JPH1081699A - How to remove color substances - Google Patents
How to remove color substancesInfo
- Publication number
- JPH1081699A JPH1081699A JP9190134A JP19013497A JPH1081699A JP H1081699 A JPH1081699 A JP H1081699A JP 9190134 A JP9190134 A JP 9190134A JP 19013497 A JP19013497 A JP 19013497A JP H1081699 A JPH1081699 A JP H1081699A
- Authority
- JP
- Japan
- Prior art keywords
- containing solution
- cation exchanger
- cationic ion
- treatment
- solution
- 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
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明はハプトグロビン（以下、
「ＨＰ」ともいう）含有画分中に夾雑する色調物質の除
去方法に関するものである。The present invention relates to haptoglobin (hereinafter referred to as "haptoglobin").
(Also referred to as "HP").
【０００２】[0002]
【従来の技術】ＨＰは、分子量８万５千〜４０万の血漿
糖蛋白質であり、血中に遊離するヘモグロビンの代謝に
重要な役割を演ずる。ＨＰはヘモグロビンと特異的に結
合し、ＨＰ−ヘモグロビン複合体を形成する特性を有す
る。血漿中のＨＰは、溶血により生じたヘモグロビンと
複合体を形成し、ヘモグロビンの正常な代謝経路である
肝に運ばれるが、ヘモグロビン量がＨＰとの結合量を上
回ったときは、余剰のヘモグロビンは遊離の状態で血漿
中に存在し、ヘモグロビン血症及び腎を経てヘモグロビ
ン尿症を引き起こす。従って、ＨＰは、熱傷、火傷、輸
血、体外循環下開心術などの溶血反応に伴うヘモグロビ
ン血症、ヘモグロビン尿症の治療に有効とされている。2. Description of the Related Art HP is a plasma glycoprotein having a molecular weight of 85,000 to 400,000, and plays an important role in the metabolism of hemoglobin released into the blood. HP has the property of specifically binding to hemoglobin and forming an HP-hemoglobin complex. HP in plasma forms a complex with hemoglobin produced by hemolysis and is transported to the liver, which is a normal metabolic pathway of hemoglobin.When the amount of hemoglobin exceeds the amount bound to HP, the excess hemoglobin becomes It is present in plasma in its free state and causes hemoglobinemia and hemoglobinuria via the kidneys. Therefore, HP is considered to be effective for the treatment of hemoglobinemia and hemoglobinuria associated with hemolysis such as burns, burns, blood transfusions, and open heart surgery under extracorporeal circulation.
【０００３】現在、ＨＰは液状製剤として発売中であ
り、商品名ハプトグロビン注−ミドリ（（株）ミドリ十
字社製）等がある。ＨＰの精製法としては、硫安分画、
アクリノール分画、陰イオン交換体処理（特開昭５０−
７７５１６）、加熱処理（同５０−７７５２７）、コロ
イド珪酸処理（特開昭６３−１７８９９）、ポリエチレ
ングリコール分画、固定化ヘパリンによるアフィニティ
処理等が知られている。しかしながら、ＨＰ含有画分中
に夾雑する色調物質の除去について言及したものはなか
った。[0003] Currently, HP is on sale as a liquid preparation, such as Haptoglobin Injection-Midori (manufactured by Midori Cross). As HP purification methods, ammonium sulfate fractionation,
Acrinol fractionation, anion exchanger treatment
77516), heat treatment (50-77527), colloidal silicic acid treatment (JP-A-63-17899), polyethylene glycol fractionation, affinity treatment with immobilized heparin, and the like. However, there was no mention of the removal of contaminant color substances in the HP-containing fraction.
【０００４】[0004]
【発明が解決しようとする課題】本発明者らは、上記の
事情を考慮して研究を重ねた結果、ＨＰ含有溶液を陽イ
オン交換体で処理することにより、ＨＰ含有画分中に夾
雑する色調物質を除去できることを見出し、本発明を完
成した。SUMMARY OF THE INVENTION The present inventors have conducted various studies in consideration of the above-mentioned circumstances. As a result, by treating an HP-containing solution with a cation exchanger, the HP-containing solution is contaminated in the HP-containing fraction. The inventors have found that the color substance can be removed, and have completed the present invention.
【０００５】[0005]
【課題を解決するための手段】本発明は、ＨＰ含有溶液
を陽イオン交換体で処理することを特徴とする、ＨＰ含
有画分中に夾雑する色調物質の除去方法である。 ＨＰ含有溶液 本発明に用いられるＨＰ含有溶液は、血漿由来、細胞培
養由来、遺伝子工学由来等のいずれにも限定されない。
血漿由来の場合は、コーンの上清II＋III 、沈澱IV、IV
−４等の各画分から調製できる。ＨＰ含有溶液は粗製段
階、精製段階のいずれの状態でもよい。精製は従来技術
の項で開示した公知の手法を用いることができる。SUMMARY OF THE INVENTION The present invention is a method for removing a color tone substance contaminating an HP-containing fraction, comprising treating an HP-containing solution with a cation exchanger. HP-containing solution The HP-containing solution used in the present invention is not limited to any one derived from plasma, cell culture, genetic engineering, and the like.
If derived from plasma, corn supernatant II + III, sediment IV, IV
-4 etc. can be prepared. The HP-containing solution may be in any of a crude stage and a purification stage. For purification, a known method disclosed in the section of the prior art can be used.
【０００６】陽イオン交換体処理 本発明の特徴はＨＰ含有溶液を陽イオン交換体で処理す
ることにある。ＨＰ含有溶液におけるＨＰ濃度は０．１
〜１０ｗ／ｖ％程度が例示される。陽イオン交換体は陽
イオン交換基を有する不溶性担体であれば、特に限定さ
れない。陽イオン交換基としてはカルボキシル基、カル
ボキシメチル基（ＣＭ）などのカルボキシアルキル基、
スルホ基、スルホプロピル基（ＳＰ）などのスルホアル
キル基、リン酸基等が例示される。また、不溶性担体と
してはデキストラン（商品名セファデックス、ファルマ
シア社製）、アガロース（商品名セファロース、ファル
マシア社製）、ビニル系親水性ポリマー（商品名トヨパ
ール、東ソー社製；フラクトゲル、メルク社製）、セル
ロース（商品名セルロファイン、生化学工業社製）、多
孔性シリカゲル（商品名セファロシル、ＳＥＰＲＯＣＯ
Ｒ社製）等が例示される。Cation exchanger treatment A feature of the present invention resides in the treatment of the HP-containing solution with a cation exchanger. The HP concentration in the HP-containing solution is 0.1
For example, about 10 to 10 w / v%. The cation exchanger is not particularly limited as long as it is an insoluble carrier having a cation exchange group. Carboxyl groups such as carboxyl groups and carboxymethyl groups (CM);
Examples thereof include a sulfo group, a sulfoalkyl group such as a sulfopropyl group (SP), and a phosphate group. Further, as the insoluble carrier, dextran (trade name: Sephadex, manufactured by Pharmacia), agarose (trade name: Sepharose, manufactured by Pharmacia), vinyl-based hydrophilic polymer (trade name: Toyopearl, Tosoh; Fractogel, manufactured by Merck), Cellulose (trade name: Cellulofine, manufactured by Seikagaku Corporation), porous silica gel (trade name: Cephalosil, SEPROCO)
R company).
【０００７】ＨＰは陽イオン交換体に接触した場合には
吸着することなく、非吸着画分として精製・回収され
る。すなわち、ＨＰ含有画分に夾雑する色調物質は吸着
したままとなり、除去される。接触条件としてはｐＨ４
〜７程度、好ましくは、５〜６程度が例示される。ま
た、伝導度としては１〜１０ｍＳ／ｃｍ程度が例示され
る。陽イオン交換体の添加量としてはＨＰ含有溶液１〜
１００容量部当たり１容量部程度が例示される。ここ
で、陽イオン交換体の容量とは、ゲル化したものの容量
を指す。[0007] When contacted with a cation exchanger, HP is purified and recovered as a non-adsorbed fraction without adsorption. That is, the color substance contaminating the HP-containing fraction remains adsorbed and is removed. The contact condition is pH4
~ 7, preferably about 5-6. The conductivity is, for example, about 1 to 10 mS / cm. The amount of the cation exchanger to be added is 1 to HP-containing solution.
About 1 volume part per 100 volume parts is exemplified. Here, the capacity of the cation exchanger refers to the capacity of the gelled product.
【０００８】本発明処理後にさらに公知の製剤化技術を
組合せることにより、臨床上適用できるＨＰ製剤を提供
することができる。例えば、安定化剤、賦形剤等の添
加、加熱処理、除菌濾過、小分け分注、透析・限外濾
過、凍結乾燥等を施すことができる。[0008] By combining known formulation techniques after the treatment of the present invention, a clinically applicable HP formulation can be provided. For example, addition of stabilizers, excipients, etc., heat treatment, sterilization filtration, aliquot dispensing, dialysis / ultrafiltration, lyophilization and the like can be performed.
【０００９】[0009]
【発明の効果】本発明の方法はＨＰ含有画分中に夾雑す
る色調物質、特にヘモグロビンまたはその複合体の除去
に極めて有用な方法である。The method of the present invention is a very useful method for removing color substances, particularly hemoglobin or a complex thereof, contaminating the HP-containing fraction.
【００１０】[0010]
【実施例】本発明をより詳細に説明するために実施例お
よび実験例を挙げるが、本発明はこれらにより何ら限定
されるものではない。 実施例１ コーンの第IV画分から特開昭５０−７７５１６に準じて
ＨＰ含有溶液（１０ｍｇ／ｍｌ）を調製した。カルボキ
シメチル−デキストラン（商品名ＣＭ−セファデック
ス、ファルマシア社製）を用い、ｐＨ５、伝導度４ｍＳ
／ｃｍ、陽イオン交換体の添加量をＨＰ含有溶液１０容
量部当たり１容量部に調整して、陽イオン交換体処理を
行い、非吸着画分を回収した。この画分はＨＰを含み、
色調物質は実質的に除去されていた。EXAMPLES The present invention will be described in more detail with reference to Examples and Experimental Examples, but the present invention is not limited thereto. Example 1 An HP-containing solution (10 mg / ml) was prepared from the corn IV fraction according to JP-A-50-77516. Using carboxymethyl-dextran (trade name CM-Sephadex, manufactured by Pharmacia), pH 5, conductivity 4 mS
/ Cm, the addition amount of the cation exchanger was adjusted to 1 part by volume per 10 parts by volume of the HP-containing solution, and a cation exchanger treatment was performed to collect a non-adsorbed fraction. This fraction contains HP,
The color material was substantially removed.
【００１１】実施例２ コーンの第II＋III 上清画分を固定化ヘパリン処理して
得られたＨＰ含有溶液を用い、カルボキシル基を有する
ビニル系親水性ポリマー（商品名フラクトゲル−ＣＯＯ
- 、メルク社製）により陽イオン交換体処理する以外は
実施例１に準じて行った。Example 2 A vinyl hydrophilic polymer having a carboxyl group (trade name: Fractogel-COO) was obtained using an HP-containing solution obtained by treating the corn II + III supernatant fraction with immobilized heparin.
-, except for processing cation exchanger was conducted according to Example 1 by Merck).
【００１２】実験例１ 本発明の方法におけるｐＨ条件の影響を確認した。ＨＰ
含有溶液を実施例に準じて、陽イオン交換体にて処理
し、非吸着画分における、ＨＰの回収率および色調物質
の除去の度合いを測定した。ＨＰの測定は、免疫比濁法
によった。色調物質の除去の度合いは、２８０ｎｍおよ
び４０３ｎｍでの吸光度Ａ280 およびＡ403 を測定し、
その比Ａ403 ／Ａ280 を算出することにより評価した。
他の処理条件は伝導度４ｍＳ／ｃｍ、陽イオン交換体の
添加量をＨＰ含有溶液１０ｍｌ当たり１ｍｌとした。結
果を表１に示す。Experimental Example 1 The effect of pH conditions on the method of the present invention was confirmed. HP
The contained solution was treated with a cation exchanger according to the examples, and the HP recovery and the degree of removal of the color substance in the non-adsorbed fraction were measured. HP was measured by immunoturbidimetry. The degree of colorant removal was determined by measuring the absorbances A 280 and A 403 at 280 nm and 403 nm,
Evaluation was made by calculating the ratio A 403 / A 280 .
Other treatment conditions were a conductivity of 4 mS / cm, and the amount of cation exchanger added was 1 ml per 10 ml of HP-containing solution. Table 1 shows the results.
【００１３】[0013]
【表１】 [Table 1]
【００１４】表１からＡ403 ／Ａ280 が陽イオン交換体
処理により低減されていることが分かる。 実験例２ 本発明の方法における陽イオン交換体の添加量の影響を
確認した。From Table 1, it can be seen that A 403 / A 280 was reduced by the cation exchanger treatment. Experimental Example 2 The effect of the amount of cation exchanger added in the method of the present invention was confirmed.
【００１５】ＨＰ含有溶液を実施例に準じて、陽イオン
交換体にて処理し、非吸着画分における、ＨＰの回収率
および色調物質の除去の度合いを測定した。ＨＰ、色調
物質の除去の度合いは実験例１の方法に準じて評価し
た。他の処理条件はｐＨ５、伝導度４ｍＳ／ｃｍとし
た。結果を表２に示す。The HP-containing solution was treated with a cation exchanger according to the examples, and the HP recovery and the degree of removal of the color substance in the non-adsorbed fraction were measured. The degree of removal of the HP and the color tone substance was evaluated according to the method of Experimental Example 1. Other treatment conditions were pH 5 and conductivity 4 mS / cm. Table 2 shows the results.
【００１６】[0016]
【表２】 表中、添加量は、ＨＰ含有溶液１２０ｍｌ当たりのｍｌ
数である。[Table 2] In the table, the addition amount is ml per 120 ml of the HP-containing solution.
Is a number.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｇ０１Ｎ 33/72 Ａ６１Ｋ 37/04 ＡＢＺ (72)発明者 三宅 正一 大阪府大阪市都島区都島中通３−５−44 株式会社ミドリ十字都島工場内 (72)発明者 橋本 元範 大阪府枚方市招提大谷２丁目25番１号 株 式会社ミドリ十字中央研究所内 (72)発明者 上村 八尋 大阪府枚方市招提大谷２丁目25番１号 株 式会社ミドリ十字中央研究所内──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 6 Identification number Agency reference number FI Technical indication location G01N 33/72 A61K 37/04 ABZ (72) Inventor Shoichi Miyake Miyakojima, Miyakojima-ku, Osaka-shi, Osaka 3-5-44 Midori Cross Miyakojima Plant Co., Ltd. (72) Inventor Motonori Hashimoto 2-25-1, Shodai Otani, Hirakata City, Osaka Prefecture Midori Cross Central Research Institute, Inc. (72) Inventor Yahiro Uemura Osaka Prefecture 2-25-1, Odani, Hirakata City
Claims (2)
体で処理することを特徴とする、ハプトグロビン含有画
分中に夾雑する色調物質の除去方法。1. A method for removing a color tone substance contaminating a haptoglobin-containing fraction, comprising treating the haptoglobin-containing solution with a cation exchanger.
ハプトグロビン含有組成物。2. A haptoglobin-containing composition having an A 403 / A 280 of 0.034 or less.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9190134A JPH1081699A (en) | 1996-07-15 | 1997-07-15 | How to remove color substances |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8-184834 | 1996-07-15 | ||
| JP18483496 | 1996-07-15 | ||
| JP9190134A JPH1081699A (en) | 1996-07-15 | 1997-07-15 | How to remove color substances |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH1081699A true JPH1081699A (en) | 1998-03-31 |
Family
ID=26502740
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP9190134A Pending JPH1081699A (en) | 1996-07-15 | 1997-07-15 | How to remove color substances |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH1081699A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003096095A (en) * | 2001-09-27 | 2003-04-03 | Nihon Pharmaceutical Co Ltd | Protein purification method |
-
1997
- 1997-07-15 JP JP9190134A patent/JPH1081699A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003096095A (en) * | 2001-09-27 | 2003-04-03 | Nihon Pharmaceutical Co Ltd | Protein purification method |
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