JP2678193B2 - Method for producing human blood plasma-derived blood coagulation factor XIII - Google Patents
Method for producing human blood plasma-derived blood coagulation factor XIIIInfo
- Publication number
- JP2678193B2 JP2678193B2 JP63080960A JP8096088A JP2678193B2 JP 2678193 B2 JP2678193 B2 JP 2678193B2 JP 63080960 A JP63080960 A JP 63080960A JP 8096088 A JP8096088 A JP 8096088A JP 2678193 B2 JP2678193 B2 JP 2678193B2
- Authority
- JP
- Japan
- Prior art keywords
- factor xiii
- coagulation factor
- blood coagulation
- plasma
- derived
- 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.)
- Expired - Lifetime
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Description
【発明の詳細な説明】 発明の目的 産業上の利用分野 本発明は、ヒト血漿由来血液凝固XIII因子の製造方法
に関する。The present invention relates to a method for producing human blood plasma-derived blood coagulation factor XIII.
従来の技術及び発明が解決しようとする問題点 ヒト血液凝固第XIII因子は、血液の止血機構において
フィブリンの安定化に寄与する重要な因子である。さら
に、近年、該因子は止血血栓機構との関連における創傷
治癒機転の一端である、フィブリン・フィブロネクチン
・コラーゲンのいわゆるフィブリンマトリックスの形成
にも関与し、創傷治癒促進作用を持つと考えられるよう
になった。実際に第XIII因子製剤は、臨床適用において
止血作用における重要因子として、先天性ないしは後天
性の血液凝固第XIII因子欠損症及び減少症への補充はも
ちろんのこと、広く一般外科手術後の創傷治癒促進に大
きな効果をもたらしている。Problems to be Solved by Conventional Techniques and Inventions Human blood coagulation factor XIII is an important factor contributing to the stabilization of fibrin in the hemostatic mechanism of blood. Furthermore, in recent years, the factor is involved in the formation of a so-called fibrin matrix of fibrin / fibronectin / collagen, which is one of the mechanisms of wound healing in relation to the hemostatic thrombotic mechanism, and has been considered to have a wound healing promoting action. It was In fact, factor XIII preparations are important factors in hemostatic activity in clinical applications, and they are widely used not only for supplementing congenital or acquired blood coagulation factor XIII deficiency and diminution but also widely for wound healing after general surgery. It has a great effect on promotion.
第XIII因子は、主に血漿、血小板及び胎盤に存在す
る。このうち、血漿由来の第XIII因子は血小板、胎盤由
来のものとはサブユニット構造が異なり、触媒サブユニ
ットａ鎖に加えて、非触媒サブユニットｂ鎖からなって
いる。このｂ鎖の役割についてはまだ十分に解明されて
いないが、触媒サブユニットａ鎖の血中での保護作用、
すなわち安定化に寄与していると考えられている。とこ
ろで、現在、製剤化されている第XIII因子製剤はａ鎖か
らのみ成り立つ胎盤由来の製剤である。従って、第XIII
因子の安定性、とりわけ血中での体内動態という観点か
ら既存の製剤は大きな問題を含んでいるものと考えられ
る。また、凝固因子以外の胎盤由来の物質で、人体に投
与した際、免疫原となる可能性のあるものの混入も完全
には否定できない。さらに、該胎盤由来凝固因子製剤に
対して混入ウイルスを不活化させる目的で加熱処理が施
されているが、この際、熱安定化剤として高濃度の糖類
を添加することが広く取り入れられている。しかしなが
ら、近年、熱安定化剤として用いられる高濃度の糖類が
不活化の対象となる混入ウイルスをも安定化してしま
い、一義的な目的であるウイルスの不活化が十分には行
われないという問題も提起されている。（宮本ら 基礎
と臨床 19巻 289ページ 1985年） 以上の理由から、血漿を由来する第XIII因子製剤の開
発、及び加熱不活化の際の適当な熱安定化方法が切望さ
れた。Factor XIII is mainly present in plasma, platelets and placenta. Of these, plasma-derived factor XIII has a subunit structure different from that of platelets and placenta, and is composed of a non-catalytic subunit b chain in addition to a catalytic subunit a chain. Although the role of this b chain has not yet been fully elucidated, the protective effect of the catalytic subunit a chain on blood,
That is, it is considered to contribute to stabilization. By the way, the currently prepared Factor XIII preparation is a placenta-derived preparation consisting only of the a chain. Therefore, the XIII
From the viewpoint of the stability of factors, especially the pharmacokinetics in blood, it is considered that the existing preparations have serious problems. In addition, it is not possible to completely rule out contamination with placenta-derived substances other than coagulation factors, which may become immunogens when administered to the human body. Further, the placenta-derived coagulation factor preparation is subjected to heat treatment for the purpose of inactivating contaminating viruses. At this time, it is widely adopted to add a high concentration of saccharide as a heat stabilizer. . However, in recent years, the high concentration of saccharides used as a heat stabilizer also stabilizes the contaminated virus that is the target of inactivation, and the problem that the virus, which is the primary purpose, is not sufficiently inactivated. Has also been raised. (Miyamoto et al., Basic and Clinical, Vol. 19, p. 289, 1985) For the above reasons, the development of a factor XIII preparation derived from plasma and a suitable heat stabilization method at the time of heat inactivation were earnestly desired.
ヒト血漿由来第XIII因子の製造方法については現在ま
でいくつか報告されているが（ローウィーA.G.等ジャー
ナル オブ バイオロジカル ケミストリー 236巻 262
5ページ 1961年；ウィンケルマン L.等 スロンボシス
アンド ヘモスタシス 55巻 402ページ 1986年）、こ
れらが実験室レベルでの製法であること、さらには該因
子の回収率の低さ及び純度の低さ等の理由から、工業的
規模で製造をおこなうには十分なものではなかった。Several methods for producing human plasma-derived factor XIII have been reported so far (Law Wee AG et al. Journal of Biological Chemistry, Vol. 236, 262.
5 Page 1961; Winkelman L. et al. Thrombosis and Hemostasis Vol. 55, page 402 1986). For reasons, it was not sufficient for production on an industrial scale.
発明の構成 問題点を解決するための手段及び作用 本発明者等は、血漿由来の第XIII因子を工業的規模で
製造できるようにするため、血漿由来の第XIII因子がグ
リシンにより沈澱しやすい性質、（ガーサル L.等 プ
ロシーディング オブ ザ ソサィアテー フォー エ
クスペリメンタル バイオロジー アンド メディシン
113巻 989ページ 1963年）に着目し、これに検討を加
え、第XIII因子の純度の上昇を伴う効率的な回収を可能
とする方法を完成した。この方法により、高純度かつ高
収量で精製されXIII因子を得ることができるようになっ
た。さらに、原料血漿の由来に対して危惧される肝炎ウ
イルス、エイズウイルス等の混入病原ウイルスの不活化
についても鋭意検討を重ね、より安全性が保証される第
XIII因子製剤を得ることのできる本発明を完成するに至
った。すなわち、胎盤由来の凝固因子製剤に適用されて
いるような種々の糖類を添加することなく、アルブミン
を添加することによって、混入病原ウイルスの不活化を
完全に成し得ることが明らかになっている液状加熱処理
条件下における熱安定性を著しく高めることを可能なら
しめることを見いだした。Structure of the Invention Means and Actions for Solving Problems The present inventors have made it possible to produce plasma-derived factor XIII on an industrial scale. , (Gersal L. etc. Proceeding of the Society for Experimental Biology and Medicine
113, page 989, 1963), and by studying this, we have completed a method that enables efficient recovery with an increase in the purity of Factor XIII. By this method, factor XIII can be obtained with high purity and high yield. Furthermore, we will continue to earnestly investigate the inactivation of hepatitis virus, AIDS virus, and other contaminating pathogenic viruses that are of concern for the origin of the raw material plasma, to ensure safety.
The present invention has been completed in which a factor XIII preparation can be obtained. That is, it has been clarified that the inactivation of the contaminating pathogenic virus can be completely achieved by adding albumin without adding various saccharides as used in the placenta-derived coagulation factor preparation. It has been found that it is possible to significantly increase the thermal stability under liquid heat treatment conditions.
本発明において、第XIII因子の精製は、血漿、クリオ
プレシピテートまたはコーン分画によるフラクションＩ
ペースト等ヒト血漿由来血液凝固第XIII因子を含むタン
パク質画分を出発材料とする。例えば、血漿からのコー
ンのアルコール分画法によるフラクションＩペーストを
用いた場合、ペーストを溶解後、グリシン法によりフィ
ブリノーゲンを含んだ粗第XIII因子画分を得る。得られ
た沈澱物を溶解後、10〜25℃、pH6.0〜7.0の条件下で、
溶液にグリシン及び塩化ナトリウムを各々終濃度1.0〜
2.0Mで添加し第XIII因子を選択的に沈澱させる。なお、
グリシン及び塩化ナトリウムの添加に先立ち、粗第XIII
因子画分を56℃で３〜30分間加熱し、夾雑しているフィ
ブリノーゲンを変性沈澱させて除去する行程を施すこと
が好ましい態様である。その後、該血液凝固第XIII因子
を常法により溶液より分離し高純度の第XIII因子濃縮画
分を収得する。本方法により、従来の方法に比べて工程
数が少なく、高収率でかつ従来法の約６倍という高純度
の第XIII因子を得ることができるようになり、さらに、
工業的規模での該因子の製造も可能となった。In the present invention, the factor XIII is purified by fractionation with plasma, cryoprecipitate or Cohn fractionation.
The starting material is a protein fraction containing human plasma-derived blood coagulation factor XIII such as paste. For example, when a fraction I paste obtained by the Cohn alcohol fractionation method from plasma is used, a crude Factor XIII fraction containing fibrinogen is obtained by the glycine method after dissolving the paste. After dissolving the obtained precipitate, under the conditions of 10 to 25 ° C. and pH 6.0 to 7.0,
Glycine and sodium chloride were added to the solution at final concentrations of 1.0-
Add 2.0 M to selectively precipitate Factor XIII. In addition,
Prior to the addition of glycine and sodium chloride, crude XIII
In a preferred embodiment, the factor fraction is heated at 56 ° C. for 3 to 30 minutes to denature and precipitate the contaminating fibrinogen to remove it. Then, the blood coagulation factor XIII is separated from the solution by a conventional method to obtain a highly purified factor XIII-enriched fraction. By this method, the number of steps is smaller than that of the conventional method, and it is possible to obtain a high-yield factor XIII having a high purity of about 6 times that of the conventional method.
It has also become possible to produce the factor on an industrial scale.
第XIII因子の加熱処理は、第XIII因子含有溶液を50〜
80℃（好ましくは60℃）で、３〜18時間（好ましくは10
時間）加熱する条件で行なう。この際の実質的な安定化
剤はアルブミンで、その添加量は0.1〜10W/V％（好まし
くは1.0W/V％）である。本加熱処理により、混入の可能
性が否定できない病原ウイルスの不活化を安全に行なう
ことができ、また、加熱による第XIII因子の物理化学的
性状、免疫学的性状にもなんら影響を及ぼさない高い安
全性が保証された凝固第XIII因子が得られる。加熱処理
を終えた第XIII因子は、除菌濾過後分注し、凍結乾燥す
る。The heat treatment of Factor XIII is performed by adding a solution containing Factor XIII
80 ℃ (preferably 60 ℃) for 3-18 hours (preferably 10 ℃)
Time) Perform under heating conditions. In this case, a substantial stabilizer is albumin, and the added amount thereof is 0.1 to 10 W / V% (preferably 1.0 W / V%). This heat treatment can safely inactivate pathogenic viruses for which the possibility of contamination cannot be ruled out, and does not affect the physicochemical or immunological properties of Factor XIII by heating. A coagulation factor XIII with a guaranteed safety is obtained. After the heat treatment, the factor XIII is sterilized, filtered, dispensed, and lyophilized.
本発明の方法によって得られる第XIII因子濃縮製剤
は、新鮮な正常ヒト血漿1mlに含まれる第XIII因子活性
量を１単位とすれば、60〜80単位/mlの活性を有し、ま
た、出発物質への夾雑ウイルスに起因する肝炎、エイズ
等の発症の危険性がないものである。さらに、本発明に
おいてはヒト血漿からアルブミン、γ−グロブリン等の
他の血漿成分の分画に支障なく、従来利用されていなか
った画分の有効利用が可能となった。従って、本発明は
血液事業によって採取された血液の有効利用という観点
からも意義深いものと考えられる。The factor XIII concentrated preparation obtained by the method of the present invention has an activity of 60 to 80 units / ml when the amount of factor XIII activity contained in 1 ml of fresh normal human plasma is 1 unit. There is no risk of the onset of hepatitis, AIDS, etc. due to contaminant viruses. Furthermore, in the present invention, fractionation of other plasma components such as albumin and γ-globulin from human plasma can be effectively performed, and fractions that have not been conventionally used can be effectively used. Therefore, the present invention is considered to be significant from the viewpoint of effective use of blood collected by the blood business.
実施例 以下、実施例を挙げて、本発明を具体的に説明する。EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples.
実施例１ ヒト血漿100からフラクションＩペーストを調製
し、ペーストをクエン酸緩衝液で溶解後1.8Mグリシンを
添加して、粗第XIII因子画分を得る。この沈澱をクエン
酸緩衝液で溶解後、56℃で30分間加熱し、フィブリノー
ゲンを除去した第XIII因子溶液を得、さらに、この溶液
に1.8Mグリシンと2M塩化ナトリウムを添加して、高純度
の第XIII因子濃縮画分を沈澱して得る。この方法によ
り、出発物である血漿からの第XIII因子の精製度が3200
倍上昇し、得られた総活性は３万単位であった。Example 1 A fraction I paste is prepared from human plasma 100, the paste is dissolved in a citrate buffer, and 1.8 M glycine is added to obtain a crude factor XIII fraction. This precipitate was dissolved in citrate buffer and heated at 56 ° C for 30 minutes to obtain a factor XIII solution from which fibrinogen was removed. Furthermore, 1.8 M glycine and 2 M sodium chloride were added to this solution to prepare a highly pure solution. The factor XIII enriched fraction is obtained by precipitation. This method provided a purification factor of 3200 from the starting plasma of 3200.
It was doubled and the total activity obtained was 30,000 units.
実施例２ 実施例１の方法で精製した高純度の第XIII因子を用い
て、各種の安定化剤による熱安定化効果を検討した。加
熱前後での第XIII因子の抗原性残存率、活性残存率の結
果を表１に示す。その結果、安定化剤無添加、マンニト
ール、ソルビトール、ショ糖を安定化剤として使用した
場合、活性残存率はいずれも加熱処理前に比較して、50
％以下に低下したにもかかわらず、アルブミンを用いた
場合は、抗原、活性残存率のいずれも高度に維持され、
顕著な熱安定化効果が認められた。Example 2 Using the highly purified factor XIII purified by the method of Example 1, the heat stabilizing effect of various stabilizers was examined. Table 1 shows the results of the antigenic residual ratio and active residual ratio of Factor XIII before and after heating. As a result, when mannitol, sorbitol, or sucrose was used as a stabilizer without adding a stabilizer, the residual activity ratios were 50% compared with those before the heat treatment.
When albumin was used, both the antigen and the residual activity ratio were maintained at a high level,
A remarkable heat stabilizing effect was recognized.
Claims (3)
血漿由来血液凝固第XIII因子含有タンパク質画分に、10
〜25℃、pH6.0〜7.0の条件下グリシン及び塩化ナトリウ
ムを各々終濃度1.0〜2.0Mの濃度で添加して該血液凝固
第XIII因子を選択的に沈澱させ、該血液凝固第XIII因子
を溶液から分離・精製することを特徴とするヒト血液凝
固第XIII因子の製造方法。1. A human plasma-derived blood coagulation factor XIII-containing protein fraction that is substantially free of fibrinogen.
-25 ° C, pH 6.0 to 7.0, glycine and sodium chloride are added at a final concentration of 1.0 to 2.0 M to selectively precipitate the blood coagulation factor XIII, and the blood coagulation factor XIII is A method for producing human blood coagulation factor XIII, which comprises separating and purifying from a solution.
血漿由来血液凝固第XIII因子含有タンパク質画分が、血
漿、クリオプレシピテートまたはコーン分画によるフラ
クションＩペーストを脱フィブリノーゲン処理したもの
である請求項１記載のヒト血液凝固第XIII因子の製造方
法。2. The human blood plasma-derived blood coagulation factor XIII-containing protein fraction substantially free of fibrinogen is a defibrinogen-treated fraction I paste from plasma, cryoprecipitate or corn fraction. 1. The method for producing human blood coagulation factor XIII according to 1.
分画によるフラクションＩペーストの脱フィブリノーゲ
ン処理が、前記原料を50〜60℃で３〜30分間加熱し夾雑
するフィブリノーゲンを沈澱除去することによって達成
される請求項２記載のヒト血液凝固第XIII因子の製造方
法。3. Defibrinogen treatment of Fraction I paste by plasma, cryoprecipitate or corn fractionation is accomplished by heating the raw material at 50-60 ° C. for 3-30 minutes to precipitate and remove contaminating fibrinogen. The method for producing human blood coagulation factor XIII according to claim 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63080960A JP2678193B2 (en) | 1988-03-31 | 1988-03-31 | Method for producing human blood plasma-derived blood coagulation factor XIII |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63080960A JP2678193B2 (en) | 1988-03-31 | 1988-03-31 | Method for producing human blood plasma-derived blood coagulation factor XIII |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01250326A JPH01250326A (en) | 1989-10-05 |
| JP2678193B2 true JP2678193B2 (en) | 1997-11-17 |
Family
ID=13733080
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63080960A Expired - Lifetime JP2678193B2 (en) | 1988-03-31 | 1988-03-31 | Method for producing human blood plasma-derived blood coagulation factor XIII |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2678193B2 (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AT359653B (en) * | 1979-02-15 | 1980-11-25 | Immuno Ag | METHOD FOR PRODUCING A TISSUE ADHESIVE |
-
1988
- 1988-03-31 JP JP63080960A patent/JP2678193B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH01250326A (en) | 1989-10-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0037078B1 (en) | Process for heat treatment of aqueous solution containing human blood coagulation factor xiii | |
| US4877608A (en) | Pharmaceutical plasma protein formulations in low ionic strength media | |
| US5118794A (en) | Process for stabilizing human albumin solutions and the solution obtained | |
| US4446134A (en) | Process for heat treatment of aqueous solution containing human blood coagulation factor VIII | |
| US4876241A (en) | Stabilization of biological and pharmaceutical products during thermal inactivation of viral and bacterial contaminants | |
| DK156698B (en) | PROCEDURE FOR PASTEURIZATION OF A MATERIAL CONTAINING A THERMALLALLY SENSITIVE, THERAPEUTIC ACTIVITY PROTEIN SELECTED WITH ALFA-L-ANTITRYPSIN, ANTITHROMOBIN-III, PRAEKALLIKRINE, ANTIHAEMOFILE FACTOR | |
| JPH01143835A (en) | Preparation of aseptic plasma-protein containing fibrinogen and factor xiii | |
| RU2144081C1 (en) | Method of large-scale production of thrombin-containing composition of therapeutic purity degree and stable in storage | |
| DK163107B (en) | PROCEDURE FOR PREPARING A HIGH PURITY ANTIHAEMOPHILIC FACTOR CONCENTRATE | |
| NO324659B1 (en) | Recovery of the von Willebrand factor by cation exchange chromatography and preparations containing such factor and its use. | |
| AU549584B2 (en) | Heat stabilization of plasma proteins | |
| JPS6160614A (en) | Pasteurized homogeneous aggulutinin- free viii factor medicine and manufacture | |
| JPH06321994A (en) | Antithrombin preparation and method of its preparation | |
| JPH0348888B2 (en) | ||
| ES2237854T5 (en) | Procedure for preparation by filtration of a virally safe factor VIII solution | |
| JPS6237010B2 (en) | ||
| JP2605102B2 (en) | Stabilization of biological and pharmaceutical products during thermal inactivation of viral and bacterial contaminants | |
| EP0011739B1 (en) | Process for obtaining blood coagulation factor xiii derived from human placenta | |
| JPS5910645B2 (en) | Method for concentrating and purifying factor 8 | |
| JPS62181220A (en) | Manufacture of antihemophilic factor for improving dissolution of cold-sedimented and collected antihemophilic factor product | |
| WO1998030230A1 (en) | Protein-containing compositions and process for producing the same | |
| JP2678193B2 (en) | Method for producing human blood plasma-derived blood coagulation factor XIII | |
| JP2001000179A (en) | Inactivation of virus | |
| DK175644B1 (en) | Process for stabilizing biological and pharmaceutical agents during inactivation of viral and bacterial contaminants | |
| JPH04502324A (en) | Pure factor I protein and method for producing the protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20080801 Year of fee payment: 11 |
|
| EXPY | Cancellation because of completion of term | ||
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20080801 Year of fee payment: 11 |