JPS6348453A - Carrier for chromatography and its production - Google Patents
Carrier for chromatography and its productionInfo
- Publication number
- JPS6348453A JPS6348453A JP61192143A JP19214386A JPS6348453A JP S6348453 A JPS6348453 A JP S6348453A JP 61192143 A JP61192143 A JP 61192143A JP 19214386 A JP19214386 A JP 19214386A JP S6348453 A JPS6348453 A JP S6348453A
- Authority
- JP
- Japan
- Prior art keywords
- porous beads
- anhydride
- amino group
- beads
- group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000004587 chromatography analysis Methods 0.000 title claims description 19
- 238000004519 manufacturing process Methods 0.000 title claims description 8
- 239000011324 bead Substances 0.000 claims abstract description 36
- 125000003277 amino group Chemical group 0.000 claims abstract description 24
- 150000008064 anhydrides Chemical class 0.000 claims abstract description 13
- 229920001661 Chitosan Polymers 0.000 claims description 22
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 claims description 8
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 claims description 8
- 229960002442 glucosamine Drugs 0.000 claims description 8
- 150000001266 acyl halides Chemical class 0.000 claims description 5
- 239000003431 cross linking reagent Substances 0.000 claims description 4
- 239000002253 acid Substances 0.000 abstract description 3
- ODIGIKRIUKFKHP-UHFFFAOYSA-N (n-propan-2-yloxycarbonylanilino) acetate Chemical group CC(C)OC(=O)N(OC(C)=O)C1=CC=CC=C1 ODIGIKRIUKFKHP-UHFFFAOYSA-N 0.000 abstract description 2
- 229920002101 Chitin Polymers 0.000 abstract description 2
- ZNZYKNKBJPZETN-WELNAUFTSA-N Dialdehyde 11678 Chemical compound N1C2=CC=CC=C2C2=C1[C@H](C[C@H](/C(=C/O)C(=O)OC)[C@@H](C=C)C=O)NCC2 ZNZYKNKBJPZETN-WELNAUFTSA-N 0.000 abstract description 2
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 abstract description 2
- 150000001335 aliphatic alkanes Chemical class 0.000 abstract description 2
- 238000004132 cross linking Methods 0.000 abstract description 2
- 125000002252 acyl group Chemical group 0.000 abstract 2
- 241000500891 Insecta Species 0.000 abstract 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 abstract 1
- 230000006196 deacetylation Effects 0.000 abstract 1
- 238000003381 deacetylation reaction Methods 0.000 abstract 1
- 238000001042 affinity chromatography Methods 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 10
- 238000001179 sorption measurement Methods 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 8
- 239000003446 ligand Substances 0.000 description 8
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 239000013076 target substance Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108010062580 Concanavalin A Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- -1 antigen-antibody Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 description 2
- 235000010582 Pisum sativum Nutrition 0.000 description 2
- 240000004713 Pisum sativum Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- WETWJCDKMRHUPV-UHFFFAOYSA-N acetyl chloride Chemical compound CC(Cl)=O WETWJCDKMRHUPV-UHFFFAOYSA-N 0.000 description 2
- 239000012346 acetyl chloride Substances 0.000 description 2
- 150000008065 acid anhydrides Chemical class 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- VANNPISTIUFMLH-UHFFFAOYSA-N glutaric anhydride Chemical compound O=C1CCCC(=O)O1 VANNPISTIUFMLH-UHFFFAOYSA-N 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- FNUFHLAIHWESDA-UHFFFAOYSA-M sodium;2,3,4-trinitrobenzenesulfonate Chemical compound [Na+].[O-][N+](=O)C1=CC=C(S([O-])(=O)=O)C([N+]([O-])=O)=C1[N+]([O-])=O FNUFHLAIHWESDA-UHFFFAOYSA-M 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 229940014800 succinic anhydride Drugs 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- HBOMLICNUCNMMY-KJFJCRTCSA-N 1-[(4s,5s)-4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1C1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-KJFJCRTCSA-N 0.000 description 1
- JPSKCQCQZUGWNM-UHFFFAOYSA-N 2,7-Oxepanedione Chemical compound O=C1CCCCC(=O)O1 JPSKCQCQZUGWNM-UHFFFAOYSA-N 0.000 description 1
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- FXXACINHVKSMDR-UHFFFAOYSA-N acetyl bromide Chemical compound CC(Br)=O FXXACINHVKSMDR-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- YHASWHZGWUONAO-UHFFFAOYSA-N butanoyl butanoate Chemical compound CCCC(=O)OC(=O)CCC YHASWHZGWUONAO-UHFFFAOYSA-N 0.000 description 1
- DVECBJCOGJRVPX-UHFFFAOYSA-N butyryl chloride Chemical compound CCCC(Cl)=O DVECBJCOGJRVPX-UHFFFAOYSA-N 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 230000000850 deacetylating effect Effects 0.000 description 1
- 125000005442 diisocyanate group Chemical group 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 229940099607 manganese chloride Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002762 monocarboxylic acid derivatives Chemical class 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- LJAGLQVRUZWQGK-UHFFFAOYSA-N oxecane-2,10-dione Chemical compound O=C1CCCCCCCC(=O)O1 LJAGLQVRUZWQGK-UHFFFAOYSA-N 0.000 description 1
- ZJHUBLNWMCWUOV-UHFFFAOYSA-N oxocane-2,8-dione Chemical compound O=C1CCCCCC(=O)O1 ZJHUBLNWMCWUOV-UHFFFAOYSA-N 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- WYVAMUWZEOHJOQ-UHFFFAOYSA-N propionic anhydride Chemical compound CCC(=O)OC(=O)CC WYVAMUWZEOHJOQ-UHFFFAOYSA-N 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000000018 receptor agonist Substances 0.000 description 1
- 229940044601 receptor agonist Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- XHFLOLLMZOTPSM-UHFFFAOYSA-M sodium;hydrogen carbonate;hydrate Chemical compound [OH-].[Na+].OC(O)=O XHFLOLLMZOTPSM-UHFFFAOYSA-M 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 230000001755 vocal effect Effects 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明は、アフィニティークロマトグラフィーをはじめ
として各種クロマトグラフィーに利用することのできる
クロマトグラフィー用担体、及び該クロマトグラフィー
用担体の製造方法に関する。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a chromatography carrier that can be used in various chromatography including affinity chromatography, and a method for producing the chromatography carrier.
従来の技術
クロマトグラフィー技術の1つとして、アフィニティー
クロマトグラフィーは互いに特異的に相互作用を及ぼし
合う物質対の親和性を利用して分離・精製を行なうもの
であシ、例えば生体物質をその生物学的特性即ち分子上
のある特定の化学構造を識別して精製する場合に有用で
ある。As a type of conventional chromatography technology, affinity chromatography is a method of separating and purifying biological materials by utilizing the affinity of pairs of substances that specifically interact with each other. It is useful for identifying and purifying specific properties, that is, specific chemical structures on molecules.
7フイニテイ一クロマトグラフイー用吸着担体(アフィ
ニティーグル)は、例えば不溶性の担体(マトリックス
)にスペーサーを結合させて得られる活性支持体の前記
ス被−サーにリガンドを結合させたものであシ、このリ
ガンドと互いに相互作用を及ぼし合う物質の組合せを選
択して吸着操作を行なう。7. An adsorption carrier (affinity group) for affinity chromatography is, for example, one in which a ligand is bound to the spacer of an active support obtained by binding a spacer to an insoluble carrier (matrix). An adsorption operation is performed by selecting a combination of substances that interact with this ligand.
リガンドと吸着目的物質の組合せとしては、例えば次の
ような組合せが挙げられる。Examples of the combination of the ligand and the adsorption target substance include the following combinations.
酵素−基質、生成物、阻害剤、補酵素。Enzymes - substrates, products, inhibitors, coenzymes.
エフェクター、 抗原−抗体、 リセプターーアゴニスト、 核酸塩基対、 レクチン−糖(蛋白)、 金属キレート−蛋白質、 疎水性基−蛋白質、 ホスト−ダスト。effector, antigen-antibody, receptor agonist, nucleobase pairs, Lectin - sugar (protein), metal chelate-protein, hydrophobic group - protein, Host - Dust.
アフィニティークロマトグラフィーによる分離・精製や
分析において、
前記アフィニティークロマトグラフィー用活性支持体に
望まれる性質としては、非特異的吸着が少ないこと、高
い多孔性を有すること、リガンドの結合が容易であシ固
定化可能容量が大きいこと、化学的に安定で声域、塩濃
度、温度の広範な条件下で十分安定であシ体積変化がな
いこと、十分な機械的強度と安定性を有し流動特性が良
いこと、生物学的汚染に耐えること、などが挙げられる
。In separation/purification and analysis by affinity chromatography, the desired properties of the active support for affinity chromatography include low nonspecific adsorption, high porosity, easy binding of ligands, and immobilization. It is chemically stable and stable enough under a wide range of vocal ranges, salt concentrations, and temperatures, has no volume change, has sufficient mechanical strength and stability, and has good flow characteristics. Examples include resistance to biological contamination.
従来よシアフィニティークロマトグラフィー用吸着担体
の基材として用いられているセルロース、デキストラン
、ポリアクリルアミド、アガロース等は、必ずしもこれ
ら望まれる性質を具有していない。とシわけ、硬さが不
足した所謂ソフトダルであるため流動特性が悪く、分離
特性が良くないという重大な欠点を有し、また寿命も短
い。Cellulose, dextran, polyacrylamide, agarose, etc., which have been conventionally used as base materials for adsorption carriers for cyaffinity chromatography, do not necessarily have these desired properties. In particular, since it is a so-called soft dull with insufficient hardness, it has serious drawbacks such as poor flow characteristics and poor separation characteristics, and also has a short lifespan.
更に、近年用いられているシリカビーズは、硬さの点で
は満足できるものの、アルカリ性条件下では使用できな
いため、分離条件や溶出・洗浄の条件の選択に大きな制
約が加わるという問題点を有していた。Furthermore, although the silica beads that have been used in recent years are satisfactory in terms of hardness, they cannot be used under alkaline conditions, which poses the problem of placing significant restrictions on the selection of separation conditions and elution/washing conditions. Ta.
発明が解決しようとする問題点
本発明の目的は、前記従来のクロマトグラフィー用担体
の欠点を克服して、アフィニティークロマトグラフィー
用活性支持体として望まれる前述した諸性質を千金に具
有するクロマトグラフィー用担体及びその製造方法を提
供することにある。Problems to be Solved by the Invention It is an object of the present invention to overcome the drawbacks of the conventional chromatography supports and to provide a chromatography support that has all of the above-mentioned properties desired as an active support for affinity chromatography. An object of the present invention is to provide a carrier and a method for producing the same.
問題点を解決するための手段
本発明によって上記目的を達成し得るクロマトグラフィ
ー用担体及びその製造方法が提供される。Means for Solving the Problems The present invention provides a chromatography carrier and a method for producing the same that can achieve the above objects.
即ち、本発明は、キトサン多孔性ビーズであって、該キ
トサンを構成するグルコサミンに基くアミノ基に、また
はキトサン多孔性ビーズを架橋処理したものであって、
該キトサンを構成するグルコサミンに基くアミノ基及び
/または架橋処理剤に基くアミノ基に、ω−カルボキシ
アルカノイル基が結合され、残アミノ基がアシル化され
ていることを特徴とするクロマトグラフィー用担体に関
する。That is, the present invention provides chitosan porous beads in which amino groups based on glucosamine constituting the chitosan or chitosan porous beads are crosslinked,
A carrier for chromatography characterized in that an ω-carboxyalkanoyl group is bonded to an amino group based on glucosamine and/or an amino group based on a crosslinking agent constituting the chitosan, and the remaining amino groups are acylated. .
また、本発明は、キトサン多孔性ビーズであって、該キ
トサンを構成するグルコサミンに基くアミノ基に、また
はキトサン多孔性ビーズを架橋処理したものであって、
該キトサンを構成するグルコサミンに基くアミノ基及び
/または架橋処理剤に基くアミノ基に、アルカフジカル
2ン酸無水物を作用させ、次いで残アミノ基にモノカル
ボン酸無水物またはハロダン化アシルを作用させること
を特徴とするクロマトグラフィー用担体の製造方法に関
する。The present invention also provides chitosan porous beads, in which amino groups based on glucosamine constituting the chitosan or chitosan porous beads are cross-linked,
The amino groups based on glucosamine and/or the amino groups based on the crosslinking treatment agent constituting the chitosan are treated with alkafudic acid anhydride, and then the remaining amino groups are treated with monocarboxylic acid anhydride or acyl halide. The present invention relates to a method for producing a chromatography carrier characterized by:
以下、本発明のクロマトグラフィー用担体、及びその製
造方法について説明する。Hereinafter, the chromatography carrier of the present invention and its manufacturing method will be explained.
本発明に係るキトサン多孔性ビーズは、甲殻類や昆虫類
などの甲皮に含まれるキチンを脱アセチル化処理して得
られるポリグルコサミンよ構成るものであシ、耐酸性を
持たせる為二官能性試薬、例えばジカルボン酸及びその
誘導体、ジアルデヒド、ジイソシアナート等を用いて架
橋処理を施したものも製造されておジ、例えば粒径0.
1〜3.0瓢、孔径500〜3000Xの多孔性ビーズ
として入手が可能である。このキトサン多孔性ビーズは
、一般に数μ〜数数10上モル当量11のグルコサミン
に基くアミ7基または架橋処理を施したものについては
架橋処理剤に基くものも含めてアミノ基を有しておシ、
これらのアミノ基はアルカンジカルボン酸無水物を作用
させることにより、カルがキシル基を有する活性支持基
に変換することができる。The chitosan porous beads according to the present invention are composed of polyglucosamine obtained by deacetylating chitin contained in the carapace of crustaceans and insects, and are bifunctional in order to have acid resistance. Products that are crosslinked using a chemical reagent such as dicarboxylic acid and its derivatives, dialdehyde, diisocyanate, etc. are also manufactured, and have a particle size of 0.
It is available as porous beads with a diameter of 1 to 3.0 mm and a pore size of 500 to 3000X. These porous chitosan beads generally have an amino group based on glucosamine with a molar equivalent of several microns to several tens of 11, or amino groups, including those based on a crosslinking agent for those that have been crosslinked. C,
These amino groups can be converted into active supporting groups in which Cal has a xyl group by the action of an alkanedicarboxylic anhydride.
反応の溶媒としては通常水が用いられるが、その他テト
ラヒドロフラン、ジオキサンなどのエーテル類、酢酸な
どのカルボン酸類、ピリジンなども用いられる。また、
特に触媒は用いないでもよいが、塩酸、硫酸などの酸や
、水酸化ナトリウムや炭酸カリウムなどのアルカリの添
加によシ反応液の−を調整することもできる。Water is usually used as a solvent for the reaction, but other solvents such as ethers such as tetrahydrofuran and dioxane, carboxylic acids such as acetic acid, and pyridine are also used. Also,
In particular, a catalyst may not be used, but the -ness of the reaction solution can also be adjusted by adding an acid such as hydrochloric acid or sulfuric acid, or an alkali such as sodium hydroxide or potassium carbonate.
反応に用いられるアルカンジカルボン酸無水物としては
、コハク酸無水物、グルタル酸無水物、アジピン酸無水
物、ピメリン酸無水物、スペリン酸無水物、アゼライン
酸無水物、セパシン酸無水物、1,10−デカンジカル
ボン酸無水物、1.12−ドデカンノカルボン酸無水物
、1.14−テトラデカンジカルボン酸無水物などを例
示することができる。Examples of the alkanedicarboxylic anhydride used in the reaction include succinic anhydride, glutaric anhydride, adipic anhydride, pimelic anhydride, speric anhydride, azelaic anhydride, cepacic anhydride, 1,10 Examples include -decanedicarboxylic anhydride, 1.12-dodecanedicarboxylic anhydride, and 1.14-tetradecanedicarboxylic anhydride.
反応条件については必ずしも制限はないが、−般に次の
ような条件を選択して反応を行なうのが好ましい。Although there are no particular restrictions on the reaction conditions, it is generally preferable to select the following conditions to carry out the reaction.
キトサン多孔質ビーズの重量(a)とアルカンジカルボ
ン酸無水物の重量(b)の比:
a : b = 1 : 0.05〜10、よシ好まし
くは、
a : b = 1 : 0.1〜3、反応温度二 0
〜150℃、よシ好ましくは室温〜100℃、反応時間
: 1〜60時間、よシ好ましくは1〜30時間、反応
圧カニ常圧〜10 atm、よシ好ましくは常圧。The ratio of the weight of chitosan porous beads (a) to the weight of alkanedicarboxylic acid anhydride (b): a: b = 1: 0.05-10, preferably a: b = 1: 0.1- 3. Reaction temperature 20
-150°C, preferably room temperature - 100°C, reaction time: 1-60 hours, preferably 1-30 hours, reaction pressure: normal pressure - 10 atm, preferably normal pressure.
反応後の後処理についても特別な要件は無く、F別、洗
浄等通常行なわれている方法にて適宜実施される。There are no special requirements for post-treatment after the reaction, and conventional methods such as separation of F and washing may be carried out as appropriate.
以上によシ得られた力、VZキシル基を有するキトサン
多孔質ビーズは、次いでモノカルボン酸無水物またはハ
ロゲン化アシルを作用させることによシ残余のアミノ基
をアシル化することができる。The chitosan porous beads having VZ xyl groups obtained above can then be treated with a monocarboxylic anhydride or an acyl halide to acylate the remaining amino groups.
このときの反応の条件は、前述のアルカンジカルボン酸
無水物の場合と同様にして行うことができるが、アシル
化剤としてハロゲン化アシルを用いる場合は溶媒として
は水板外のものを用いた方がよく、また触媒としてはア
ルカリが好ましい。The reaction conditions at this time can be the same as those for the alkane dicarboxylic anhydride described above, but when using an acyl halide as the acylating agent, it is preferable to use a solvent other than water. is preferred, and an alkali is preferred as the catalyst.
反応に用いられるモノカルボン酸無水物としては、無水
酢酸、無水プロピオン酸、無水酪酸などが例示され、ま
た、ハロダン化アシルとしては、塩化アセチル、臭化ア
セチル、塩化グロピオニル、塩化ブチリルなどを例示す
ることができる。Examples of the monocarboxylic anhydride used in the reaction include acetic anhydride, propionic anhydride, butyric anhydride, and examples of the acyl halide include acetyl chloride, acetyl bromide, glopionyl chloride, butyryl chloride, etc. be able to.
発明の効果
本発明によシ提供されるクロマトグラフィー用担体は、
既に結合されているカルがキシル基を有する活性支持基
を介してアフィニティクロマトグラフィー用配位子(リ
ガンド)を容易に共有結合させることが可能であシ、従
って7フイニテイクロマトグラフイー用の水不溶性活性
支持体として非常に有用なものである。Effects of the Invention The chromatography carrier provided by the present invention is
It is possible to easily covalently bond an affinity chromatography ligand (ligand) to an already bound carbon via an active support group having a xyl group. It is very useful as an insoluble active support.
その上、前述したアフィニティークロマトグラフィー用
活性支持体に望まれる性質については、本発明に係るキ
トサン多孔質ビーズのもたらす親水的性質は例えば蛋白
質等と接触する際の疎水的非特異的吸着を抑え、またそ
の有孔構造はリガンドと吸着目的物質の相互作用の発現
に有利に作用しうろことが明らかである。Furthermore, regarding the properties desired in the above-mentioned active support for affinity chromatography, the hydrophilic properties provided by the chitosan porous beads according to the present invention suppress hydrophobic non-specific adsorption when coming into contact with proteins, etc. It is also clear that the porous structure has an advantageous effect on the interaction between the ligand and the adsorbed target substance.
また本発明によシ提供されるクロマトグラフィー用担体
は、キトサン多孔質ビーズの有するアミノ基を完全にア
シル化することによシ、例えば蛋白質等と接触する際の
イオン的な非特異的吸着をも排除している為、リガンド
と吸着目的物質の選択性を更に高めたものとすることが
できる。Furthermore, the chromatography carrier provided by the present invention completely acylates the amino groups of chitosan porous beads, thereby preventing ionic nonspecific adsorption when coming into contact with proteins, etc. Also, the selectivity between the ligand and the target substance to be adsorbed can be further improved.
更に本発明により提供されるクロマトグラフィー用担体
は、化学的に中性で安定な共有結合によシリガントを結
合せしめうる為、通常供せられる臭化シアンやグルタル
アルデヒドを用いたものに比してアフィニティクロマト
グラフィー用吸着担体として目的物質の分離、溶出およ
び洗浄の条件下で全く何らの影響を受けることがないこ
とは大きな利点となる。これらの性質は特に高速アフィ
ニティクロマトグラフィー用の担体及び工業的分離精製
プロセス用の担体としては欠くべからざるものであシ、
本発明によシ提供されるクロマトグラフィー用担体の効
果は非常に大きい。Furthermore, the chromatography carrier provided by the present invention is capable of bonding a siligant with a chemically neutral and stable covalent bond, compared to the commonly available carriers using cyanogen bromide or glutaraldehyde. As an adsorption carrier for affinity chromatography, it has a great advantage that it is completely unaffected by the conditions of separation, elution, and washing of the target substance. These properties are indispensable especially as a carrier for high-speed affinity chromatography and a carrier for industrial separation and purification processes.
The effects of the chromatography carrier provided by the present invention are very large.
実施例
本発明によシ提供されるクロマトグラフィー用担体は、
前述の如くアフイニティクロマトグラフィー用の活性支
持体として有用なものであるが、以下に本発明のクロマ
トグラフィー用担体の製造法について代表的な例を示し
、更に具体的に説明する。但し、これらは説明の為の単
なる例示であって、本発明はこれらに何ら制限されない
ことは言うまでもない。Examples The chromatographic carrier provided by the present invention is
As mentioned above, it is useful as an active support for affinity chromatography, and below, representative examples of the method for producing the chromatography support of the present invention will be shown and more specifically explained. However, these are merely examples for explanation, and it goes without saying that the present invention is not limited thereto.
実施例1
粒径061mのキトサン多孔性ビーズ(商品名ショウデ
ックスキトノセール、芳香族系架橋品)1.OIに無水
グルタル酸1.0gの水5−における溶液を加え、4規
定水酸化ナトリウム水溶液を加えて−を6に調整した後
、室温で一日振盪した。ビーズを戸数し、0.1規定炭
酸水素ナトリウム水溶液、0.1規定塩酸及び水の順に
洗浄した。次いで、ビーズに0.2モル酢酸ナトリウム
水溶液5−を加え、無水酢酸0.5gを加えた後、室温
で2時間振盪した。ビーズを戸数し大量の水で洗い乾燥
させた。Example 1 Chitosan porous beads with a particle size of 061 m (trade name Shodex Chitonosele, aromatic crosslinked product) 1. A solution of 1.0 g of glutaric anhydride in water was added to OI, and after adjusting the - to 6 by adding 4N aqueous sodium hydroxide solution, it was shaken at room temperature for one day. The beads were separated and washed in the order of 0.1N aqueous sodium bicarbonate solution, 0.1N hydrochloric acid, and water. Next, a 0.2 molar aqueous sodium acetate solution 5- was added to the beads, and 0.5 g of acetic anhydride was added thereto, followed by shaking at room temperature for 2 hours. The beads were washed in large amounts of water and dried.
コノビーズはトリニトロベンゼンスルホン酸ナトリウム
による比色法でアミノを含まないことが確められた。中
和滴定によるカルボン酸の定量値は乾燥ビーズ1g当り
0.35ミリモルであった。Conobeads were confirmed to be amino-free using a colorimetric method using sodium trinitrobenzenesulfonate. The quantitative value of carboxylic acid determined by neutralization titration was 0.35 mmol per gram of dry beads.
実施例2
粒径0.3簡のキトサン多孔性ビーズ(商品名ショウデ
ックスキトノソール、脂肪族系架橋品)1.OJに無水
コハク酸0.11のジオキサン5づにおける溶液を加え
、次いでピリジン0.1gを加えて室温で6時間振盪し
た。ビーズを戸数口、ジオキサンで洗浄した後、塩化ア
セチル0.5Fのジオキサン5Tntにおける溶液を加
え、トリエチルアミノ0.5gを加えて室温で30分振
盪した。ビーズをF取しジオキサンで洗浄した後、大量
の水で洗い乾燥すせた。このビーズはトリニトロベンゼ
ンスルホン酸ナトリウムによる比色法でアミノを含まな
いことが確められた。中和滴定によるカルボン酸の定量
値は乾燥ピーズ1g当、?0.80ミ’、1モルであっ
た。Example 2 Chitosan porous beads with a particle size of 0.3 mm (trade name Shodex Chitonosol, aliphatic crosslinked product) 1. A solution of 0.11 succinic anhydride in 5 parts of dioxane was added to the OJ, followed by 0.1 g of pyridine and shaken at room temperature for 6 hours. After washing the beads several times with dioxane, a solution of 0.5 F of acetyl chloride in 5 Tnt of dioxane was added, 0.5 g of triethylamino was added, and the mixture was shaken for 30 minutes at room temperature. The beads were taken out and washed with dioxane, then washed with a large amount of water and dried. The beads were confirmed to be amino-free using a colorimetric method using sodium trinitrobenzenesulfonate. What is the quantitative value of carboxylic acid by neutralization titration per 1g of dry peas? It was 0.80 m', 1 mole.
参考例1
実施例1によシ得られたビーズ1.OFを無水ジオキサ
ンで充分洗浄した後、無水ジオキサン4 ml中に加え
、更にN−ヒドロキシコハク酸イミド801M9及びジ
シクロへキシルカルボジイミド144■を加えて室温で
2時間振盪した。次いでビーズを戸数し無水ジオキサン
20−、メタノール6ゴ、冷水3mlの順で素早く洗浄
した。このビーズをコンカナバリンA 307n9及び
メチル−α−マンノピラノシド401n9の0.1ミリ
モル塩化カルシウム、0.1ミりモル塩化マンガン及び
o、oiモル炭酸水素ナトリウムの水2−による溶液に
加え、室温で2時間振盪後、4℃で1夜放置した。ビー
ズを戸取し、1モル塩化ナトリウム水溶液及び水で洗浄
した後、1モルトリス−塩酸緩衝液(pH8,0)2−
に加え室温で1時間振盪した後、再びビーズを戸数して
水洗した。こうして得られた吸着担体は乾fflビーズ
1g当シコンカナバリンAを15m9担持させているこ
とが未反応による回収コンカナバリンAの量から確かめ
られた。Reference Example 1 Beads obtained according to Example 1 1. After thoroughly washing the OF with anhydrous dioxane, it was added to 4 ml of anhydrous dioxane, 801M9 of N-hydroxysuccinimide and 144 ml of dicyclohexylcarbodiimide were added, and the mixture was shaken at room temperature for 2 hours. The beads were then washed quickly in the following order: 20 ml of anhydrous dioxane, 6 ml of methanol, and 3 ml of cold water. The beads were added to a solution of concanavalin A 307n9 and methyl-α-mannopyranoside 401n9 in 0.1 mmol calcium chloride, 0.1 mmol manganese chloride and o, oi moles sodium bicarbonate in water for 2 hours at room temperature. After shaking, it was left at 4°C overnight. After taking the beads and washing them with a 1M aqueous sodium chloride solution and water, they were added to a 1M Tris-HCl buffer (pH 8,0) 2-
After shaking at room temperature for 1 hour, the beads were washed again with water. It was confirmed from the amount of unreacted concanavalin A that the adsorption carrier thus obtained supported 15 m9 of concanavalin A per gram of dry ffl beads.
参考例2
実施例2により得られたビーズについて参考例1のコン
カナバリンA30■の溶液の代りに、プロティンA10
m9の0.01モル炭酸水素ナトリウムの水2rnlK
おける溶液を用い、その他は同様の操作を行うことによ
り、乾燥ピーズ1g白シプロナインAを6. Orn9
担持させている吸着担体を得ることができた。Reference Example 2 Regarding the beads obtained in Example 2, protein A10 was added instead of the concanavalin A30 solution of Reference Example 1.
m9 0.01 mol sodium bicarbonate water 2rnlK
By using the solution in 6.5 hours and performing the same procedure as above, 1 g of dried peas white cypronaine A was added. Orn9
It was possible to obtain an adsorption carrier on which the particles were supported.
特許出臥 昭和電工株式会社Patent issued: Showa Denko Co., Ltd.
Claims (2)
成するグルコサミンに基くアミノ基に、またはキトサン
多孔性ビーズを架橋処理したものであって、該キトサン
を構成するグルコサミンに基くアミノ基及び/または架
橋処理剤に基くアミノ基に、ω−カルボキシアルカノイ
ル基が結合され、残アミノ基がアシル化されていること
を特徴とするクロマトグラフィー用担体。(1) Chitosan porous beads, in which the amino groups based on glucosamine constituting the chitosan are cross-linked, or chitosan porous beads are cross-linked, and the amino groups based on glucosamine constituting the chitosan and/or A chromatography carrier characterized in that an ω-carboxyalkanoyl group is bonded to an amino group based on a crosslinking agent, and the remaining amino group is acylated.
成するグルコサミンに基くアミノ基に、またはキトサン
多孔性ビーズを架橋処理したものであって、該キトサン
を構成するグルコサミンに基くアミノ基及び/または架
橋処理剤に基くアミノ基に、アルカンジカルボン酸無水
物を作用させ、次いで残アミノ基にモノカルボン酸無水
物またはハロゲン化アシルを作用させることを特徴とす
るクロマトグラフィー用担体の製造方法。(2) Chitosan porous beads, in which amino groups based on glucosamine constituting the chitosan are cross-linked; A method for producing a chromatography carrier, which comprises reacting an alkanedicarboxylic anhydride with an amino group based on a crosslinking agent, and then reacting a monocarboxylic anhydride or an acyl halide with the remaining amino group.
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP61192143A JPS6348453A (en) | 1986-08-19 | 1986-08-19 | Carrier for chromatography and its production |
GB8719348A GB2195344B (en) | 1986-08-19 | 1987-08-14 | Adsorbent composed of porous beads of chitosan and adsorption method using same |
DE19873727707 DE3727707A1 (en) | 1986-08-19 | 1987-08-19 | ADSORPTION AGENT, COMPOSED FROM POROUS CHITOSAN SEEDS, AND ADSORPTION METHOD USING THIS ADSORPTION AGENT |
US07/086,989 US4879340A (en) | 1986-08-19 | 1987-08-19 | Adsorbent composed of porous beads of chitosan and adsorption method using same |
GB9016548A GB2232984B (en) | 1986-08-19 | 1990-07-27 | Method of adsorbing immunoglobulin by using porous beads of chitosan |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP61192143A JPS6348453A (en) | 1986-08-19 | 1986-08-19 | Carrier for chromatography and its production |
Publications (1)
Publication Number | Publication Date |
---|---|
JPS6348453A true JPS6348453A (en) | 1988-03-01 |
Family
ID=16286408
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP61192143A Pending JPS6348453A (en) | 1986-08-19 | 1986-08-19 | Carrier for chromatography and its production |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS6348453A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH01248057A (en) * | 1988-03-29 | 1989-10-03 | Fuji Spinning Co Ltd | Manufacture of support for affinity chromatography |
JP2019513549A (en) * | 2016-04-12 | 2019-05-30 | ザルトリウス ステディム ビオテック ゲーエムベーハー | Multimodal adsorption medium with multimodal ligands, process for its preparation and its use |
-
1986
- 1986-08-19 JP JP61192143A patent/JPS6348453A/en active Pending
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH01248057A (en) * | 1988-03-29 | 1989-10-03 | Fuji Spinning Co Ltd | Manufacture of support for affinity chromatography |
JP2019513549A (en) * | 2016-04-12 | 2019-05-30 | ザルトリウス ステディム ビオテック ゲーエムベーハー | Multimodal adsorption medium with multimodal ligands, process for its preparation and its use |
JP2021058882A (en) * | 2016-04-12 | 2021-04-15 | ザルトリウス ステディム ビオテック ゲーエムベーハー | Multimodal adsorption medium with multimodal ligands, method for production and use thereof |
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