides. The body is more preferred.
また、支持体は、担体表面の表面積が大きく、多孔質であることが好ましい。また、支持体の形態は、粒子状、モノリス状、板状、チップ状、繊維状、膜状（中空糸を含む）等のいずれでもよく、任意の形態でよいが、標的物の動的結合容量の観点から、粒子状、モノリス状、板状、繊維状、膜状が好ましく、粒子状がより好ましい。 The support preferably has a large surface area on the carrier surface and is porous. Further, the form of the support may be any of a particle form, a monolith form, a plate form, a chip form, a fibrous form, a film form (including a hollow fiber) and the like, and may be any form. From the viewpoint of capacity, a particle shape, a monolith shape, a plate shape, a fiber shape, and a film shape are preferable, and the particle shape is more preferable.
支持体は、市販品を用いても、常法に従い合成したものを用いてもよい。市販品としては、多孔質セルロースゲルであるＧＣＬ２０００（生化学工業株式会社製）、架橋アガロース系の支持体であるＳｅｐｈａｒｏｓｅ ＣＬ４Ｂ（ＧＥヘルスケア・ジャパン株式会社製）、架橋セルロース系の支持体であるＣｅｌｌｕｆｉｎｅ（ＪＮＣ株式会社製）、デキストランであるＳｅｐｈａｄｅｘ（ＧＥヘルスケア・ジャパン株式会社製）、アリルデキストランとメチレンビスアクリルアミドを共有結合で架橋したＳｅｐｈａｃｒｙｌ Ｓ−１０００（ＧＥヘルスケア・ジャパン株式会社製）、アクリレート系の支持体であるＴｏｙｏｐｅａｒｌ（東ソー株式会社製）等が挙げられる。 As the support, a commercially available product or a support synthesized according to a conventional method may be used. Commercially available products include porous cellulose gel GCL2000 (manufactured by Seikagaku Corporation), crosslinked agarose-based support Sepharose CL4B (GE Healthcare Japan Co., Ltd.), and crosslinked cellulose-based support. Cellufine (manufactured by JNC), dextran Sephadex (manufactured by GE Healthcare Japan KK), Sephacryl S-1000 (manufactured by GE Healthcare Japan KK) obtained by covalently crosslinking allyldextran and methylenebisacrylamide, Toyopearl (manufactured by Tosoh Corporation), which is an acrylate-based support, may be used.
＜酸性基を含む基＞
本発明の担体は、酸性基を含む基が、支持体表面上に結合しているものである。これによって、優れた防汚性が得られる。
酸性基としては、カルボキシ基、スルホ基、リン酸基、ホスホジエステル基、スルフィノ基等が挙げられ、これらが解離したイオン性の官能基及びその塩も含むものとする。これらの中では、酸性基の化学的安定性の観点から、カルボキシ基、スルホ基、リン酸基、ホスホジエステル基、これらが解離したイオン性の官能基又はその塩が好ましい。
<Group containing acidic group>
The carrier of the present invention is a carrier in which a group containing an acidic group is bonded on the surface of a support. Thereby, excellent antifouling property is obtained.
Examples of the acidic group include a carboxy group, a sulfo group, a phosphoric acid group, a phosphodiester group, a sulfino group, and the like, and include an ionic functional group dissociated therefrom and a salt thereof. Among these, from the viewpoint of the chemical stability of the acidic group, a carboxy group, a sulfo group, a phosphoric acid group, a phosphodiester group, an ionic functional group in which these are dissociated, or a salt thereof are preferable.
酸性基としては、具体的には、−Ｃ（＝Ｏ）ＯＨ、−Ｃ（＝Ｏ）Ｏ-Ｍ+、−Ｃ（＝Ｏ）Ｏ-、−Ｓ（＝Ｏ）2ＯＨ、−Ｓ（＝Ｏ）2Ｏ-Ｍ+、−Ｓ（＝Ｏ）2Ｏ-、−Ｏ−Ｐ（＝Ｏ）（ＯＨ）2、−Ｏ−Ｐ（＝Ｏ）（Ｏ-Ｍ+）2、−Ｏ−Ｐ（＝Ｏ）（Ｏ-）2、−Ｏ−Ｐ（＝Ｏ）（ＯＨ）（Ｏ-Ｍ+）、−Ｏ−Ｐ（＝Ｏ）（ＯＨ）（Ｏ-）等の１価の酸性基；−Ｏ−（Ｏ＝Ｐ−ＯＨ）−Ｏ−、−Ｏ−（Ｏ＝Ｐ−Ｏ-Ｍ+）−Ｏ−、−Ｏ−（Ｏ＝Ｐ−Ｏ-）−Ｏ−等の２価の酸性基が挙げられる。なお、上記１価の酸性基は、結合手が１つの酸性基を意味し、２価の酸性基は、結合手が２つの酸性基を意味する。
上記Ｍ+は、それぞれ対イオンを示す。対イオンとしては、例えば、ナトリウムイオン、カリウムイオン等のアルカリ金属イオン；マグネシウムイオン、カルシウムイオン等のアルカリ土類金属イオン；アンモニウムイオン；有機アンモニウムイオン等が挙げられる。
これら酸性基の中でも、上記１価の酸性基が好ましく、防汚性の観点から、−Ｃ（＝Ｏ）ＯＨ、−Ｃ（＝Ｏ）Ｏ-Ｍ+、−Ｃ（＝Ｏ）Ｏ-、−Ｓ（＝Ｏ）2ＯＨ、−Ｓ（＝Ｏ）2Ｏ-Ｍ+、−Ｓ（＝Ｏ）2Ｏ-がより好ましく、リガンドから標的物を解離する際に少ない解離液の使用量で解離させ、効率よく標的物を分離精製、定量又は検出する観点から、−Ｓ（＝Ｏ）2ＯＨ、−Ｓ（＝Ｏ）2Ｏ-Ｍ+、−Ｓ（＝Ｏ）2Ｏ-が特に好ましい。
As the acidic group, specifically, -C (= O) OH, -C (= O) O - M +, -C (= O) O -, -S (= O) 2 OH, -S ( = O) 2 O - M + , -S (= O) 2 O -, -O-P (= O) (OH) 2, -O-P (= O) (O - M +) 2, -O -P (= O) (O - ) 2, -O-P (= O) (OH) (O - M +), - O-P (= O) (OH) (O -) 1 monovalent etc. Acidic groups; such as -O- (O = P-OH) -O-, -O- (O = P-O - M + )-O-, -O- (O = P-O - ) - O- A divalent acidic group is mentioned. In addition, the said monovalent acidic group means one acidic group of a bond, and the bivalent acidic group means two acidic groups of a bond.
M + represents a counter ion. Examples of the counter ion include an alkali metal ion such as a sodium ion and a potassium ion; an alkaline earth metal ion such as a magnesium ion and a calcium ion; an ammonium ion; and an organic ammonium ion.
Among these acidic groups, the monovalent acidic group is preferable, from the viewpoint of antifouling property, -C (= O) OH, -C (= O) O - M +, -C (= O) O -, -S (= O) 2 OH, -S (= O) 2 O - M +, -S (= O) 2 O - , more preferably, in the amount of less dissociation solution when dissociating targets from the ligand dissociated efficiently separating and purifying the target product, in terms of quantifying or detecting, -S (= O) 2 OH , -S (= O) 2 O - M +, -S (= O) 2 O - particularly preferable.
酸性基を含む基としては、リガンドから標的物を解離する際に少ない解離液の使用量で解離させ、効率よく標的物を分離精製、定量又は検出する観点から、ｐＫａが−３．０〜５．０の範囲内の基であるのが好ましい。特に、ｐＫａが−３．０〜１．０の範囲内であると、標的物を解離させる際に使用する解離液として一般的に用いられているような解離液を用いた場合でも解離液の量を少なく抑えることができ、標的物を高濃度で回収することができる。 The group containing an acidic group may have a pKa of -3.0 to 5 from the viewpoint of efficiently dissociating, purifying, quantifying, or detecting the target substance by using a small amount of a dissociating solution when dissociating the target substance from the ligand. It is preferred that the group be in the range of 0.0. In particular, when the pKa is within the range of -3.0 to 1.0, even when a dissociation solution that is generally used as a dissociation solution used for dissociating a target substance is used, The amount can be kept low, and the target can be recovered at a high concentration.
酸性基を含む基としては、酸性基を含む有機基が挙げられ、好適な例としては、以下の式（１）で表される１価の基が挙げられる。 Examples of the group containing an acidic group include an organic group containing an acidic group, and preferable examples thereof include a monovalent group represented by the following formula (1).
〔式（１）中、Ｒ1は、炭素数１〜１２の２価の有機基を示し、Ｘは酸性基を示す。〕 [In the formula (1), R 1 represents a divalent organic group having 1 to 12 carbon atoms, and X represents an acidic group. ]
上記式（１）中のＸで示される酸性基は、上記１価の酸性基と同様である。中でも、防汚性、動的結合容量の観点から、−Ｃ（＝Ｏ）ＯＨ、−Ｃ（＝Ｏ）Ｏ-Ｍ+、−Ｃ（＝Ｏ）Ｏ-、−Ｓ（＝Ｏ）2ＯＨ、−Ｓ（＝Ｏ）2Ｏ-Ｍ+、−Ｓ（＝Ｏ）2Ｏ-が好ましく、リガンドから標的物を解離する際に少ない解離液の使用量で解離させ、効率よく標的物を分離精製、定量又は検出する観点から、−Ｓ（＝Ｏ）2ＯＨ、−Ｓ（＝Ｏ）2Ｏ-Ｍ+、−Ｓ（＝Ｏ）2Ｏ-が特に好ましい。 The acidic group represented by X in the above formula (1) is the same as the above-mentioned monovalent acidic group. Among them, antifouling properties, in terms of dynamic binding capacity, -C (= O) OH, -C (= O) O - M +, -C (= O) O -, -S (= O) 2 OH , —S (＝ O) 2 O − M + , and —S (） O) 2 O − are preferable, and when the target is dissociated from the ligand, it is dissociated with a small amount of a dissociating solution to efficiently separate the target. purification, from the viewpoint of quantifying or detecting, -S (= O) 2 OH , -S (= O) 2 O - M +, -S (= O) 2 O - is particularly preferred.
また、Ｒ1で示される２価の有機基の炭素数は、好ましくは１〜８であり、より好ましくは１〜６であり、更に好ましくは１〜４である。
Ｒ1で示される２価の有機基としては、以下の式（２）で表される２価の基が好ましい。
Further, the carbon number of the divalent organic group represented by R 1 is preferably 1 to 8, more preferably 1 to 6, and still more preferably 1 to 4.
As the divalent organic group represented by R 1 , a divalent group represented by the following formula (2) is preferable.
〔式（２）中、Ｒ2は、炭素数１〜１２の２価の炭化水素基を示し、Ｙ1は、＞Ｓ、＞Ｓ＝Ｏ、＞Ｓ（＝Ｏ）2、＞ＮＨ、又は＞Ｏを示し、＊は、式（１）中のＸとの結合位置を示す。〕 [In the formula (2), R 2 represents a divalent hydrocarbon group having 1 to 12 carbon atoms, and Y 1 represents>S,> S = O,> S (＝ O) 2 ,> NH, or > O, and * indicates the bonding position to X in the formula (1). ]
Ｙ1としては、＞Ｓ、＞ＮＨが好ましく、＞Ｓがより好ましい。 The Y 1,> S,> NH are preferred,> S is more preferable.
Ｒ2で示される２価の炭化水素基は、直鎖状でも分岐鎖状でもよい。２価の炭化水素基の炭素数は、好ましくは１〜８であり、より好ましくは１〜６であり、更に好ましくは１〜４である。また、２価の炭化水素基は、好ましくは２価の脂肪族炭化水素基であり、より好ましくはアルカンジイル基である。アルカンジイル基の具体例としては、メタン−１，１−ジイル基、エタン−１，１−ジイル基、エタン−１，２−ジイル基、プロパン−１，１−ジイル基、プロパン−１，２−ジイル基、プロパン−１，３−ジイル基、プロパン−２，２−ジイル基、ブタン−１，４−ジイル基、ペンタン−１，５−ジイル基、ヘキサン−１，６−ジイル基等が挙げられる。 The divalent hydrocarbon group represented by R 2 may be linear or branched. The carbon number of the divalent hydrocarbon group is preferably 1 to 8, more preferably 1 to 6, and still more preferably 1 to 4. Further, the divalent hydrocarbon group is preferably a divalent aliphatic hydrocarbon group, and more preferably an alkanediyl group. Specific examples of the alkanediyl group include a methane-1,1-diyl group, an ethane-1,1-diyl group, an ethane-1,2-diyl group, a propane-1,1-diyl group, and a propane-1,2. -Diyl group, propane-1,3-diyl group, propane-2,2-diyl group, butane-1,4-diyl group, pentane-1,5-diyl group, hexane-1,6-diyl group and the like. No.
酸性基を含む基の含有量としては、防汚性の観点から、担体の表面積１ｍ2あたり、０．０５μｍｏｌ以上が好ましく、０．５〜３０μｍｏｌがより好ましく、１．０〜１０μｍｏｌがさらに好ましく、２．０〜５．０μｍｏｌが特に好ましい。
酸性基を含む基の含有量は、後述する実施例と同様の方法で測定すればよい。
From the viewpoint of antifouling properties, the content of the group containing an acidic group is preferably 0.05 μmol or more, more preferably 0.5 to 30 μmol, even more preferably 1.0 to 10 μmol, per 1 m 2 of the surface area of the carrier. 2.0 to 5.0 μmol is particularly preferred.
The content of the group containing an acidic group may be measured by the same method as in Examples described later.
＜リガンド＞
本発明の担体は、酸性基を含む基を介さずに支持体表面上にリガンドが結合したものである。これによって、リガンドの多点結合が抑えられ、リガンドの三次元構造が維持されるため、リガンドの活性が保たれ、標的物へのアフィニティー低下を防ぐことができる。
<Ligand>
The carrier of the present invention is a carrier in which a ligand is bound on the surface of a support without the intervention of a group containing an acidic group. As a result, the multipoint binding of the ligand is suppressed, and the three-dimensional structure of the ligand is maintained, so that the activity of the ligand is maintained and a decrease in affinity for the target can be prevented.
本発明において、リガンドは、標的物と特異的に結合するものであればよいが、例えば、タンパク質、核酸、ペプチド、酵素、キレート化合物、レセプター、アプタマー、抗体、抗原、ビタミン、金属イオン等が挙げられる。
タンパク質としては、プロテインＡ、プロテインＧ、アビジン、レクチン等が挙げられる。核酸としては、ＤＮＡ、ＲＮＡが挙げられる。ペプチドとしては、インシュリン、グルタチオン等が挙げられる。酵素としては、グルタチオン−Ｓ−トランスフェラーゼ等が挙げられる。キレート化合物としては、ニトリロ三酢酸等が挙げられる。レセプターとしては、ホルモンレセプター、サイトカインレセプター等が挙げられる。アプタマーとしては、ＤＮＡアプタマー、ＲＮＡアプタマー等が挙げられる。抗体としては、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤ、ＩｇＥ、ＩｇＹ等が挙げられる。ビタミンとしては、ビオチン等が挙げられる。金属イオンとしては、Ｎｉ2+、Ｃｏ2+、Ｃｕ2+、Ｚｎ2+、Ｆｅ3+等が挙げられる。
斯様なリガンドの中でも、タンパク質、核酸、ペプチド、酵素、キレート化合物が好ましく、タンパク質、ペプチドがより好ましい。中でも、精製の標的物が抗体である場合は、タンパク質が好ましく、イムノグロブリン結合タンパク質がより好ましく、プロテインＡが特に好ましい。
In the present invention, any ligand may be used as long as it specifically binds to a target, and examples thereof include proteins, nucleic acids, peptides, enzymes, chelating compounds, receptors, aptamers, antibodies, antigens, vitamins, and metal ions. Can be
Proteins include protein A, protein G, avidin, lectin and the like. Examples of the nucleic acid include DNA and RNA. Examples of the peptide include insulin and glutathione. Examples of the enzyme include glutathione-S-transferase. Examples of the chelate compound include nitrilotriacetic acid. Receptors include hormone receptors, cytokine receptors and the like. Aptamers include DNA aptamers, RNA aptamers, and the like. Antibodies include IgG, IgM, IgA, IgD, IgE, IgY, and the like. Examples of vitamins include biotin. Examples of the metal ion include Ni 2+ , Co 2+ , Cu 2+ , Zn 2+ , and Fe 3+ .
Among such ligands, proteins, nucleic acids, peptides, enzymes, and chelating compounds are preferred, and proteins and peptides are more preferred. Among them, when the target substance for purification is an antibody, a protein is preferable, an immunoglobulin binding protein is more preferable, and protein A is particularly preferable.
リガンド結合量としては、動的結合容量の観点から、担体の乾燥重量１ｇあたり、好ましくは１０〜２００ｍｇ、より好ましくは２５〜１００ｍｇ、更に好ましくは３０〜１００ｍｇ、特に好ましくは５０〜１００ｍｇである。
リガンド結合量は、後述する実施例と同様の方法で測定すればよい。
The ligand binding amount is preferably from 10 to 200 mg, more preferably from 25 to 100 mg, still more preferably from 30 to 100 mg, particularly preferably from 50 to 100 mg, per 1 g of the dry weight of the carrier, from the viewpoint of the dynamic binding capacity.
The ligand binding amount may be measured by a method similar to the example described later.
＜ヒドロキシ基を含む基＞
また、本発明の担体は、ヒドロキシ基を含む基が、支持体表面上に結合していてもよい。酸性基が、−Ｓ（＝Ｏ）2ＯＨ、−Ｓ（＝Ｏ）2Ｏ-Ｍ+、−Ｓ（＝Ｏ）2Ｏ-等のｐＫａが低い酸由来である場合において、更にヒドロキシ基を含む基が支持体表面上に結合していると、標的物を解離させる際のテーリングを抑えることができる。
<Group containing hydroxy group>
In the carrier of the present invention, a group containing a hydroxy group may be bonded on the surface of the support. Acidic groups, -S (= O) 2 OH , -S (= O) 2 O - M +, -S (= O) 2 O - in the case pKa is from lower acid such as, a further hydroxy group When the containing group is bonded to the surface of the support, tailing when dissociating the target can be suppressed.
ヒドロキシ基を含む基としては、ヒドロキシ基を含む有機基が挙げられ、好適な例としては、以下の式（３）で表される１価の基が挙げられ、より好ましくは以下の式（４）で表される１価の基である。 Examples of the group containing a hydroxy group include an organic group containing a hydroxy group, and preferred examples include a monovalent group represented by the following formula (3), and more preferably the following formula (4) )).
〔式（３）中、Ｒ3は、炭素数１〜１２の２価又は３価の有機基を示し、ｎは、１又は２を示す。〕 [In the formula (3), R 3 represents a divalent or trivalent organic group having 1 to 12 carbon atoms, and n represents 1 or 2. ]
〔式（４）中、Ｒ4は、炭素数１〜１２の２価又は３価の炭化水素基を示し、Ｙ2は、＞Ｓ、＞Ｓ＝Ｏ、＞Ｓ（＝Ｏ）2、＞ＮＨ、又は＞Ｏを示し、ｎは、１又は２を示す。〕 [In the formula (4), R 4 represents a divalent or trivalent hydrocarbon group having 1 to 12 carbon atoms, and Y 2 represents>S,> S = O,> S (= O) 2,> NH represents> or O, and n represents 1 or 2. ]
式（３）中、Ｒ3で示される２価又は３価の有機基の炭素数は、好ましくは１〜８であり、より好ましくは１〜６であり、更に好ましくは１〜４である。 In the formula (3), the carbon number of the divalent or trivalent organic group represented by R 3 is preferably 1 to 8, more preferably 1 to 6, and still more preferably 1 to 4.
式（４）中、Ｙ2としては、＞Ｓが好ましい。 In the formula (4), the Y 2,> S are preferred.
Ｒ4で示される２価又は３価の炭化水素基は、直鎖状でも分岐鎖状でもよい。２価又は３価の炭化水素基の炭素数は、好ましくは１〜８であり、より好ましくは１〜６であり、更に好ましくは１〜４である。
また、２価又は３価の炭化水素基は、好ましくは２価又は３価の脂肪族炭化水素基であり、より好ましくはアルカンジイル基、アルカントリイル基である。
上記アルカンジイル基の具体例としては、メタン−１，１−ジイル基、エタン−１，１−ジイル基、エタン−１，２−ジイル基、プロパン−１，１−ジイル基、プロパン−１，２−ジイル基、プロパン−１，３−ジイル基、プロパン−２，２−ジイル基、ブタン−１，４−ジイル基、ペンタン−１，５−ジイル基、ヘキサン−１，６−ジイル基等が挙げられる。
上記アルカントリイル基の具体例としては、メタン−１，１，１−トリイル基、エタン−１，１，２−トリイル基、プロパン−１，２，３−トリイル基、プロパン−１，２，２−トリイル基等が挙げられる。
The divalent or trivalent hydrocarbon group represented by R 4 may be linear or branched. The carbon number of the divalent or trivalent hydrocarbon group is preferably 1 to 8, more preferably 1 to 6, and still more preferably 1 to 4.
The divalent or trivalent hydrocarbon group is preferably a divalent or trivalent aliphatic hydrocarbon group, and more preferably an alkanediyl group or an alkanetriyl group.
Specific examples of the alkanediyl group include a methane-1,1-diyl group, an ethane-1,1-diyl group, an ethane-1,2-diyl group, a propane-1,1-diyl group, a propane-1, 2-diyl group, propane-1,3-diyl group, propane-2,2-diyl group, butane-1,4-diyl group, pentane-1,5-diyl group, hexane-1,6-diyl group, etc. Is mentioned.
Specific examples of the alkanetriyl group include a methane-1,1,1-triyl group, an ethane-1,1,2-triyl group, a propane-1,2,3-triyl group, and propane-1,2,2. And a 2-triyl group.
酸性基を含む基の含有量（α）とヒドロキシ基を含む基の含有量（β）とのモル比〔（α）：（β）〕は特に限定されないが、好ましくは１００：０〜５：９５、より好ましくは９９：１〜２５：７５、更に好ましくは９０：１０〜５０：５０、更に好ましくは８５：１５〜５５：４５、特に好ましくは８５：１５〜６０：４０とした場合には、酸性基が、−Ｓ（＝Ｏ）2ＯＨ、−Ｓ（＝Ｏ）2Ｏ-Ｍ+、−Ｓ（＝Ｏ）2Ｏ-等のｐＫａが低い酸由来であるときでも、標的物を解離させる際のテーリングを抑えながら、優れた防汚性を得ることができる。 The molar ratio [(α) :( β)] between the content (α) of the group containing an acidic group and the content (β) of the group containing a hydroxy group is not particularly limited, but is preferably 100: 0 to 5: 95, more preferably 99: 1 to 25:75, still more preferably 90:10 to 50:50, still more preferably 85:15 to 55:45, and particularly preferably 85:15 to 60:40. , acidic groups, -S (= O) 2 OH , -S (= O) 2 O - M +, -S (= O) 2 O - even when pKa of such is from low acid, the target product Excellent antifouling property can be obtained while suppressing tailing at the time of dissociation.
本発明の担体において、リガンド、酸性基を含む基、ヒドロキシ基を含む基は、担体の安定性の観点から、環状エーテル基が開環してなる基、ホルミル基が還元してなる基、トシル基が求核置換されてなる基等のような、反応性基が開環、還元又は求核置換等してなる基を介して、支持体表面上に共有結合しているものが好ましく、環状エーテル基が開環してなる基を介して支持体表面上に結合しているものがより好ましい。環状エーテル基が開環してなる基としては、開環エポキシ基が好ましい。 In the carrier of the present invention, the ligand, the group containing an acidic group, and the group containing a hydroxy group are, from the viewpoint of the stability of the carrier, a group formed by opening a cyclic ether group, a group formed by reducing a formyl group, and tosyl. A group in which a reactive group is covalently bonded to a support surface through a group obtained by ring opening, reduction or nucleophilic substitution, such as a group obtained by nucleophilic substitution, is preferred. Those in which the ether group is bonded to the support surface via a ring-opened group are more preferred. As a group formed by ring opening of a cyclic ether group, a ring-opened epoxy group is preferable.
ここで、「環状エーテル基」としては、環を構成する原子数が３〜７個の環状エーテル基が好ましい。環状エーテル基は、置換基としてアルキル基を有していてもよい。環状エーテル基の具体例としては、以下の式（５）〜（１０）で表される環状エーテル基が挙げられ、式（５）で表される環状エーテル基がより好ましい。 Here, as the “cyclic ether group”, a cyclic ether group having 3 to 7 atoms constituting a ring is preferable. The cyclic ether group may have an alkyl group as a substituent. Specific examples of the cyclic ether group include the cyclic ether groups represented by the following formulas (5) to (10), and the cyclic ether group represented by the formula (5) is more preferable.
〔式中、Ｒ5〜Ｒ8は、それぞれ独立して、水素原子又はアルキル基を示し、＊は結合位置を示す。〕 [Wherein, R 5 to R 8 each independently represent a hydrogen atom or an alkyl group, and * indicates a bonding position. ]
Ｒ5〜Ｒ8で示される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又は２である。アルキル基は直鎖状でも分岐鎖状でもよく、例えば、メチル基、エチル基、ｎ−プロピル基、イソプロピル基、ｎ−ブチル基、ｓｅｃ−ブチル基、ｔｅｒｔ−ブチル基等が挙げられる。
また、Ｒ5〜Ｒ8としては、水素原子が好ましい。
The carbon number of the alkyl group represented by R 5 to R 8 is preferably 1 to 4, more preferably 1 or 2. The alkyl group may be linear or branched, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group and a tert-butyl group.
Further, as R 5 to R 8 , a hydrogen atom is preferable.
〔担体の製造方法〕
本発明の担体の製造方法は、以下の工程１及び２を含むことを特徴とする。斯かる方法によれば、簡便且つ容易に、上記本発明の担体を得ることができる。
（工程１）リガンドと結合可能な反応性基（以下、単に反応性基ともいう）を表面に有する支持体（以下、原料支持体ともいう）の前記反応性基の一部に、リガンドを結合する工程
（工程２）残りの反応性基に、当該反応性基と反応可能な官能基及び酸性基を有する化合物を反応させる工程
(Method for producing carrier)
The method for producing a carrier of the present invention is characterized by comprising the following steps 1 and 2. According to such a method, the carrier of the present invention can be obtained simply and easily.
(Step 1) A ligand is bound to a part of the reactive group of a support (hereinafter, also referred to as a raw material support) having on its surface a reactive group capable of binding to the ligand (hereinafter, also simply referred to as a reactive group). (Step 2) a step of reacting the remaining reactive group with a compound having a functional group capable of reacting with the reactive group and an acidic group
＜工程１＞
工程１は、原料支持体の反応性基の一部に、リガンドを結合する工程である。反応性基に対してリガンドを結合させる手法は特に限定されるものではなく、例えば、リガンドに存在するアミノ基やカルボキシ基、チオール基等を利用した、従来のカップリング法で行えばよい。カップリング法としては、（ｉ）臭化シアン、エピクロロヒドリン、ジグリシジルエーテル、トシルクロライド、トレシルクロライド、ヒドラジン、過ヨウ素酸ナトリウム等を用いて原料支持体を活性化し、リガンドとカップリング反応を行い固定する方法、（ii）原料支持体とリガンドが存在する系に、カルボジイミドのような縮合試薬やグルタルアルデヒドのような分子中に複数の反応性基を持つ試薬を加えて、縮合・架橋することにより固定する方法、（iii）原料支持体に反応性基をあらかじめ導入しておき、原料支持体の活性化を経ずに、カップリング反応を行い固定する方法等が挙げられる。
<Step 1>
Step 1 is a step of binding a ligand to a part of the reactive group of the raw material support. The method of binding the ligand to the reactive group is not particularly limited, and may be, for example, a conventional coupling method using an amino group, a carboxy group, a thiol group, or the like present in the ligand. As the coupling method, (i) the raw material support is activated using cyanogen bromide, epichlorohydrin, diglycidyl ether, tosyl chloride, tresyl chloride, hydrazine, sodium periodate, etc., and the coupling with the ligand is performed. (Ii) adding a condensing reagent such as carbodiimide or a reagent having a plurality of reactive groups in a molecule such as glutaraldehyde to a system in which a raw material support and a ligand are present; A method of fixing by cross-linking, and (iii) a method of introducing a reactive group into a raw material support in advance and performing a coupling reaction and fixing without activating the raw material support, and the like.
なお、原料支持体として多孔質粒子を用いる場合には、公知のシード重合、懸濁重合等で多孔質粒子を合成すればよい。重合に際しては、モノマーに加えて、重合開始剤、多孔化剤、水系媒体、分散安定剤、界面活性剤、重合調整剤、重合禁止剤、シード粒子等を必要に応じて使用する。モノマーとしては、反応性基を有するモノマーに加え、必要に応じて当該モノマー以外のモノマーを用いてよい。これらモノマーとしては、いずれも、エチレン性不飽和モノマーが挙げられる。具体的には、スチレン系モノマー、ビニルケトン系モノマー、（メタ）アクリロニトリル系モノマー、（メタ）アクリレート系モノマー及び（メタ）アクリルアミド系モノマーから選ばれる１種又は２種以上のモノマーを例示できる。 When porous particles are used as a raw material support, the porous particles may be synthesized by known seed polymerization, suspension polymerization, or the like. Upon polymerization, in addition to the monomer, a polymerization initiator, a porogen, an aqueous medium, a dispersion stabilizer, a surfactant, a polymerization regulator, a polymerization inhibitor, seed particles, and the like are used as necessary. As the monomer, a monomer other than the monomer may be used, if necessary, in addition to the monomer having a reactive group. All of these monomers include an ethylenically unsaturated monomer. Specifically, one or more monomers selected from styrene monomers, vinyl ketone monomers, (meth) acrylonitrile monomers, (meth) acrylate monomers, and (meth) acrylamide monomers can be exemplified.
また、原料支持体が有する反応性基としては、水系中でも安定してカップリング反応を実施できる観点から、環状エーテル基が好ましく、環を構成する原子数が３〜７個の環状エーテル基がより好ましい。環状エーテル基は、置換基としてアルキル基を有していてもよい。環状エーテル基の具体例としては、上記式（５）〜（１０）で表される環状エーテル基が挙げられる。 In addition, as the reactive group of the raw material support, a cyclic ether group is preferable from the viewpoint of stably performing a coupling reaction even in an aqueous system, and a cyclic ether group having 3 to 7 atoms constituting a ring is more preferable. preferable. The cyclic ether group may have an alkyl group as a substituent. Specific examples of the cyclic ether group include the cyclic ether groups represented by the above formulas (5) to (10).
また、原料支持体に結合させるリガンドは上記と同様であり、上記でも述べたとおり、精製の標的物が抗体である場合は、プロテインＡが特に好ましい。
リガンドの合計使用量は、原料支持体１ｇに対し、通常５０〜３００ｍｇ程度であるが、好ましくは１２０〜１８０ｍｇである。
Further, the ligand to be bound to the raw material support is the same as described above, and as described above, when the target substance for purification is an antibody, protein A is particularly preferable.
The total amount of the ligand used is usually about 50 to 300 mg, preferably 120 to 180 mg, per 1 g of the raw material support.
また、工程１は、塩を添加したバッファー存在下で行うのが好ましい。塩の種類としては、クエン酸三ナトリウム、硫酸ナトリウム等が挙げられ、バッファーとしては、リン酸ナトリウムバッファー、リン酸カリウムバッファー、ホウ酸バッファー等が挙げられる。バッファーの合計使用量は、原料支持体に対し、通常２０〜８０質量倍程度であるが、好ましくは３５〜４５質量倍である。
また、工程１の反応時間は特に限定されないが、通常０．５〜７２時間程度であり、反応温度は通常１〜６０℃程度である。
Step 1 is preferably performed in the presence of a buffer to which a salt has been added. Examples of the type of salt include trisodium citrate, sodium sulfate, and the like, and examples of the buffer include a sodium phosphate buffer, a potassium phosphate buffer, and a borate buffer. The total amount of the buffer used is usually about 20 to 80 times by mass, preferably 35 to 45 times by mass, relative to the raw material support.
The reaction time of Step 1 is not particularly limited, but is usually about 0.5 to 72 hours, and the reaction temperature is usually about 1 to 60 ° C.
＜工程２＞
工程２は、残りの反応性基に、当該反応性基と反応可能な官能基及び酸性基を有する化合物（以下、酸性基含有化合物ともいう）を反応させる工程である。
<Step 2>
Step 2 is a step of reacting the remaining reactive group with a compound having a functional group capable of reacting with the reactive group and an acidic group (hereinafter also referred to as an acidic group-containing compound).
酸性基含有化合物としては、メルカプト酢酸（ｐＫａ＝３．５〜４．０）、３−メルカプトプロピオン酸（ｐＫａ＝３．５〜４．０）、４−メルカプトブタン酸（ｐＫａ＝３．５〜４．０）等のメルカプトカルボン酸；グリシン（ｐＫａ＝２．０〜２．５）、β−アラニン（ｐＫａ＝２．０〜２．５）等のアミノ酸；２−メルカプトエタンスルホン酸（ｐＫａ＝−２．５〜−２．０）、３−メルカプト−１−プロパンスルホン酸（ｐＫａ＝−２．５〜−２．０）等のメルカプトスルホン酸；アミノメタンスルホン酸（ｐＫａ＝−２．０〜−１．０）、２−アミノエタンスルホン酸（ｐＫａ＝−２．０〜−１．０）、３−アミノ−１−プロパンスルホン酸（ｐＫａ＝−２．３〜−１．３）等のアミノ基含有スルホン酸；これらの塩等が挙げられる。塩としては、ナトリウム塩、カリウム塩等のアルカリ金属塩；マグネシウム塩、カルシウム塩等のアルカリ土類金属塩；アンモニウム塩；有機アンモニウム塩等が挙げられる。
酸性基含有化合物の合計使用量は、残留反応性基１モルに対し、通常０．１〜４０モル当量であるが、リガンドの多点結合を防ぐ観点から、２〜４０モル当量の過剰量にて残留反応性基をブロッキングすることが好ましい。
Examples of the acidic group-containing compound include mercaptoacetic acid (pKa = 3.5-4.0), 3-mercaptopropionic acid (pKa = 3.5-4.0), and 4-mercaptobutanoic acid (pKa = 3.5-4.0). 4.0); amino acids such as glycine (pKa = 2.0-2.5) and β-alanine (pKa = 2.0-2.5); 2-mercaptoethanesulfonic acid (pKa = -2.5 to -2.0), mercaptosulfonic acid such as 3-mercapto-1-propanesulfonic acid (pKa = -2.5 to -2.0); aminomethanesulfonic acid (pKa = -2.0) To -1.0), 2-aminoethanesulfonic acid (pKa = -2.0 to -1.0), 3-amino-1-propanesulfonic acid (pKa = -2.3 to -1.3) and the like Amino group-containing sulfonic acids; and salts thereof. . Examples of the salt include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; ammonium salt; organic ammonium salt and the like.
The total amount of the acidic group-containing compound used is usually 0.1 to 40 molar equivalents relative to 1 mol of the remaining reactive group, but from the viewpoint of preventing multipoint binding of the ligand, the excess amount is 2 to 40 molar equivalents. To block residual reactive groups.
なお、ヒドロキシ基を含む基を導入する場合は、メルカプトエタノール、チオグリセロール等のメルカプト基を含むアルコール等を酸性基含有化合物と組み合わせて添加すればよい。 When a group containing a hydroxy group is introduced, an alcohol containing a mercapto group such as mercaptoethanol and thioglycerol may be added in combination with an acidic group-containing compound.
また、工程２の反応時間は特に限定されないが、通常０．５〜７２時間程度であり、好ましくは１〜４８時間である。また、反応温度は、溶媒の沸点以下で適宜選択すればよいが、通常２〜１００℃程度である。
なお、上記工程１と工程２は、効率やコストの面から工程１から工程２の順に行うのが好ましい。
The reaction time of Step 2 is not particularly limited, but is usually about 0.5 to 72 hours, preferably 1 to 48 hours. The reaction temperature may be appropriately selected below the boiling point of the solvent, but is usually about 2 to 100 ° C.
Steps 1 and 2 are preferably performed in the order of step 1 to step 2 in terms of efficiency and cost.
そして、上記のようにして得られる本発明の担体は、標的物の動的結合容量が高く、且つ非特異吸着が起こり易い支持体を用いた場合であったとしても、防汚性に優れる。したがって、本発明の担体は、支持体の組成等に依存することなく、宿主細胞に含まれるＨＣＰ（Ｈｏｓｔ Ｃｅｌｌ Ｐｒｏｔｅｉｎ）やＤＮＡ等の生体由来不純物等に対して優れた防汚性を示し、極めて高い分離選択性を有する。したがって、本発明の担体は、クロマトグラフィー用担体として特に有用である。 Further, the carrier of the present invention obtained as described above has a high dynamic binding capacity for a target substance and is excellent in antifouling properties even when using a support in which nonspecific adsorption is likely to occur. Therefore, the carrier of the present invention exhibits excellent antifouling properties against biological impurities such as HCP (Host Cell Protein) and DNA contained in host cells without depending on the composition of the support and the like. Has high separation selectivity. Therefore, the carrier of the present invention is particularly useful as a carrier for chromatography.
〔クロマトグラフィーカラム〕
本発明のクロマトグラフィーカラムは、上記本発明の担体がカラム容器に充填されているものである。該カラムはアフィニティクロマトグラフィーへの使用に適する。
[Chromatography column]
The chromatography column of the present invention is a column in which the carrier of the present invention is packed in a column container. The column is suitable for use in affinity chromatography.
〔標的物の精製方法〕
本発明の試料中の標的物を精製する方法は、上記本発明の担体を用いることを特徴とする。具体的には、以下の工程Ａ及びＢを含む方法が挙げられる。
（工程Ａ）本発明の担体と、前記リガンドに結合する標的物を含む試料とを接触させる工程
（工程Ｂ）前記リガンドと前記標的物とを解離させる解離液と、前記工程Ａで標的物を捕捉した担体とを接触させる工程
精製は、本発明の担体を用いる以外は常法に従い行えばよいが、解離液としては、標的物の解離性の観点から、酸性溶液が好ましく、２５℃におけるｐＨが２．５を超える酸性溶液がより好ましい。上記ｐＨは、より好ましくは３．０〜５．０である。
なお、標的物としては、タンパク質、抗体等の生体関連物質が挙げられる。
(Target method for purification)
A method of purifying a target in a sample of the present invention is characterized by using the above-mentioned carrier of the present invention. Specifically, a method including the following steps A and B is exemplified.
(Step A) a step of bringing the carrier of the present invention into contact with a sample containing a target substance that binds to the ligand; (Step B) a dissociation solution for dissociating the ligand and the target substance; Step of Contacting with Captured Carrier Purification may be performed according to a conventional method except for using the carrier of the present invention. As the dissociation solution, an acidic solution is preferable from the viewpoint of dissociation of the target substance, and the pH at 25 ° C. Are more preferred. The above pH is more preferably 3.0 to 5.0.
In addition, examples of the target include biological substances such as proteins and antibodies.
以下、実施例を挙げて本発明を詳細に説明するが、本発明はこれら実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.
実施例における各分析条件を以下に示す。
＜エポキシ基含有量＞
重合で使用したエポキシ基含有モノマー量より計算されるエポキシ基のモル数が１．００ｍｍｏｌとなるように、支持体水分散体をポリエチレンボトルに正確に測り取り、これに塩化カルシウム３７．５質量％水溶液２５ｍＬ及び０．２規定の塩酸１０ｍＬを加えて、７５℃で１時間撹拌することによりエポキシ基を開環し、冷却後、０．２規定の水酸化ナトリウム水溶液１０ｍＬで中和し、さらにｐＨメーターでｐＨをモニターしながら０．１規定の塩酸で逆滴定することにより、リガンド結合前の支持体のエポキシ基含有量を定量した。
Each analysis condition in the examples is shown below.
<Epoxy group content>
The aqueous dispersion of the support was accurately measured in a polyethylene bottle so that the mole number of the epoxy group calculated from the amount of the epoxy group-containing monomer used in the polymerization was 1.00 mmol, and 37.5% by mass of calcium chloride was added thereto. 25 mL of an aqueous solution and 10 mL of 0.2 N hydrochloric acid were added, and the mixture was stirred at 75 ° C. for 1 hour to open the epoxy group. After cooling, the solution was neutralized with 10 mL of a 0.2 N aqueous sodium hydroxide solution. The epoxy group content of the support before ligand binding was quantified by back titration with 0.1 N hydrochloric acid while monitoring the pH with a meter.
＜プロテインＡ結合量＞
プロテインＡ固定粒子（Ｓ−４）、アフィニティクロマトグラフィー用担体７のプロテインＡの結合量は、ビシンコニン酸（ＢＣＡ）試薬を用いた試薬セットで定量した。具体的には、固形分換算で１ｍｇの粒子（Ｓ−４）、アフィニティクロマトグラフィー用担体７をテストチューブに採取し、これをＴｈｅｒｍｏＦｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社のＢＣＡ Ｐｒｏｔｅｉｎ Ａｓｓａｙ Ｋｉｔで定量した。反応は、３７℃で３０分間、転倒混和することによって行った。検量線は、担体に結合させたプロテインＡと同一のロットのものを用いて作成した。
<Protein A binding amount>
The amount of protein A immobilized on the protein A-immobilized particles (S-4) and the carrier 7 for affinity chromatography was quantified using a reagent set using a bicinchoninic acid (BCA) reagent. Specifically, 1 mg of the particles (S-4) in terms of solid content and the carrier 7 for affinity chromatography were collected in a test tube, and quantified with a BCA Protein Assay Kit manufactured by ThermoFisher Scientific. The reaction was performed by inverting at 37 ° C. for 30 minutes. The calibration curve was prepared using the same lot as Protein A bound to the carrier.
＜酸性基の結合量＞
固形分換算で０．５ｇのアフィニティクロマトグラフィー用担体１〜７を正確に測り取り、電気伝導度測定計（Ｍｅｔｒｏｈｍ製 ７９４ Ｂａｓｉｃ Ｔｉｔｒｉｎｏ）を用いて、酸性基の結合量を算出した。
<Binding amount of acidic group>
0.5 g of the affinity chromatography supports 1 to 7 were accurately measured in terms of solid content, and the amount of acid groups bonded was calculated using an electric conductivity meter (794 Basic Titrino manufactured by Metrohm).
（実施例１）
（１）３６０ｇの純水にポリビニルアルコール（クラレ社製 ＰＶＡ−２１７）０．７２ｇを添加し、加熱撹拌しポリビニルアルコールを溶解させ、冷却した後、ドデシル硫酸ナトリウム（花王社製 エマール１０Ｇ）０．１８ｇ、炭酸ナトリウム０．３６ｇ及び亜硝酸ナトリウム０．１８ｇを添加し、撹拌して、水溶液（Ｓ−１）を調製した。
一方、グリシジルメタクリレート（三菱レーヨン社製）６．８８ｇ、グリセロールモノメタクリレート（日油社製）１．３７ｇ、トリメチロールプロパントリメタクリレート（サートマー社製）４．１２ｇ及びポリエチレングリコール＃４００ジメタクリレート（新中村化学社製、９Ｇ）１．３７ｇからなる単量体組成物を、２−オクタノン（東洋合成社製）２０．６３ｇ及びアセトフェノン（井上香料製造所社製）５．３０ｇの混液に溶解させ、単量体溶液（Ｓ−２）を調製した。
次いで、前記水溶液（Ｓ−１）を、５００ｍＬセパラブルフラスコ内に全量投入し、温度計、撹拌翼及び冷却管を装着して、温水バスにセットし、窒素雰囲気下で撹拌を開始した。セパラブルフラスコ内に前記単量体溶液（Ｓ−２）を全量投入して、温水バスにより加温し内温が８５℃に到達したところで２，２’−アゾイソブチロニトリル（和光純薬工業社製）０．５３ｇを添加し、８６℃に温度を維持した。
（２）その後、８６℃に温度を維持したまま３時間撹拌を行い、反応液を冷却した後、斯かる反応液をろ過し、純水とエタノールで洗浄した。洗浄した粒子を純水に分散させてデカンテーションを３回行い、小粒子を除いた。次いで、粒子の濃度が１０質量％となるように粒子を純水に分散させ、多孔質粒子分散液（Ｓ−３）を得た。この多孔質粒子のエポキシ基含有量は、粒子の乾燥重量１ｇあたり１．５７ｍｍｏｌであった。
（３）次に、改変プロテインＡ（Ｒｅｐｌｉｇｅｎ製 ｒＳＰＡ）０．１５ｇを、１．０Ｍクエン酸三ナトリウム／０．１Ｍリン酸ナトリウムバッファー（ｐＨ６．６）４０ｍＬに分散させプロテインＡ分散液を得、このプロテインＡ分散液に、前記多孔質粒子分散液（Ｓ−３）を粒子乾燥重量換算で１ｇ添加した。この分散液を２５℃で５時間振とう撹拌し、プロテインＡを粒子に固定した。この粒子を、プロテインＡ固定粒子（Ｓ−４）とする。プロテインＡ固定粒子（Ｓ−４）のプロテインＡ結合量は、粒子の乾燥重量１ｇあたり６３．２ｍｇであった。
（４）前記プロテインＡ固定粒子（Ｓ−４）を、０．１Ｍリン酸ナトリウムバッファー（ｐＨ７．６）で洗浄した後、１．０Ｍメルカプト酢酸（和光純薬工業社製）／０．１Ｍ硫酸ナトリウム水溶液（ｐＨ８．３）４０ｍＬに分散させ、２５℃で１７時間振とう撹拌することで、未反応のエポキシ基を完全に開環（ブロッキング）させた。
その後、０．１Ｍリン酸ナトリウムバッファー（ｐＨ７．６）、０．５Ｍ水酸化ナトリウム水溶液、０．１Ｍクエン酸ナトリウムバッファー（ｐＨ３．２）で洗浄し、アフィニティクロマトグラフィー用担体１を得た。メルカプト酢酸の結合量は、担体の表面積１ｍ2あたり、２．４８μｍｏｌであった。
(Example 1)
(1) To 360 g of pure water, 0.72 g of polyvinyl alcohol (PVA-217, manufactured by Kuraray Co., Ltd.) was added, and the mixture was heated and stirred to dissolve the polyvinyl alcohol. After cooling, sodium dodecyl sulfate (Emal 10G, manufactured by Kao Corporation) was added. 18 g, 0.36 g of sodium carbonate and 0.18 g of sodium nitrite were added and stirred to prepare an aqueous solution (S-1).
On the other hand, 6.88 g of glycidyl methacrylate (manufactured by Mitsubishi Rayon Co.), 1.37 g of glycerol monomethacrylate (manufactured by NOF CORPORATION), 4.12 g of trimethylolpropane trimethacrylate (manufactured by Sartomer) and polyethylene glycol # 400 dimethacrylate (Shinnakamura) A monomer composition composed of 1.37 g of 9G) was dissolved in a mixture of 20.63 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) and 5.30 g of acetophenone (manufactured by Inoue Perfumery Co., Ltd.) A monomer solution (S-2) was prepared.
Next, the whole amount of the aqueous solution (S-1) was put into a 500 mL separable flask, fitted with a thermometer, a stirring blade and a cooling tube, set in a hot water bath, and started stirring under a nitrogen atmosphere. The whole amount of the monomer solution (S-2) was put into a separable flask, heated in a hot water bath, and when the internal temperature reached 85 ° C., 2,2′-azoisobutyronitrile (Wako Pure Chemical Industries, Ltd.) 0.53 g (manufactured by Kogyo Co., Ltd.) was added, and the temperature was maintained at 86 ° C.
(2) Thereafter, stirring was performed for 3 hours while maintaining the temperature at 86 ° C., and after cooling the reaction solution, the reaction solution was filtered and washed with pure water and ethanol. The washed particles were dispersed in pure water and decanted three times to remove small particles. Next, the particles were dispersed in pure water so that the concentration of the particles became 10% by mass, to obtain a porous particle dispersion (S-3). The epoxy group content of the porous particles was 1.57 mmol per 1 g of dry weight of the particles.
(3) Next, 0.15 g of modified protein A (rSPA manufactured by Repligen) was dispersed in 40 mL of 1.0 M trisodium citrate / 0.1 M sodium phosphate buffer (pH 6.6) to obtain a protein A dispersion, To the protein A dispersion, 1 g of the porous particle dispersion (S-3) was added in terms of particle dry weight. This dispersion was shaken and stirred at 25 ° C. for 5 hours to fix protein A to the particles. The particles are referred to as Protein A-fixed particles (S-4). The amount of protein A bound to the protein A-immobilized particles (S-4) was 63.2 mg per 1 g of dry weight of the particles.
(4) After washing the protein A-fixed particles (S-4) with a 0.1 M sodium phosphate buffer (pH 7.6), 1.0 M mercaptoacetic acid (manufactured by Wako Pure Chemical Industries) /0.1 M sulfuric acid It was dispersed in 40 mL of a sodium aqueous solution (pH 8.3) and shaken and stirred at 25 ° C. for 17 hours to completely open (block) unreacted epoxy groups.
Thereafter, the resultant was washed with a 0.1 M sodium phosphate buffer (pH 7.6), a 0.5 M sodium hydroxide aqueous solution, and a 0.1 M sodium citrate buffer (pH 3.2) to obtain a carrier 1 for affinity chromatography. The amount of mercaptoacetic acid bound was 2.48 μmol per 1 m 2 of the surface area of the carrier.
（実施例２）
実施例１のエポキシ開環工程に用いる溶液を、１．０Ｍメルカプト酢酸（和光純薬工業社製）／０．１Ｍ硫酸ナトリウム水溶液（ｐＨ８．３）から、１．０Ｍ ３−メルカプト−１−プロパンスルホン酸ナトリウム（和光純薬工業社製）／０．１Ｍ硫酸ナトリウム水溶液（ｐＨ８．３）に変更した以外は実施例１と同様の操作を行い、アフィニティクロマトグラフィー用担体２を得た。３−メルカプト−１−プロパンスルホン酸ナトリウムの結合量は、担体の表面積１ｍ2あたり、３．１２μｍｏｌであった。
(Example 2)
The solution used in the epoxy ring-opening step of Example 1 was prepared from 1.0 M mercaptoacetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) / 0.1 M aqueous sodium sulfate solution (pH 8.3) in 1.0 M 3-mercapto-1-propane. A carrier 2 for affinity chromatography was obtained in the same manner as in Example 1 except that sodium sulfonate (manufactured by Wako Pure Chemical Industries, Ltd.) / 0.1 M aqueous sodium sulfate solution (pH 8.3) was used. The amount of sodium 3-mercapto-1-propanesulfonate bound was 3.12 μmol per 1 m 2 of the surface area of the carrier.
（実施例３）
実施例１のエポキシ開環工程に用いる溶液を、１．０Ｍメルカプト酢酸（和光純薬工業社製）／０．１Ｍ硫酸ナトリウム水溶液（ｐＨ８．３）から、０．８０Ｍ ３−メルカプト−１−プロパンスルホン酸ナトリウム（和光純薬工業社製）／０．２０Ｍ チオグリセロール（旭化学工業社製）／０．１Ｍ硫酸ナトリウム水溶液（ｐＨ８．３）に変更した以外は実施例１と同様の操作を行い、アフィニティクロマトグラフィー用担体３を得た。３−メルカプト−１−プロパンスルホン酸ナトリウムの結合量は、担体の表面積１ｍ2あたり、２．８８μｍｏｌであった。
(Example 3)
The solution used in the epoxy ring-opening step of Example 1 was prepared from 1.0 M mercaptoacetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) / 0.1 M aqueous sodium sulfate solution (pH 8.3) using 0.80 M 3-mercapto-1-propane. The same operation as in Example 1 was performed except that sodium sulfonate (manufactured by Wako Pure Chemical Industries, Ltd.) / 0.20 M thioglycerol (manufactured by Asahi Chemical Industry) /0.1 M sodium sulfate aqueous solution (pH 8.3) was used. The carrier 3 for affinity chromatography was obtained. The amount of sodium 3-mercapto-1-propanesulfonate bound was 2.88 μmol per 1 m 2 of the surface area of the carrier.
（実施例４）
実施例１のエポキシ開環工程に用いる溶液を、１．０Ｍメルカプト酢酸（和光純薬工業社製）／０．１Ｍ硫酸ナトリウム水溶液（ｐＨ８．３）から、０．５０Ｍ ３−メルカプト−１−プロパンスルホン酸ナトリウム（和光純薬工業社製）／０．５０Ｍ チオグリセロール（旭化学工業社製）／０．１Ｍ硫酸ナトリウム水溶液（ｐＨ８．３）に変更した以外は実施例１と同様の操作を行い、アフィニティクロマトグラフィー用担体４を得た。３−メルカプト−１−プロパンスルホン酸ナトリウムの結合量は、担体の表面積１ｍ2あたり、１．８３μｍｏｌであった。
(Example 4)
The solution used in the epoxy ring-opening step of Example 1 was prepared from 1.0 M mercaptoacetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) / 0.1 M sodium sulfate aqueous solution (pH 8.3) using 0.50 M 3-mercapto-1-propane. The same operation as in Example 1 was performed except that sodium sulfonate (manufactured by Wako Pure Chemical Industries, Ltd.) / 0.50 M thioglycerol (manufactured by Asahi Chemical Industry) /0.1 M aqueous sodium sulfate solution (pH 8.3) was used. The carrier 4 for affinity chromatography was obtained. The amount of sodium 3-mercapto-1-propanesulfonate bound was 1.83 μmol per 1 m 2 of the surface area of the carrier.
（比較例１）
実施例１のエポキシ開環工程に用いる溶液を、１．０Ｍメルカプト酢酸（和光純薬工業社製）／０．１Ｍ硫酸ナトリウム水溶液（ｐＨ８．３）から、１．０Ｍ チオグリセロール（旭化学工業社製）／０．１Ｍ硫酸ナトリウム水溶液（ｐＨ８．３）に変更した以外は実施例１と同様の操作を行い、アフィニティクロマトグラフィー用担体５を得た。酸性基の結合量は、担体の表面積１ｍ2あたり、＜０．０５μｍｏｌであった。
(Comparative Example 1)
The solution used in the epoxy ring opening step of Example 1 was prepared from 1.0 M mercaptoacetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) / 0.1 M sodium sulfate aqueous solution (pH 8.3), and then 1.0 M thioglycerol (Asahi Chemical Industry Co., Ltd.) ) /0.1 M aqueous sodium sulfate solution (pH 8.3), and the same operation as in Example 1 was carried out to obtain affinity chromatography carrier 5. The binding amount of the acidic group was <0.05 μmol / m 2 of the surface area of the carrier.
（比較例２）
実施例１のエポキシ開環工程に用いる溶液を、１．０Ｍメルカプト酢酸（和光純薬工業社製）／０．１Ｍ硫酸ナトリウム水溶液（ｐＨ８．３）から、０．１Ｍ硫酸ナトリウム水溶液（ｐＨ８．３）に変更した以外は実施例１と同様の操作を行い、アフィニティクロマトグラフィー用担体６を得た。酸性基の結合量は、担体の表面積１ｍ2あたり、＜０．０５μｍｏｌであった。
(Comparative Example 2)
The solution used in the epoxy ring-opening step of Example 1 was changed from 1.0 M mercaptoacetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) / 0.1 M sodium sulfate aqueous solution (pH 8.3) to 0.1 M sodium sulfate aqueous solution (pH 8.3). ) Was performed in the same manner as in Example 1, except that the carrier for affinity chromatography 6 was obtained. The binding amount of the acidic group was <0.05 μmol / m 2 of the surface area of the carrier.
（比較例３）
特開２０１０−１３３７３４号公報の実施例３を再現して多孔質粒子（Ｓ−５）分散液を得た。多孔質粒子（Ｓ−５）のエポキシ基含有量は、粒子の乾燥重量１ｇあたり０．２５ｍｍｏｌであった。次いで、ｒＰｒｏｔｅｉｎＡＴＭから、改変プロテインＡ（Ｒｅｐｌｉｇｅｎ製 ｒＳＰＡ）に変更した以外は特開２０１０−１３３７３４号公報の実施例６と同様の操作を行うことで、上記多孔質粒子（Ｓ−５）に改変プロテインＡを固定し、アフィニティクロマトグラフィー用担体７を得た。このアフィニティクロマトグラフィー用担体７のプロテインＡ結合量は、粒子の乾燥重量１ｇあたり２７．７ｍｇであった。また、酸性基の結合量は、担体の表面積１ｍ2あたり、＜０．０５μｍｏｌであった。
(Comparative Example 3)
Example 3 of JP-A-2010-133734 was reproduced to obtain a dispersion liquid of porous particles (S-5). The epoxy group content of the porous particles (S-5) was 0.25 mmol per 1 g of dry weight of the particles. Next, the same procedure as in Example 6 of JP-A-2010-133732 was performed except that rProteinATM was changed to modified protein A (rSPA manufactured by Repligen), whereby the modified protein was added to the porous particles (S-5). A was immobilized to obtain an affinity chromatography carrier 7. The protein A binding amount of the affinity chromatography support 7 was 27.7 mg / g of the dry weight of the particles. The amount of acidic groups bonded was <0.05 μmol per 1 m 2 of the surface area of the carrier.
（試験例１ 動的結合容量（ＤＢＣ）測定試験）
ＧＥヘルスケア社製ＡＫＴＡｐｒｉｍｅ ｐｌｕｓを用いて、線流速６０ｃｍ／ｈｒにおけるタンパク質（ヒトＩｇＧ抗体、Ｅｑｕｉｔｅｃｈ Ｂｉｏ社製 ＨＧＧ−１０００）に対する実施例１〜４及び比較例１〜３で得られた担体１〜７のＤＢＣを測定した。カラムは容量４ｍＬ（５ｍｍφ×２００ｍｍ長）のものを、タンパク質は２０ｍＭリン酸ナトリウム／１５０ｍＭ塩化ナトリウムバッファー（ｐＨ７．５）で２５ｍｇ／ｍＬに希釈したものをそれぞれ使用し、溶出先端１０％ブレークスルーのときのタンパク質捕捉量とカラム充填体積からＤＢＣを求めた。結果を表１に示す。
(Test Example 1 Dynamic coupling capacity (DBC) measurement test)
Using the AKTAprime plus manufactured by GE Healthcare, carriers 1 to 4 obtained in Examples 1 to 4 and Comparative Examples 1 to 3 for a protein (human IgG antibody, HGG-1000 manufactured by Equitech Bio) at a linear flow rate of 60 cm / hr. The DBC of 7 was measured. The column used had a capacity of 4 mL (5 mmφ × 200 mm length), and the protein used was diluted to 25 mg / mL with 20 mM sodium phosphate / 150 mM sodium chloride buffer (pH 7.5). The DBC was determined from the amount of protein captured at this time and the column packing volume. Table 1 shows the results.
また、実施例１〜２で得られた担体１〜２から、捕捉したタンパク質を５０ｍＭクエン酸緩衝液（ｐＨ３．２）で解離させ、その際の解離応答性をＧＥヘルスケア社製ＡＫＴＡｐｒｉｍｅ ｐｌｕｓで３ＣＶ（３ Ｃｏｌｕｍｎ Ｖｏｌｕｍｅ）まで測定した。結果を図１に示す。
図１に示すように、３−メルカプト−１−プロパンスルホン酸ナトリウム（３−メルカプト−１−プロパンスルホン酸のｐＫａ＝−２．５〜−２．０）で表面修飾した実施例２の担体２は、メルカプト酢酸（ｐＫａ＝３．５〜４．０）で表面修飾した実施例１の担体１より少ない量の解離液でタンパク質の解離が始まった。
また、実施例３〜４で得られた担体３〜４から、捕捉したタンパク質を５０ｍＭクエン酸緩衝液（ｐＨ３．２）で解離させ、その際の解離応答性をＧＥヘルスケア社製ＡＫＴＡｐｒｉｍｅ ｐｌｕｓで３ＣＶ（３Ｃｏｌｕｍｎ Ｖｏｌｕｍｅ）まで測定したところ、テーリングがほとんどみられなかった。
Further, the captured protein was dissociated from the carriers 1 and 2 obtained in Examples 1 and 2 with a 50 mM citrate buffer (pH 3.2), and the dissociation responsiveness at that time was measured using AKTAprime plus manufactured by GE Healthcare. The measurement was performed up to 3 CV (3 Column Volume). The results are shown in FIG.
As shown in FIG. 1, the carrier 2 of Example 2 surface-modified with sodium 3-mercapto-1-propanesulfonic acid (pKa of 3-mercapto-1-propanesulfonic acid = −2.5 to −2.0) The protein dissociation started with a smaller amount of dissociation solution than the carrier 1 of Example 1 surface-modified with mercaptoacetic acid (pKa = 3.5-4.0).
The captured proteins were dissociated from the carriers 3 and 4 obtained in Examples 3 and 4 with a 50 mM citrate buffer (pH 3.2), and the dissociation responsiveness at that time was measured using AKTAprime plus manufactured by GE Healthcare. When measured up to 3 CV (3 Column Volume), almost no tailing was observed.
（試験例２ ＨＣＰ測定試験）
実施例１で得られた担体１を７０μＬ（湿潤状態）秤取り、１．０ｍｇ／ｍＬモノクローナル抗体溶液（モノクローナル抗体Ｔｒａｓｔｕｚｕｍａｂのバイオシミラーを含有するＣＨＯ細胞培養上清）２．４５ｍＬを加えて、室温で１．５時間撹拌振とうして、抗体を担体に捕捉した。
次いで、前記抗体を捕捉させた担体の全量をスピンカラム（Ｔｈｅｒｍｏ製）に移し、遠心分離（１０００ｒｐｍ）によって抗体溶液を除いた。
次に、２０ｍＭリン酸ナトリウム／１５０ｍＭ ＮａＣｌバッファー（ｐＨ７．５）をスピンカラムに加え、合計８４０μＬを遠心分離によって通液し、担体を洗浄した。
次いで、２０ｍＭリン酸ナトリウム／１．０Ｍ ＮａＣｌバッファー（ｐＨ７．５）をスピンカラムに加え、合計８４０μＬを遠心分離によって通液し、担体を洗浄した。
次に、２０ｍＭリン酸ナトリウム／１５０ｍＭ ＮａＣｌバッファー（ｐＨ７．５）をスピンカラムに加え、合計８４０μＬを遠心分離によって通液し、担体を洗浄した。
その後、５０ｍＭクエン酸緩衝液（ｐＨ３．２）をスピンカラムに加え、合計７００μＬを遠心分離によって通液し、担体に捕捉されていたモノクローナル抗体を解離した。解離液中の抗体濃度を、ＢｉｏＲＡＤ製Ｓｍａｒｔｓｐｅｃ Ｐｌｕｓを使用して吸光度から算出した。
そして、Ｃｙｇｎｕｓ Ｔｅｃｈｎｏｌｏｇｉｅｓ社製 ＣＨＯ ＨＣＰ ＥＬＩＳＡ ｋｉｔ，３Ｇを用い、Ｈｏｓｔ Ｃｅｌｌ Ｐｒｏｔｅｉｎ（ＨＣＰ）濃度を測定し、抗体濃度でスタンダード化した。
また、実施例２〜４及び比較例１〜３で得られた担体２〜７についても、上記と同様にしてＨＣＰ測定試験を行った。
結果を表１に示す。
(Test Example 2 HCP measurement test)
70 μL (wet state) of the carrier 1 obtained in Example 1 was weighed, and 2.45 mL of a 1.0 mg / mL monoclonal antibody solution (a CHO cell culture supernatant containing a biosimilar of the monoclonal antibody Trastuzumab) was added thereto. The antibody was captured on the carrier by stirring and shaking at room temperature for 1.5 hours.
Next, the entire amount of the carrier capturing the antibody was transferred to a spin column (manufactured by Thermo), and the antibody solution was removed by centrifugation (1000 rpm).
Next, 20 mM sodium phosphate / 150 mM NaCl buffer (pH 7.5) was added to the spin column, and a total of 840 μL was passed by centrifugation to wash the carrier.
Next, 20 mM sodium phosphate / 1.0 M NaCl buffer (pH 7.5) was added to the spin column, and a total of 840 μL was passed by centrifugation to wash the carrier.
Next, 20 mM sodium phosphate / 150 mM NaCl buffer (pH 7.5) was added to the spin column, and a total of 840 μL was passed by centrifugation to wash the carrier.
Thereafter, 50 mM citrate buffer (pH 3.2) was added to the spin column, and a total of 700 μL was passed by centrifugation to dissociate the monoclonal antibody captured by the carrier. The antibody concentration in the dissociation solution was calculated from the absorbance using SmartSpec Plus manufactured by BioRAD.
Then, the host cell protein (HCP) concentration was measured using a CHO HCP ELISA kit, 3G manufactured by Cygnus Technologies, and standardized by the antibody concentration.
In addition, the carriers 2 to 7 obtained in Examples 2 to 4 and Comparative Examples 1 to 3 were also subjected to the HCP measurement test in the same manner as described above.
Table 1 shows the results.
酸性基を有する化合物で表面を修飾した実施例１〜４の担体１〜４は、防汚性に優れるためＨＣＰ値が低く、また、ＤＢＣも高かった。一方、水酸基を有する化合物のみで表面を修飾した比較例１の担体５は、防汚性が不十分であるためＨＣＰ値が高くなった。また、表面を修飾していない比較例２〜３の担体６〜７は、防汚性が不十分であるためＨＣＰ値が高く、また、ＤＢＣが低かった。
このように、実施例１〜４の担体１〜４は、標的物の動的結合容量が高く、且つ優れた防汚性を有するものであった。
Carriers 1 to 4 of Examples 1 to 4 whose surfaces were modified with a compound having an acidic group had excellent antifouling properties, and therefore had a low HCP value and a high DBC. On the other hand, the carrier 5 of Comparative Example 1, in which the surface was modified only with the compound having a hydroxyl group, had a high HCP value due to insufficient antifouling properties. In addition, the carriers 6 to 7 of Comparative Examples 2 and 3 whose surfaces were not modified had an insufficient antifouling property, and thus had a high HCP value and a low DBC.
As described above, the carriers 1 to 4 of Examples 1 to 4 had a high dynamic binding capacity of the target substance and had excellent antifouling properties.
Claims (19)
酸性基を含む基と、
当該酸性基を含む基を介さずに前記支持体表面上に結合したリガンドと、を有し、
前記リガンドが、イムノグロブリン結合タンパク質であり、
前記酸性基を含む基が、前記支持体表面上に結合しており、前記酸性基を含む基の含有量が、担体の表面積１ｍ2あたり、１．０μｍｏｌ以上であり、前記酸性基が、−Ｃ（＝Ｏ）ＯＨ、−Ｃ（＝Ｏ）Ｏ-Ｍ+、−Ｃ（＝Ｏ）Ｏ-、−Ｓ（＝Ｏ）2ＯＨ、−Ｓ（＝Ｏ）2Ｏ-Ｍ+、又は−Ｓ（＝Ｏ）2Ｏ-である（Ｍ+は、対イオンを示す）ことを特徴とする、
担体。 A support,
A group containing an acidic group,
A ligand bound on the support surface without a group containing the acidic group,
The ligand is an immunoglobulin binding protein,
The group containing the acidic group is bonded on the surface of the support, the content of the group containing the acidic group is 1.0 μmol or more per 1 m 2 of the surface area of the carrier, and the acidic group is -C (= O) OH, -C (= O) O - M +, -C (= O) O -, -S (= O) 2 OH, -S (= O) 2 O - M +, or -S (= O) 2 O - an (M + represents a counter ion), characterized in that,
Carrier.
（工程Ａ）請求項１〜１４のいずれか１項に記載の担体と、前記リガンドに結合する標的物を含む試料とを接触させる工程
（工程Ｂ）前記リガンドと前記標的物とを解離させる解離液と、前記工程Ａで標的物を捕捉した担体とを接触させる工程 A method for purifying a target, comprising the following steps A and B:
(Step A) a step of bringing the carrier according to any one of claims 1 to 14 into contact with a sample containing a target that binds to the ligand (Step B) dissociation that dissociates the ligand from the target Contacting the liquid with the carrier capturing the target in step A
（工程１）イムノグロブリン結合タンパク質リガンドと結合可能な反応性基を表面に有する支持体の前記反応性基の一部に、イムノグロブリン結合タンパク質リガンドを結合する工程
（工程２）残りの反応性基に、当該反応性基と反応可能な官能基及び酸性基を有し、前記酸性基が、−Ｃ（＝Ｏ）ＯＨ、−Ｃ（＝Ｏ）Ｏ-Ｍ+、−Ｃ（＝Ｏ）Ｏ-、−Ｓ（＝Ｏ）2ＯＨ、−Ｓ（＝Ｏ）2Ｏ-Ｍ+、又は−Ｓ（＝Ｏ）2Ｏ-である（Ｍ+は、対イオンを示す）化合物を反応させる工程 Characterized by comprising the following steps 1 and 2, wherein the content of the group containing an acidic group is 1.0 μmol or more per 1 m 2 of the surface area of the carrier, and the acidic group is -C (＝ O) OH, -C (= O) O - M +, -C (= O) O -, -S (= O) 2 OH, -S (= O) 2 O - M +, or -S (= O) 2 O - a (M + represents a counter ion) method for producing a carrier.
(Step 1) A step of binding an immunoglobulin-binding protein ligand to a part of the reactive group of a support having a reactive group capable of binding to an immunoglobulin-binding protein ligand on the surface (Step 2) Remaining reactive groups Has a functional group capable of reacting with the reactive group and an acidic group, wherein the acidic group is -C (= O) OH, -C (= O) O - M + , -C (= O) O -, -S (= O) 2 OH, -S (= O) 2 O - M +, or -S (= O) 2 O - a is (M + represents a counter ion) reacting the compound
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