JPS6288961A - Separation of albumin and glogulin - Google Patents
Separation of albumin and glogulinInfo
- Publication number
- JPS6288961A JPS6288961A JP60228967A JP22896785A JPS6288961A JP S6288961 A JPS6288961 A JP S6288961A JP 60228967 A JP60228967 A JP 60228967A JP 22896785 A JP22896785 A JP 22896785A JP S6288961 A JPS6288961 A JP S6288961A
- Authority
- JP
- Japan
- Prior art keywords
- albumin
- ion exchange
- exchange group
- separation
- gel
- 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.)
- Granted
Links
- 102000009027 Albumins Human genes 0.000 title claims abstract description 35
- 108010088751 Albumins Proteins 0.000 title claims abstract description 35
- 238000000926 separation method Methods 0.000 title description 16
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 13
- 238000005342 ion exchange Methods 0.000 claims abstract description 11
- 230000001476 alcoholic effect Effects 0.000 claims abstract description 7
- 210000001124 body fluid Anatomy 0.000 claims abstract description 7
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 6
- 229920001577 copolymer Polymers 0.000 claims abstract description 6
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 26
- 102000006395 Globulins Human genes 0.000 claims description 10
- 108010044091 Globulins Proteins 0.000 claims description 10
- 239000010839 body fluid Substances 0.000 claims description 5
- 238000005349 anion exchange Methods 0.000 abstract description 5
- 125000001664 diethylamino group Chemical group [H]C([H])([H])C([H])([H])N(*)C([H])([H])C([H])([H])[H] 0.000 abstract description 4
- KOMNUTZXSVSERR-UHFFFAOYSA-N 1,3,5-tris(prop-2-enyl)-1,3,5-triazinane-2,4,6-trione Chemical compound C=CCN1C(=O)N(CC=C)C(=O)N(CC=C)C1=O KOMNUTZXSVSERR-UHFFFAOYSA-N 0.000 abstract description 3
- 229920006037 cross link polymer Polymers 0.000 abstract description 3
- 150000002500 ions Chemical class 0.000 abstract 2
- 238000004132 cross linking Methods 0.000 abstract 1
- 239000007788 liquid Substances 0.000 abstract 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 11
- 238000004587 chromatography analysis Methods 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 8
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- 238000007796 conventional method Methods 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 102000004506 Blood Proteins Human genes 0.000 description 3
- 108010017384 Blood Proteins Proteins 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 238000005341 cation exchange Methods 0.000 description 2
- 208000019425 cirrhosis of liver Diseases 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 208000006454 hepatitis Diseases 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 208000019423 liver disease Diseases 0.000 description 2
- 230000005976 liver dysfunction Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- 230000003169 placental effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000005185 salting out Methods 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical group C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 description 1
- UCBVELLBUAKUNE-UHFFFAOYSA-N 1,3-bis(prop-2-enyl)-1,3,5-triazinane-2,4,6-trione Chemical compound C=CCN1C(=O)NC(=O)N(CC=C)C1=O UCBVELLBUAKUNE-UHFFFAOYSA-N 0.000 description 1
- VAYTZRYEBVHVLE-UHFFFAOYSA-N 1,3-dioxol-2-one Chemical compound O=C1OC=CO1 VAYTZRYEBVHVLE-UHFFFAOYSA-N 0.000 description 1
- BJELTSYBAHKXRW-UHFFFAOYSA-N 2,4,6-triallyloxy-1,3,5-triazine Chemical compound C=CCOC1=NC(OCC=C)=NC(OCC=C)=N1 BJELTSYBAHKXRW-UHFFFAOYSA-N 0.000 description 1
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- 208000027932 Collagen disease Diseases 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 108091006629 SLC13A2 Proteins 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- XTXRWKRVRITETP-UHFFFAOYSA-N Vinyl acetate Chemical compound CC(=O)OC=C XTXRWKRVRITETP-UHFFFAOYSA-N 0.000 description 1
- 208000033559 Waldenström macroglobulinemia Diseases 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 239000012052 hydrophilic carrier Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000000564 macroglobulinemia Diseases 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- -1 polyethylene Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 125000000542 sulfonic acid group Chemical group 0.000 description 1
- 208000006379 syphilis Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
Landscapes
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
Description
【発明の詳細な説明】
Ｃ産業上の利用分野）
本発明は、クロマトグラフィーを用いたアルブミンの迅
速分離法に関する。DETAILED DESCRIPTION OF THE INVENTION C. Industrial Application Field The present invention relates to a method for rapid separation of albumin using chromatography.
最近、血液の有効利用と輸血による副作用の防止の両面
から、全血をそのまま保存血液として１史用するので汀
なく、できるだけ細かい成分に分けて患者にとって必要
な成分のみを号・貰注し、他の成分はそれを必要とする
別の馨者に役立てようという方式、すなわち、成分製剤
としての＋１１用が急増してきた。Recently, in order to effectively utilize blood and prevent side effects from blood transfusions, whole blood is used as preserved blood for a period of time, so it is divided into as small components as possible and only the components necessary for the patient are ordered. The use of +11 as an ingredient preparation has rapidly increased, with other ingredients being used to help other people who need them.
アルブミン製剤は最も広く用いられている成分製剤の一
つで、厚生省薬務局の生物学的製剤基準によれば、「加
熱人血漿蛋白」と［人ｉｎ ｔｉＩアルブミン］とがあ
り、浮腫、腹水の防止に、また、出血性ショックや火傷
の救急治療、さらには１ｍ漿交換時の輸液として有用で
ある、
アルブミン製剤の需要針は、その供給計をはるかに超え
、このアンバランスは、今ｔＳ：、ますます深刻化する
可能性が強い。当然ながら供給量には限度があるので、
効率のよい、留製法の開発が熱望されている。Albumin preparations are one of the most widely used component preparations, and according to the Biological Products Standards of the Pharmaceutical Affairs Bureau of the Ministry of Health, Labor and Welfare, they contain "heated human plasma protein" and [human in tiI albumin], and are known to cause edema and ascites. The demand for albumin preparations far exceeds the supply, and this imbalance is currently occurring. : There is a strong possibility that the situation will become even more serious. Naturally, there is a limit to the supply, so
The development of efficient distillation methods is eagerly awaited.
一方、アルブミンは臨床検査の分野でも極めて重要であ
る。On the other hand, albumin is also extremely important in the field of clinical testing.
一般に、多くの疾患でアルブミンＦｉ減少することが多
く、アルブミンとグロブリンの油化、いわゆるＡ／Ｇ比
は低下することが多い。例えば、肝硬変、慢性肝炎、肝
癌１、色性肝炎、肝委酪等の肝機能障害にともなうアル
ブミンの減少、多発性骨髄腫、マクログロブリン血症、
悪性リンパ腫、膠原病、肝硬変、慢性感染症、梅毒、関
節リウマチ、マラリア等にともなうグロブリンの減少を
反映する、このような理由で、Ａ／Ｇ比は最も頻繁に行
なわれる健康診断（スクリーニング）の項目の一つであ
る。Generally, albumin Fi often decreases in many diseases, and the conversion of albumin and globulin into oil, the so-called A/G ratio, often decreases. For example, liver cirrhosis, chronic hepatitis, liver cancer1, chromatic hepatitis, decreased albumin due to liver dysfunction such as liver dysfunction, multiple myeloma, macroglobulinemia,
For this reason, the A/G ratio is one of the most frequently performed medical examinations (screenings), reflecting the decrease in globulin associated with malignant lymphoma, collagen disease, liver cirrhosis, chronic infections, syphilis, rheumatoid arthritis, malaria, etc. This is one of the items.
（従来の技術および発明が解決しようとする問題点）
！Ｔｒｉ嘴蛋白質の従来の分画法には、塩析法、コーン
（Ｃｏｈｎ ）分別法、ポリエチレングリコール法等が
あるが、中でも１９４０年代に開発された冷エタノール
を使用するコーン法が最も幅広く使用されている。にも
かかわらず、この方法には多くの欠点がある。一つは、
高濃度のエタノールを使用するため、多くの貴重な生物
学的に活性な血漿蛋白の変性と不活性化をまねく。さら
に、この方法は、多数の処理工程からなり、煩雑で長時
間を要し、かつ、バッチ方式でのみ実施可能な方法であ
る。(Problems to be solved by conventional techniques and inventions)! Conventional methods for fractionating Tri-beak proteins include the salting-out method, the Cohn fractionation method, and the polyethylene glycol method, among which the Cohn method, which uses cold ethanol and was developed in the 1940s, is the most widely used. ing. Nevertheless, this method has many drawbacks. one,
The use of high concentrations of ethanol leads to denaturation and inactivation of many valuable biologically active plasma proteins. Furthermore, this method consists of a large number of processing steps, is complicated and takes a long time, and can only be carried out in a batch manner.
半透膜による分離法は、操作性は高いが、満足できる分
離能を有するものは未だない。Separation methods using semipermeable membranes have high operability, but none yet have satisfactory separation performance.
また、最近では、ゲル濾過法、イオン交換クロマトグラ
フィー法等が開発されている。Furthermore, recently, gel filtration methods, ion exchange chromatography methods, and the like have been developed.
例えば、カチオン交検体とアニオン交換体を組み合わせ
て、血漿製品からアルブミンを単離する方法（特開昭５
２−４７９１５）が開発されているが、凝血因子Ｉ、
ＩＩ、■、■、ＩＸ、Ｓ−よびＸｌおよびＩｇＧの主
要部分を実質的に含まない血漿フラクションにしか適用
できず、また、操作が簡便であるとけ言えない。For example, a method for isolating albumin from plasma products by combining a cation exchanger and an anion exchanger (Japanese Patent Laid-open No.
2-47915) has been developed, but clotting factor I,
This method can only be applied to plasma fractions that do not substantially contain major portions of II, ■, ■, IX, S- and Xl, and IgG, and it cannot be said that it is easy to operate.
また、一方、親水性担体を用いた分子篩クロマトグラフ
ィーによるσ１漿蛋白質の分離方法Ｃ％開昭５４−４６
８１３）が開発されている。この方法によれば、α、β
、ｒの各グロブリンおよびアルブミンの相互分離が従来
法と比較すると良好であるが、アルブミンと他の成分と
の分離は不充分である。On the other hand, a method for separating σ1 serum protein by molecular sieve chromatography using a hydrophilic carrier
813) has been developed. According to this method, α, β
, r and albumin are better separated from each other than conventional methods, but the separation of albumin from other components is insufficient.
例えば、ＩｇＧの一部およびトランスフェリンのほぼ全
量がアルブミン分画に混入してしまう。また、これらの
分離に３０分以上を要する。For example, part of the IgG and almost all of the transferrin end up contaminating the albumin fraction. Moreover, these separations require 30 minutes or more.
一方、Ａ／Ｇ比の測定は、従来、塩析法、ｐ紙電気泳励
法、セルロースアセテート膜法、屈折法等があるが、各
々の測定法による正常値が、それぞれ１．０〜１．５．
１．６ Ｓ−１，９，１，６〜２．４．１．５〜２．４
と大きくばらつく。したがって、Ａ／Ｇ比ｄ１１１定の
信頓できる標準法の開発が待たれる。On the other hand, conventional methods for measuring the A/G ratio include the salting-out method, p-paper electrophoresis method, cellulose acetate membrane method, and refraction method, but the normal values for each measurement method are 1.0 to 1. .5.
1.6 S-1,9,1,6~2.4.1.5~2.4
It varies widely. Therefore, the development of a reliable standard method with a constant A/G ratio of d111 is awaited.
（問題点を解決するための手段）
本発明者らは、前記の問題点を克服するため鋭意研究の
結果、迅速かつ１ｍ便な操作で、アルブミンを含む体液
または１ｍ漿製品から、アルブミンと他の成分全効率よ
く分離する方法を見出し、本発明を完成するに至った。(Means for Solving the Problems) In order to overcome the above-mentioned problems, the present inventors have conducted intensive research and found that albumin and other substances can be extracted from albumin-containing body fluids or 1 m serum products by a quick and 1 m stool operation. We have discovered a method for efficiently separating all of the components, and have completed the present invention.
すなわち、本発明は、主鎖に直接結合したアルコール性
水酸基とイオン交換基とを有する硬質の全多孔性粒状架
橋共重合体を固定相とし、ｐＨ６，５〜１０．０、イオ
ン強度０．０５〜１．５の移動相を用いた液体クロマト
グラフィーによって、アルブミンを含む体液またはｌ［
ｉｌ漿堰品から、従来法と比較してはるかに短時間のう
ちに、また、簡便に、さらに効率よく、アルブミンと他
の成分とを分離する方法を提供するものである。That is, the present invention uses a hard fully porous granular crosslinked copolymer having an alcoholic hydroxyl group and an ion exchange group directly bonded to the main chain as a stationary phase, and has a pH of 6.5 to 10.0 and an ionic strength of 0.05. Albumin-containing body fluids or l[
The object of the present invention is to provide a method for separating albumin and other components from an il serum product in a much shorter time, more easily, and more efficiently than conventional methods.
以下、本発明の詳細な説明する。The present invention will be explained in detail below.
本発明で固定相として用いられる硬質の全多値性粒状架
橋共重合体ｃ以下、単にゲルと表わす）は、少なくとも
主鎖に結合したアルコール性水酸基を有する。主鎖に直
接結合したアルコール性水酸基とは、例えば、酢酸ビニ
ルや炭酸ビニレンの重合体をケン化して得られる水酸基
のことである。The hard fully multivalued particulate crosslinked copolymer c (hereinafter simply referred to as gel) used as the stationary phase in the present invention has at least an alcoholic hydroxyl group bonded to its main chain. The alcoholic hydroxyl group directly bonded to the main chain is, for example, a hydroxyl group obtained by saponifying a polymer of vinyl acetate or vinylene carbonate.
水酸基の量は、ゲル乾燥重叶当り５〜９ ｍｅｑ／？の
範囲にあるのがよい。ここで、水酸基の前は、例えば、
水酸基を無水酢酸と反応させて、消費した無水酢酸の！
またはゲルの重量変化を６１１１定することで求めるこ
とができる。The amount of hydroxyl groups is 5 to 9 meq/? It is good that it is within the range of . Here, before the hydroxyl group, for example,
Of the consumed acetic anhydride by reacting the hydroxyl group with acetic anhydride!
Alternatively, it can be determined by determining the weight change of the gel.
また、本発明のゲルは、水酸基の他にイオン交換基を有
する。例えば、アミン基、ジエチルアミノ基等の弱塩基
性アニオン交換基、４級アンモニウム基等の強塩基性ア
ニオン交換基、あるいはカルボキシル基、リン酸基等の
弱酸性カチオン交換基、スルホン酸基のような！ｉｌｉ
酸性カチオン交換基が好ましい。中でも塩基性アニオン
交換基が好運しく、特に、ジエチルアミノ基等の弱塩基
性アニオン交する基が好ましい。また、イオン交換基の
量は、０．１〜３ 、０ ｍｅｑ ／ｆの範囲にあるの
が好ましい。イオン交換基の情は、通常のイオン交換樹
脂の交摸容看の測定方法で求めることができる。Moreover, the gel of the present invention has ion exchange groups in addition to hydroxyl groups. For example, weakly basic anion exchange groups such as amine groups and diethylamino groups, strongly basic anion exchange groups such as quaternary ammonium groups, weakly acidic cation exchange groups such as carboxyl groups and phosphoric acid groups, and sulfonic acid groups. ! ili
Acidic cation exchange groups are preferred. Among these, basic anion exchange groups are preferred, and weakly basic anion exchange groups such as diethylamino groups are particularly preferred. Further, the amount of ion exchange groups is preferably in the range of 0.1 to 3.0 meq/f. The information on ion-exchange groups can be determined by the usual method of measuring the exchange capacity of ion-exchange resins.
上記の水酸基およびイオン交換基は、ゲル全体に分布し
ても、また、ボア形成部分すなわちボア＆面のみに分布
してもよい。The above-mentioned hydroxyl groups and ion exchange groups may be distributed throughout the gel, or may be distributed only in the bore-forming portion, that is, the bore & surface.
本発明の架橋共重合体の架喬構造は特に限定されないが
、例えば、トリアリルイソシアヌレート、ジアリルイソ
シアヌレートやトリアリルシアヌレート等のトリシアヌ
レート環やトリアジン環を有する架橋性単忙体単位によ
って架橋された構造等を挙げることができる。なかでも
トリアリルイソシアヌレートによって架橋された構造が
好ましい。The crosslinked structure of the crosslinked copolymer of the present invention is not particularly limited, but for example, crosslinked by a crosslinkable monomeric unit having a tricyanurate ring or a triazine ring such as triallyl isocyanurate, diallyl isocyanurate, and triallyl cyanurate. Examples of such structures include: Among these, a structure crosslinked with triallyl isocyanurate is preferred.
本発明で用いる硬質のゲルとは、機械的強度が大で、し
かも内部までボアが分布した構造を有するゲルのことで
ある。このようなゲルは、前述したセファデックスのよ
うな軟質のゲルと異なり、乾燥状態でも膨潤時のボア構
造を実質的に維持するため、乾燥状態での比表面積が大
である。本発明のゲルは、通常、乾燥ゲル重ｔ＋ｔ ３
Ｆ） ２ ｒｎ”／ １以上、好ましくは５〜ｔｏｏ
ｏｒＩＬ２／ｙの比表面積を有する。一方、軟質ゲルの
乾燥時の比表面積１ηＬ２／ｙ以下の小さい僅を示す。The hard gel used in the present invention is a gel that has high mechanical strength and has a structure in which bores are distributed throughout the interior. Unlike the above-mentioned soft gel such as Sephadex, such a gel substantially maintains the swollen bore structure even in a dry state, and therefore has a large specific surface area in a dry state. The gel of the present invention typically has a dry gel weight t+t 3
F) 2rn”/1 or more, preferably 5 to too
It has a specific surface area of orIL2/y. On the other hand, the dry specific surface area of the soft gel is less than 1ηL2/y.
比表面積が本発明の範囲にあるゲルは、機械的強度が大
きいので、クロマトグラフィー用の４ｐ体として用いた
ときに、溶ｍ浴媒を高流速で通液することができ、迅速
な分離・分析が可能となる。ゲル中のボアの大きさは、
少なくともアルブミンが浸透できる程度の大きさであれ
ばよい。ボアの大きさは、デキストラン、ポリエチレン
グリフール等の分子全既知の標準サンプルヲ用いてゲル
パーミェーションクロマトグラフィーを行ない、得られ
た検量線から公知の方法で推定することができる。A gel with a specific surface area within the range of the present invention has high mechanical strength, so when used as a 4p body for chromatography, it allows the solution solution to be passed through at a high flow rate, resulting in rapid separation and separation. Analysis becomes possible. The size of the bore in the gel is
It is sufficient that the size is at least large enough to allow albumin to penetrate. The bore size can be estimated by a known method from a calibration curve obtained by performing gel permeation chromatography using a standard sample of all known molecules such as dextran or polyethylene glyfur.
本発明のゲルの粒径は、通常は１〜２０００μｍの範囲
にあるのがよい。高速液体クロマトグラフィー用充填削
として用いる場合は、平均粒径が２〜１５μｍｎの範囲
にあるのが好ましい。人前サンプルの分離ｆＣ目的とす
る場合は、より大きい粒径でよい。The particle size of the gel of the present invention is usually in the range of 1 to 2000 μm. When used as a filler for high performance liquid chromatography, the average particle size is preferably in the range of 2 to 15 μm. For the purpose of separation fC of human samples, a larger particle size may be used.
本発明の硬質ゲルの一例は、特開昭５９−８２６１号公
報に示されている。An example of the hard gel of the present invention is shown in JP-A-59-8261.
アルブミンの分離は、前記ゲルを通常ステンレスやガラ
スのカラムに充填し、いわゆる液体クロマトグラフィー
によって行なわれるが、薄層クロマトグラフィーとして
行なってもよい。Separation of albumin is usually carried out by filling a stainless steel or glass column with the gel and performing so-called liquid chromatography, but it may also be carried out by thin layer chromatography.
本発明のクロマトグラフィーで用いる移動相のｐＨは６
．５〜１０．０である。さらに好ましくは７．０〜９．
５である。また、移動相のイオン強度は０．０５〜１．
５、さらに好ましくは０．５〜１．０である。The pH of the mobile phase used in the chromatography of the present invention is 6.
．． 5 to 10.0. More preferably 7.0 to 9.
It is 5. In addition, the ionic strength of the mobile phase is 0.05 to 1.
5, more preferably 0.5 to 1.0.
サラに、エタノール、メタノール、アセトニトリル、エ
チレングリコール等の水溶性の有機溶媒を少量加えるこ
とにより、クロマトグラムの形状はシャープになり、蛋
白質の回収率を上げることができる。By adding a small amount of a water-soluble organic solvent such as ethanol, methanol, acetonitrile, or ethylene glycol to Sara, the shape of the chromatogram becomes sharper and the recovery rate of protein can be increased.
通常、クロマトグラフィーを行なう間、移動相の組成は
変化させることなく一定組成のままでよいが、必要によ
っては、段階的または連続的に変化させてもよい。Generally, the composition of the mobile phase may remain constant during chromatography, but it may be changed stepwise or continuously, if necessary.
本発明で対象とするアルブミンを含む体液または皿泉製
品とは、血漿、血清、胎盤血清、胎盤エキス、髄液等の
ような人間および人間以外の（動物の体液またはそれら
の分１ｌｉｌｉｌｉ物のうちアルブミンを含むものを言
う。The albumin-containing body fluids or dish spring products targeted by the present invention include human and non-human (animal body fluids) such as plasma, serum, placental serum, placental extract, cerebrospinal fluid, etc. Refers to substances containing albumin.
（実施例）
以下に本発明の実施例を示すが、本発明のめ囲は、これ
らの実施例により限定されるものではない。(Example) Examples of the present invention are shown below, but the scope of the present invention is not limited to these Examples.
実施例１
アルブミンの分離は、匂下に示すような条件の液体クロ
マトグラフィーで行なった。なお、カラム（Ａｓａｈｉ
ｐａｋ ＥＳ−５０２Ｎ ）は、主鎖に直接結合したア
ルコール性水酸基とイオン交閑）＆（ジエチルアミン基
）とを４１する硬質の全多孔性粒状架篇重合体を固定相
とした高速液体クロマトグラフィー用充填カラムである
。Example 1 Albumin was separated by liquid chromatography under the conditions shown below. In addition, the column (Asahi
pak ES-502N) is for high performance liquid chromatography with a stationary phase made of a hard fully porous granular cross-linked polymer with alcoholic hydroxyl groups directly bonded to the main chain and ion exchange) & (diethylamine groups). It is a packed column.
カラム：旭化成工業：閑 商品名
Ａｓａｈｉｐａｋ ＥＳ−５０２Ｎ
内径７．６＋ｎｔｘ、長さ１０Ｇ
移動相： ５０ ｍＮビストリｘ−ＨＣ１＋５００ｒｎ
ＭＮａＣ１＋１０％エタノール（ｐＨ７，０）
ポンプ二日本分光工業１掬 商品名
ＴＲＩ ＲＯＴＡＲ−Ｖ（流ｔ、ｔ ： １．Ｏｔａｐ
／ｒｔａｔ ）検出器：日本分光工業］閑 商品名
ＵＶＩＤＥＣ−１００−ＶＩ （波長：２８０＋ａｍ）
温 度： ３５Ｃ
（結 果）
血清（オーフ・ダイアグノスティック・システムズ丁−
夷のノーマルコントロール［ｍｔｆり（７）分１１！１
１Ｊｆｃ＠１図に示した。クロマトグラムかられかるよ
うに、アルブミンとグロブリンの分離は約４分で完了し
た。Column: Asahi Kasei Kogyo: Blank Product name Asahipak ES-502N Inner diameter 7.6+ntx, length 10G Mobile phase: 50 mN Bistrix-HC1+500rn
MNaC1 + 10% ethanol (pH 7.0) Pump Nihon Bunko Kogyo 1 scoop Product name TRI ROTAR-V (flow t, t: 1.Otap
/rtat) Detector: JASCO Corporation] Product name: UVIDEC-100-VI (Wavelength: 280+am)
Temperature: 35C (Results) Serum (Off Diagnostic Systems)
Yi's normal control [mtfri (7) min 11!1
1Jfc@1 shown in figure. As seen from the chromatogram, the separation of albumin and globulin was completed in about 4 minutes.
実施ｌ＋１１２
移動相：５０ｒｒＬＭエタノールアミｙ −ＨＣ／−＋
５００ ｒｎＭＮａＣ１＋４％エタノール（ｐＨ９，
５）その他は実施例１とまったく同一の条件で血清の分
離を行なった。Implementation 1+112 Mobile phase: 50rrLM ethanolamine -HC/-+
500 rnM NaC1 + 4% ethanol (pH 9,
5) Serum was otherwise separated under exactly the same conditions as in Example 1.
（結 果）
得られたクロマトグラムを第２図に示した。図かられか
るように、アルブミンとグロブリンの分離は約５分で完
了し、その分離は極めて良好であった。(Results) The obtained chromatogram is shown in Figure 2. As can be seen from the figure, the separation of albumin and globulin was completed in about 5 minutes, and the separation was extremely good.
（発明の効果）
本発明のクロマトグラフィーによるアルブミンとグロブ
リンの分離方法においては、主鎖に直接結合したアルコ
ール性水酸基の他に、イオン交換基を導入した硬質の全
多孔性粒状架橋共重合体を固定相とし、ｐ Ｈ６，５〜
１０．０、イオン強度０．０５〜１．５の移動相を用い
ることによって、アルブミンとグロブリンの分子量、等
電点、親疎水性等の物性の差を利用し、かつ、それらの
ゲルへの吸着をできるだけ押えることによって、アルブ
ミンとグロブリンの分離を短時間のうちに効率よく行な
うことができるようになった。しかも、クロマトグラフ
ィーを行なう間、移動相の組成は変化させることなく一
定組成のままでもよいので、操作も極めて簡便である。(Effects of the Invention) In the method for separating albumin and globulin by chromatography of the present invention, a hard fully porous granular crosslinked copolymer having an ion exchange group introduced in addition to the alcoholic hydroxyl group directly bonded to the main chain is used. Fixed phase, pH 6.5~
By using a mobile phase with an ionic strength of 10.0 and an ionic strength of 0.05 to 1.5, the differences in physical properties such as molecular weight, isoelectric point, and hydrophilicity between albumin and globulin can be utilized, and their adsorption onto the gel can be achieved. By suppressing the amount of globulin as much as possible, albumin and globulin can now be separated efficiently in a short period of time. Furthermore, since the composition of the mobile phase may remain constant during chromatography without being changed, the operation is extremely simple.
本発明の比較例として、前述したイオン交換クロマトグ
ラフィー法（特開昭５２−４７９１５）と、分子篩クロ
マトグラフィー法（特開昭５４−４６８１５）が挙げら
れるが、前者は、擬餌因子とＩｇＧを含まない血漿フラ
クションにしか適用できないという条件付きで、分離度
と操作性に問題がある。後者は、やはり分離度と迅速性
に問題がある。Comparative examples of the present invention include the aforementioned ion exchange chromatography method (Japanese Unexamined Patent Publication No. 52-47915) and molecular sieve chromatography method (Japanese Unexamined Patent Publication No. 54-46815), but the former contains a bait factor and IgG. However, it has problems with resolution and ease of use, with the proviso that it can only be applied to plasma fractions that do not exist. The latter also has problems with the degree of separation and speed.
以上述べたように、本発明におけるアルブミンとそれ以
外の成分（グロブリン）の分離は、従来のいかなる方法
と比較しても、分離能、迅速性、簡便性等のあらゆる点
においてけるかに優れている。As mentioned above, the separation of albumin and other components (globulins) in the present invention is superior to any conventional method in all respects such as separation ability, speed, and simplicity. There is.
第１図は実施例１によって健常人血清を分だした結果を
示すクロマトグラム、第２図は実施例２によって同じく
健常人血清を分離した結果を示すクロマトグラムである
。FIG. 1 is a chromatogram showing the results of separating healthy human serum according to Example 1, and FIG. 2 is a chromatogram showing the results of separating healthy human serum according to Example 2.
Claims (1)
とを有する硬質の全多孔性粒状架橋共重合体を固定相と
し、ｐＨ６．５〜１０．０、イオン強度０．０５〜１．
５の移動相を用いた液体クロマトグラフィーによつて、
アルブミンを含む体液または血漿製品から、アルブミン
と他の成分とを分離することを特徴とするアルブミンと
グロブリンの分離方法。The stationary phase is a hard fully porous granular crosslinked copolymer having alcoholic hydroxyl groups and ion exchange groups directly bonded to the main chain, pH 6.5-10.0, ionic strength 0.05-1.
By liquid chromatography using a mobile phase of 5.
A method for separating albumin and globulin, which comprises separating albumin from other components from a body fluid or plasma product containing albumin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60228967A JP2562576B2 (en) | 1985-10-16 | 1985-10-16 | Method for separating albumin and globulin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60228967A JP2562576B2 (en) | 1985-10-16 | 1985-10-16 | Method for separating albumin and globulin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6288961A true JPS6288961A (en) | 1987-04-23 |
| JP2562576B2 JP2562576B2 (en) | 1996-12-11 |
Family
ID=16884670
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP60228967A Expired - Fee Related JP2562576B2 (en) | 1985-10-16 | 1985-10-16 | Method for separating albumin and globulin |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2562576B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990015617A1 (en) * | 1989-06-15 | 1990-12-27 | The Green Cross Corporation | Albumin preparation and method of producing the same |
| US5277818A (en) * | 1988-10-31 | 1994-01-11 | The Green Cross Corporation | Albumin preparation and process for preparing the same |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5967456A (en) * | 1982-10-12 | 1984-04-17 | Asahi Chem Ind Co Ltd | Separation of albumin by chromatography |
-
1985
- 1985-10-16 JP JP60228967A patent/JP2562576B2/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5967456A (en) * | 1982-10-12 | 1984-04-17 | Asahi Chem Ind Co Ltd | Separation of albumin by chromatography |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5277818A (en) * | 1988-10-31 | 1994-01-11 | The Green Cross Corporation | Albumin preparation and process for preparing the same |
| WO1990015617A1 (en) * | 1989-06-15 | 1990-12-27 | The Green Cross Corporation | Albumin preparation and method of producing the same |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2562576B2 (en) | 1996-12-11 |
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