JP4606524B2 - Polylysine, polylysine production method, polylysine composition, and pharmaceutical production method for removing endotoxin - Google Patents
Polylysine, polylysine production method, polylysine composition, and pharmaceutical production method for removing endotoxin Download PDFInfo
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Description
【０００１】
【発明の属する技術分野】
本発明は発熱原因物質であるエンドトキシンの吸着能を有するポリリジン、及びその製造方法、更に、該ポリリジンを含むエンドトキシン除去用ポリリジン組成物、及び該ポリリジンを用いて、エンドトキシンを除去する医薬品の生産方法に関する。
【０００２】
【従来の技術】
エンドトキシンは細菌の菌体成分中にある毒性物質の総称であり、菌が死滅する際に遊離してくるものである。エンドトキシンの構造成分はリポサッカライドであり、医薬品の生産プロセスに中に生息する細菌が菌体内に生産し、死滅することによって、医薬品に混入する。
エンドトキシンの除去方法としては、従来から、活性炭やイオン交換体による吸着法、膜やメンブレンフィルター等を使うろ過法、高温・高圧処理または酸・アルカリを使う分解法が知られている。何れの方法も一長一短があり、工業的に用いる上には問題があった。例えば、医薬品製造におけるエンドトキシンの除去は、本来の医薬品の安定性維持の面から、過酷な条件下で行うことが出来なかったり、存在するエンドトキシンの量が極微量であるため、実験室的には吸着がうまく行えても、実際の工業スケールでは満足に吸着が行えなかったり、吸着体にエンドトキシン以外に医薬品そのものが吸着されたりして決して満足の行くものがなかった。
これに対し近年、工業的にも満足のいくエンドトキシンの吸着を可能にすべく、幾つかの吸着体が開示されている。特公平6-16843号公報には、側鎖及び／又は主鎖の末端に、脂肪族基及び／又はアリール基を有する修飾基を含むポリアミノ酸より構成される吸着体が開示され、特開平1-127039号公報には、ポリアミノ酸球状粒子を担体とし、これにイミダゾール誘導体を結合させた吸着体が開示されている。
【０００３】
【発明が解決しようとする課題】
しかしながら残念なことに、これらの吸着体には低エンドトキシン含有医薬品から、エンドトキシンのみを除去するには選択性が不足しており、本来吸着されるべきではない酸性蛋白質等がエンドトキシンとともに吸着されるという問題点があった。
更に、従来の吸着体は、高イオン強度域では選択的に吸着できるが、低イオン強度域では選択的に吸着できない、若しくはその逆のものであり、何れのイオン強度域においても、選択性に影響を受けないものはなかった。
本発明者らは従来技術の問題点に鑑み、鋭意検討を重ねた結果、橋かけ構造を有するポリリジンに、低エンドトキシン含有医薬品溶液からのエンドトキシンの除去において、何れのイオン強度域においても高い選択性があることを知見し、本発明者らはこの知見に基づき本発明を完成させた。
【０００４】
【課題を解決するための手段】
本発明は下記の構成からなる。
（１）ポリリジンを水溶液中で橋かけ剤と懸濁重合させることにより得られた橋かけ構造ポリリジンを用いて、エンドトキシンを除去することを特徴とする、血液製剤、酵素医薬品、または蛋白質医薬品の生産方法。
（２）エンドトキシンの除去が、橋かけ構造ポリリジンを充填したカラムにエンドトキシンを含む目的物を通液させ、その素通り画分を回収することにより達成される、前記第１項１に記載の血液製剤、酵素医薬品、または蛋白質医薬品の生産方法。
（３）橋かけ構造ポリリジンの形状が粒子状である、前記第１項または第２項に記載の血液製剤、酵素医薬品、または蛋白質医薬品の生産方法。
（４）橋かけ構造ポリリジンの形状が平板状である、前記第１項または第２項に記載の血液製剤、酵素医薬品、または蛋白質医薬品の生産方法。
（５）橋かけ剤がエポキシ化合物である、前記第１項に記載の血液製剤、酵素医薬品、または蛋白質医薬品の生産方法。
（６）橋かけ剤がエピクロロヒドリンである、前記第５項に項記載の血液製剤、酵素医薬品、または蛋白質医薬品の生産方法。
（７）ポリリジンがポリ（ε−リジン）である、前記第１項に記載の血液製剤、酵素医薬品、または蛋白質医薬品の生産方法。
（８）ポリリジンがストレプトマイセス属細菌により生産された微生物由来のものである、前記第１項に記載の血液製剤、酵素医薬品、または蛋白質医薬品の生産方法。
【０００５】
以下、本発明を詳細に説明する。
本願第１の発明は橋かけ剤により橋かけ構造とされたポリリジンである。本発明に用いるポリリジンは、構造上α、ε型のどちらでも良いが、ε型はα型に比べカチオン性（pKa7.6）であることから、好ましくε型の方が良い。また、ストレプトマイセス属細菌により生産された微生物由来のものであっても、化学合成によって得られたものであっても構わないが、ストレプトマイセス属細菌により生産された微生物由来のもは、ε型であり、エンドトキシンの選択吸着に優れ、また、生分解性があることから生体適合性が高く、更に、安価で大量入手が可能であり特に好ましい。
【０００６】
本発明に用いるポリリジンの分子量は５００〜１,０００,０００の範囲のものが使用でき、特に限定されるものではないが、粒子化する場合にはその容易さの面から１,０００〜１０,０００の範囲であることが好ましい。
本発明においては、上記ポリリジンの単品を橋かけ構造としたものであってもよく、上記ポリリジンの２種以上を任意の配合比で混合したものであっても良い。
【０００７】
本発明の橋かけ構造を有するポリリジンは、橋かけ剤によって該構造をとるものであれば、何れの橋かけ剤を用いたものであってもよい。本発明に用いる橋かけ剤は、特に限定されるものではないが、ヘキサメチレンジイソシアネート等の２官能性試薬、ジグリシジルエーテル等のジエポキシ、一端がハロゲン化されたエピクロロヒドリンなどを挙げることができる。その中でもエポキシは容易に開環し安価であることから特に好ましい橋かけ剤であり、更にエポキシの中でもエピクロロヒドリンを架橋剤に用いた場合には、橋かけ反応が容易であり、且つ適度な疎水性をポリリジン粒子に持たせることになるため、エンドトキシンの吸着能力が顕著に向上するため、特に好ましい橋かけ剤である。
【０００８】
該橋かけ剤を用いた反応方法、条件は、使用する橋かけ剤毎に好ましいものを選択すべきであるが、一般的には不活性の分散媒中で攪拌しながら行うことにより、あるいは水溶液中で橋かけ剤と懸濁重合させることにより、ポリリジンを橋かけ構造とすることが出来る。
また、その際の橋かけ剤の添加量も特に限定されるものではないが、２〜１００ｍｏｌ％であればエンドトキシンの極めて高い吸着率を示し、更に１０〜３０ｍｏｌ％の範囲であれば、使用時の吸水率を顕著に押さえることが出来、機械的強度が飛躍的に向上するので特に好ましい。
【０００９】
本発明においては、上記橋かけ剤によって橋かけ構造としたポリリジンであれば、何れの形状のものであっても本発明の効果を得ることが出来る。その形状は板やフィルムのような平板状であってもよく、粒子状であってもよく、また、薬液が通過するためのポーラスを有する塊状であっても良い。
本発明のポリリジンをカラムに充填させる場合には粒状が好ましい。その際の粒径は特に限定されるものではないが、通常１〜１,０００μｍの範囲であることが好ましい。
【００１０】
また、平板状のものであれば、フィルターとして用いることも可能であり、多量に医薬品を処理する際には特に好ましい形状である。平板状にする際の厚さは、薬液の透過速度との関係もあり一概に特定できるものではない。一方、透過速度は吸着能力との関係から一概に特定できるものではないが、通常１０〜１,０００Ｌ／ｈｒ／ｍ2である。この透過速度であれば、厚さは１００〜１０００μｍの範囲とすることが好ましい。
【００１１】
本発明の橋かけ構造を有するポリリジンに、エンドトキシンの高い吸着能力がある理由は現在のところ特定出来ていないが、橋かけ構造にすることにより増大した疎水性と、ポリリジンのアミノ基による静電性との相乗効果によると考えられる。
【００１２】
本願第２の発明は、本願第１の発明である、橋かけ剤により橋かけ構造とされたポリリジンの製造方法に関するものであり、具体的にはポリリジンの水溶液に橋かけ剤を添加することを特徴とする該ポリリジンの製造方法である。ポリリジンは本来水溶性であり、ポリリジン水溶液に橋かけ剤を添加することで橋かけ構造となり不溶化する。
用いるポリリジンは前述の通りであり、水溶液中のポリリジン濃度は特に限定されるものではないが、好ましくは１０〜７０重量％である。
橋かけ剤は前述のものを用いることができる。反応温度及び反応時間は用いる橋かけ剤の反応性、更に添加量によって異なるため、一概に特定できないが、本発明の好ましい橋かけ剤であるエピクロロヒドリンを用いた場合には、４０℃前後の反応温度で１６時間程度攪拌することで架橋が終了する。
【００１３】
本願第３の発明は、本願第１の発明である橋かけ剤により橋かけ構造とされたポリリジンを含むエンドトキシン除去用ポリリジン組成物である。該エンドトキシン除去組成物は、本発明のポリリジンを含むものであれば、何れの組成であってもよい。
例えば、粒子状の吸着体をカラムに充填して用いる場合には、本発明の効果を損なわない範囲で、本発明以外のエンドトキシン吸着体を混合してもよく、エンドトキシン以外の病原物質を吸着する吸着体を混合したものであってもよい。本発明以外のエンドトキシン吸着体としては、ポリミキシンＢを固定化したもの、ヒスタミン等の含イミダゾール化合物を固定化したものなどを挙げることが出来、エンドトキシン以外の病原物質を吸着する吸着体としては、ウイルスを吸着する硫酸化セルロース、低密度コレステロールを吸着するデキストラン硫酸を固定化したものなどを挙げることができる。また、吸着体以外に、ろ過速度を上げる効果のある大粒子の不活性担体や、水平方向の拡散効果のある多孔性粒子を混合してもよい。
粒子状の吸着体は通常カラムに充填して用いるものであり、本発明のエンドトキシン除去組成物は、その最終製品形態として粒子の混合物であっても、カラムに充填されたものであってもよい。
【００１４】
また、平板状の吸着体の場合には、フィルターとして用いることができ、本発明の平板状、若しくはフィルター状のポリリジンを基盤とする不活性もの、例えばセルロースのフィルターと一体成型したものであってもよい。
本発明のエンドトキシン除去組成物を用いれば、何れのイオン強度域においても低エンドトキシン含有医薬品溶液から、選択的にエンドトキシンを除去することが出来る。
【００１５】
本願第４の発明は、本願第１の発明である橋かけ剤により橋かけ構造とされたポリリジンを用いて、エンドトキシンを除去することを特徴とする医薬品の生産方法である。該エンドトキシンが除去された医薬品の生産方法は、本発明のポリリジンを用いた生産方法であれば、何れの生産方法であってもよい。
通常、エンドトキシンの除去は、吸着体を充填したカラムを用いたクロマトグラフィー法またはバッチ法によるか、フィルターに薬液を通すろ過法で行われる。カラム法で行う場合は、本発明のエンドトキシン吸着体をカラムに充填し、適当な緩衝液で洗浄したあとエンドトキシンを含む医薬品等の目的物を通液させ、素通り画分を回収することにより達成される。また、バッチ法で行う場合は、本発明のエンドトキシン吸着体を適当な緩衝液で洗浄したあとエンドトキシンを含む医薬品等の目的物の溶液中に添加し攪拌したあと、吸着剤をろ別等により分離除去することにより達成される。
本発明で生産する医薬品は特に限定されるものではないが、例えば血液製剤、生理活性物質、蛋白質・核酸・酵素等を挙げることができる。
【００１６】
【実施例】
以下、本発明について実施例及び比較例を用いて詳細にに説明するが、本発明はこれらの実施例に限定されるものではない。
【００１７】
エンドトキシン吸着剤の調製
実施例１
ε-ポリリジン２５％水溶液（チッソ株式会社製）６０mlにエピクロロヒドリン１０ｇおよび５０％ＮａＯＨ ５mlを添加し４０℃で１６時間攪拌し、水不溶性の塊状重合物を得た。得られた塊状重合物を濾別しエーテルで洗浄したのち、乾燥した。適当な大きさに粉砕したのち篩により１００〜２００μｍの大きさの粒子を３０ｇ得た。こうして得られた粒子を吸着体Ａと称する。
【００１８】
実施例２
流動パラフィン１０００ｍｌ中に、ε-ポリリジン２５％水溶液（チッソ株式会社製）３０mlを添加し攪拌し液滴を形成したのちエピクロロヒドリン５ｇおよびＫＯＨを２ｇ添加し、５０℃で１６時間攪拌して水不溶性の球状粒子を得た。得られた球状粒子を水メタノールで洗浄したのち篩により分級し５３〜１２５μｍの大きさの粒子を２０ｍｌほど得た。こうして得られた粒子を吸着体Ｂと称する。
【００１９】
実施例３
α-ポリリジン（和光純薬製）３０ｇを５％ＮａＯＨ水溶液50ml中に溶解し、o-ジクロルベンゼン２０００ｍｌ中に懸濁させた。ヘキサメチレンジイソシアネート５ｇを添加し９０℃で１６時間攪拌して水不溶性の球状粒子を得た。得られた球状粒子をメタノールを用いてソックスレー抽出器で洗浄し、乾燥して真球状の粒子２１ｇを得た。こうして得られた粒子を吸着体Ｃと称する。
【００２０】
実施例４
飽和無水硫酸ナトリウム水溶液２０００ｍｌ中に、ε-ポリリジン２５％水溶液（チッソ株式会社製）３０mlを添加し攪拌し液滴を形成したのちエピクロロヒドリン２５ｍｌ添加し、５０℃で１６時間攪拌して水不溶性の球状粒子を得た。得られた球状粒子を水メタノールで洗浄したのち篩により分級し５３〜１２５μｍの大きさの粒子を３５ｍｌ得た。こうして得られた粒子を吸着体Ｄと称する。
【００２１】
実施例５
実施例１〜４より得られた各吸着剤Ａ、Ｂ、Ｃ、Ｄ及び特開平1-127039の実施例１と同じ方法で得られた吸着剤（以下吸着剤Ｅと呼ぶ）、特公平6-16843の実施例１と同じ方法で得られた吸着剤（以下吸着剤とＦ呼ぶ）ならびに市販のエンドトキシン吸着剤パイロセップ（ダイセル社製）、デトキシゲル（ピアス社製）を用い以下の条件で吸着試験を行った。
各吸着剤をエンドトキシンをほとんど含まない０．０２M、０．１Ｍ、０．２Ｍの各リン酸バファー（ｐＨ7.0）を用いて洗浄し、乾熱滅菌したカラム（内径4mm、長さ80mm）に充填した。このカラムに大腸菌由来のエンドトキシン（ＬＳＰ）500ng/mlを含む牛血清アルブミン（BSA）500μ/mlを１０ｍｌ通液させ、さらに０．０２M、０．１Ｍ、０．２Ｍリン酸バファー（ｐＨ7.0）を２ｍｌ通液させた。
通液を全て集め、ＬＳＰの残存量及びＢＳＡの回収量を測定し各吸着剤のＬＳＰとＢＳＡの吸着率を求めた。その結果を表1〜３に示す。
【００２２】
【発明の効果】
本発明の橋かけ剤により橋かけ構造とされたポリリジン、該ポリリジンからなるエンドトキシン除去組成物であれば、低エンドトキシン含有医薬品溶液からのエンドトキシンの除去において、何れのイオン強度域においても高い選択性があり、極めて効率よくエンドトキシンを除去することができる。この特異的な吸着能力により、従来困難であった工業的大スケールでの利用が可能となる。
また、本発明の該ポリリジンを用いた、エンドトキシンが除去された医薬品の生産方法であれば、エンドトキシン含有量が極めて少ない医薬品を生産することが出来る。
【００２３】
【表１】
【表２】
【表３】
BACKGROUND OF THE INVENTION
The present invention relates to a polylysine capable of adsorbing endotoxin that is a fever-causing substance, a method for producing the same, a polylysine composition for removing endotoxin containing the polylysine, and a method for producing a pharmaceutical product that removes endotoxin using the polylysine. .
[0002]
[Prior art]
Endotoxin is a general term for toxic substances in bacterial cell components, and is released when the bacteria die. The structural component of endotoxin is liposaccharide, and bacteria that inhabit the production process of the drug are produced in the microbial cells and are killed to be mixed into the drug.
Conventionally known methods for removing endotoxin include adsorption using activated carbon and ion exchangers, filtration using membranes and membrane filters, and high-temperature / high-pressure treatment or decomposition using acid / alkali. Each method has advantages and disadvantages, and has a problem in industrial use. For example, removal of endotoxins in pharmaceutical production cannot be performed under harsh conditions from the standpoint of maintaining the stability of the original drug, or the amount of endotoxin present is extremely small. Even if the adsorption could be performed successfully, the actual industrial scale could not perform the adsorption satisfactorily, or the adsorbent had adsorbed the drug itself in addition to the endotoxin.
On the other hand, several adsorbents have been disclosed in recent years to enable endotoxin adsorption which is industrially satisfactory. Japanese Patent Publication No. 6-18683 discloses an adsorbent comprising a polyamino acid containing a modifying group having an aliphatic group and / or an aryl group at the end of the side chain and / or main chain. -127039 discloses an adsorbent in which polyamino acid spherical particles are used as a carrier and an imidazole derivative is bound thereto.
[0003]
[Problems to be solved by the invention]
Unfortunately, however, these adsorbents are not selective enough to remove only endotoxins from low endotoxin-containing drugs, and acidic proteins that should not be adsorbed are adsorbed together with endotoxins. There was a problem.
Furthermore, conventional adsorbents can selectively adsorb in the high ionic strength region, but cannot selectively adsorb in the low ionic strength region, or vice versa. There was nothing unaffected.
As a result of intensive studies in view of the problems of the prior art, the inventors of the present invention have high selectivity in any ionic strength region in removing endotoxin from a low endotoxin-containing pharmaceutical solution to polylysine having a crosslinked structure. Based on this finding, the present inventors have completed the present invention.
[0004]
[Means for Solving the Problems]
The present invention has the following configuration.
( 1 ) Production of blood products, enzyme drugs, or protein drugs characterized by removing endotoxin using a crosslinked polylysine obtained by suspension polymerization of polylysine with a crosslinking agent in an aqueous solution. Method.
( 2 ) The blood product according to item 1 above, wherein removal of endotoxin is achieved by passing a target product containing endotoxin through a column packed with a crosslinked polylysine and collecting the flow-through fraction. , Production methods for enzyme drugs, or protein drugs.
( 3 ) The method for producing a blood product, enzyme drug product, or protein drug product according to item 1 or 2, wherein the cross-linked polylysine has a particulate shape.
( 4 ) The method for producing a blood product, enzyme drug product, or protein drug product according to item 1 or 2, wherein the cross-linked polylysine has a flat plate shape.
( 5 ) The method for producing a blood product, enzyme pharmaceutical product, or protein pharmaceutical product according to item 1, wherein the crosslinking agent is an epoxy compound .
( 6 ) The method for producing a blood product, enzyme pharmaceutical product, or protein pharmaceutical product according to item 5, wherein the crosslinking agent is epichlorohydrin .
( 7 ) The method for producing a blood product, enzyme pharmaceutical product, or protein pharmaceutical product according to item 1, wherein the polylysine is poly (ε-lysine) .
( 8 ) The method for producing a blood product, enzyme drug, or protein drug according to the above item 1, wherein the polylysine is derived from a microorganism produced by a Streptomyces bacterium .
[0005]
Hereinafter, the present invention will be described in detail.
1st invention of this application is the polylysine made into the crosslinked structure by the crosslinking agent. The polylysine used in the present invention may be either α or ε in terms of structure, but since the ε type is more cationic (pKa7.6) than the α type, the ε type is preferred. In addition, it may be derived from microorganisms produced by Streptomyces bacteria or may be obtained by chemical synthesis, but those derived from microorganisms produced by Streptomyces bacteria, It is particularly preferable because it is ε-type, excellent in selective adsorption of endotoxin, biodegradable and highly biocompatible, and inexpensive and available in large quantities.
[0006]
The molecular weight of polylysine used in the present invention may be in the range of 500 to 1,000,000, and is not particularly limited. However, when it is made into particles, it is 1,000 to 10,000 in view of ease. A range of 000 is preferred.
In the present invention, a single product of the above polylysine may have a crosslinked structure, or two or more of the above polylysine may be mixed at an arbitrary blending ratio.
[0007]
The polylysine having a crosslinking structure according to the present invention may be one using any crosslinking agent as long as the structure is formed by a crosslinking agent. The crosslinking agent used in the present invention is not particularly limited, and examples thereof include bifunctional reagents such as hexamethylene diisocyanate, diepoxy such as diglycidyl ether, and epichlorohydrin having one end halogenated. it can. Among them, epoxy is a particularly preferable crosslinking agent because it is easy to open a ring and is inexpensive. Further, among the epoxies, when epichlorohydrin is used as a crosslinking agent, the crosslinking reaction is easy and moderate. Since the polylysine particles are imparted with such a hydrophobic property, the adsorption ability of endotoxin is remarkably improved. Therefore, this is a particularly preferred crosslinking agent.
[0008]
The reaction method and conditions using the cross-linking agent should be preferably selected for each cross-linking agent to be used. In general, the reaction is carried out with stirring in an inert dispersion medium, or an aqueous solution. Polylysine can be made into a crosslinked structure by suspension polymerization with a crosslinking agent.
In addition, the addition amount of the crosslinking agent at that time is not particularly limited, but 2 to 100 mol% shows an extremely high adsorption rate of endotoxin. This is particularly preferable because the water absorption can be remarkably suppressed and the mechanical strength is remarkably improved.
[0009]
In the present invention, the effect of the present invention can be obtained in any shape as long as it is a polylysine having a crosslinked structure with the above-mentioned crosslinking agent. The shape may be a flat shape such as a plate or a film, may be a particle shape, or may be a lump shape having a porous material for allowing a chemical solution to pass therethrough.
Granularity is preferred when the polylysine of the present invention is packed in a column. The particle size at that time is not particularly limited, but it is usually preferably in the range of 1 to 1,000 μm.
[0010]
Moreover, if it is a flat thing, it can also be used as a filter and is a particularly preferable shape when processing a large amount of pharmaceuticals. The thickness at the time of forming into a flat plate shape cannot be generally specified because of the relationship with the permeation rate of the chemical solution. On the other hand, the permeation rate is not generally specified from the relationship with the adsorption capacity, but is usually 10 to 1,000 L / hr / m 2 . With this transmission rate, the thickness is preferably in the range of 100 to 1000 μm.
[0011]
The reason why the polylysine having a crosslinked structure of the present invention has a high endotoxin adsorption ability has not been identified at present, but the increased hydrophobicity due to the crosslinked structure and the electrostatic property due to the amino group of polylysine. This is thought to be due to a synergistic effect.
[0012]
The second invention of the present application relates to a method for producing polylysine having a crosslinked structure by a crosslinking agent, which is the first invention of the present application, and specifically, adding a crosslinking agent to an aqueous solution of polylysine. This is a method for producing the polylysine. Polylysine is inherently water-soluble and becomes a crosslinked structure and becomes insoluble when a crosslinking agent is added to an aqueous polylysine solution.
The polylysine to be used is as described above, and the concentration of polylysine in the aqueous solution is not particularly limited, but is preferably 10 to 70% by weight.
The aforementioned crosslinking agents can be used. The reaction temperature and reaction time vary depending on the reactivity of the crosslinking agent used, and the amount added, and therefore cannot be specified. However, when epichlorohydrin, which is the preferred crosslinking agent of the present invention, is used, it is around 40 ° C. Crosslinking is completed by stirring for about 16 hours at this reaction temperature.
[0013]
The third invention of the present application is a polylysine composition for removing endotoxin, which comprises polylysine having a crosslinked structure by the crosslinking agent according to the first invention of the present application. The endotoxin removal composition may be any composition as long as it contains the polylysine of the present invention.
For example, when a column-shaped adsorbent is packed and used, an endotoxin adsorbent other than the present invention may be mixed and a pathogen other than endotoxin is adsorbed within a range not impairing the effects of the present invention. It may be a mixture of adsorbents. Examples of endotoxin adsorbents other than those of the present invention include those immobilized with polymyxin B, those immobilized with imidazole-containing compounds such as histamine, and the like. Adsorbents that adsorb pathogens other than endotoxins include viruses. And sulfated cellulose that adsorbs dextran sulfate that adsorbs low-density cholesterol. In addition to the adsorbent, an inert carrier of large particles having an effect of increasing the filtration rate or porous particles having a horizontal diffusion effect may be mixed.
The particulate adsorbent is usually used by being packed in a column, and the endotoxin removal composition of the present invention may be a mixture of particles as the final product form or may be packed in a column. .
[0014]
Further, in the case of a flat adsorbent, it can be used as a filter, and is formed integrally with an inactive one based on the flat or filter-like polylysine of the present invention, for example, a cellulose filter. Also good.
If the endotoxin removing composition of the present invention is used, endotoxin can be selectively removed from a low endotoxin-containing pharmaceutical solution in any ionic strength region.
[0015]
A fourth invention of the present application is a method for producing a pharmaceutical product characterized by removing endotoxin using polylysine having a crosslinked structure by the crosslinking agent of the first invention of the present application. The production method of the pharmaceutical from which the endotoxin has been removed may be any production method as long as it is a production method using the polylysine of the present invention.
Usually, endotoxin is removed by a chromatography method using a column packed with an adsorbent or a batch method, or a filtration method in which a chemical solution is passed through a filter. In the case of the column method, the endotoxin adsorbent of the present invention is packed in a column, washed with an appropriate buffer, passed through a target product such as a pharmaceutical product containing endotoxin, and the flow-through fraction is collected. The When the batch method is used, the endotoxin adsorbent of the present invention is washed with an appropriate buffer solution, added to a solution of a target product such as a drug containing endotoxin and stirred, and then the adsorbent is separated by filtration or the like. This is achieved by removing.
The pharmaceutical product produced in the present invention is not particularly limited, and examples thereof include blood products, physiologically active substances, proteins / nucleic acids / enzymes and the like.
[0016]
【Example】
Hereinafter, although the present invention is explained in detail using an example and a comparative example, the present invention is not limited to these examples.
[0017]
Preparation Example 1 of Endotoxin Adsorbent
To 60 ml of 25% aqueous solution of ε-polylysine (manufactured by Chisso Corporation), 10 g of epichlorohydrin and 5 ml of 50% NaOH were added and stirred at 40 ° C. for 16 hours to obtain a water-insoluble bulk polymer. The resulting bulk polymer was filtered off, washed with ether and dried. After pulverizing to an appropriate size, 30 g of particles having a size of 100 to 200 μm were obtained with a sieve. The particles thus obtained are called adsorbent A.
[0018]
Example 2
In 1000 ml of liquid paraffin, 30 ml of ε-polylysine 25% aqueous solution (manufactured by Chisso Corporation) was added and stirred to form droplets, then 5 g of epichlorohydrin and 2 g of KOH were added and stirred at 50 ° C. for 16 hours. Water-insoluble spherical particles were obtained. The obtained spherical particles were washed with water methanol and then classified by a sieve to obtain about 20 ml of particles having a size of 53 to 125 μm. The particles thus obtained are referred to as adsorbent B.
[0019]
Example 3
30 g of α-polylysine (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 50 ml of 5% NaOH aqueous solution and suspended in 2000 ml of o-dichlorobenzene. 5 g of hexamethylene diisocyanate was added and stirred at 90 ° C. for 16 hours to obtain water-insoluble spherical particles. The obtained spherical particles were washed with a Soxhlet extractor using methanol and dried to obtain 21 g of true spherical particles. The particles thus obtained are referred to as adsorbent C.
[0020]
Example 4
In 2000 ml of saturated anhydrous sodium sulfate aqueous solution, 30 ml of ε-polylysine 25% aqueous solution (manufactured by Chisso Corporation) was added and stirred to form droplets, and then 25 ml of epichlorohydrin was added and stirred at 50 ° C. for 16 hours. Insoluble spherical particles were obtained. The obtained spherical particles were washed with water methanol and then classified with a sieve to obtain 35 ml of particles having a size of 53 to 125 μm. The particles thus obtained are called adsorbent D.
[0021]
Example 5
Each of the adsorbents A, B, C, D obtained from Examples 1 to 4 and the adsorbent obtained by the same method as Example 1 of JP-A-1-27039 (hereinafter referred to as adsorbent E), -16843 adsorption test (hereinafter referred to as "adsorbent F") obtained in the same manner as in Example 1 and a commercially available endotoxin adsorbent Pyrosep (Daicel) or Detoxygel (Pierce) under the following conditions Went.
Each adsorbent was washed with 0.02M, 0.1M, and 0.2M phosphate buffers (pH 7.0) containing almost no endotoxin and sterilized by dry heat (4mm ID, 80mm length). Filled. 10 ml of bovine serum albumin (BSA) containing 500 ng / ml of E. coli-derived endotoxin (LSP) was passed through this column, and 0.02M, 0.1M, 0.2M phosphate buffer (pH 7.0). Was passed through 2 ml.
All the flow-throughs were collected, the remaining amount of LSP and the recovered amount of BSA were measured, and the adsorption rate of LSP and BSA of each adsorbent was determined. The results are shown in Tables 1-3.
[0022]
【The invention's effect】
The polylysine having a crosslinked structure by the crosslinking agent of the present invention, and an endotoxin removal composition comprising the polylysine, have high selectivity in any ionic strength range in removing endotoxin from a low endotoxin-containing pharmaceutical solution. And endotoxin can be removed very efficiently. Due to this specific adsorption capacity, it is possible to use it on a large industrial scale, which has been difficult in the past.
Moreover, if it is the manufacturing method of the pharmaceutical from which the endotoxin was removed using this polylysine of this invention, a pharmaceutical with very little endotoxin content can be produced.
[0023]
[Table 1]
[Table 2]
[Table 3]
Claims (8)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33502197A JP4606524B2 (en) | 1997-11-19 | 1997-11-19 | Polylysine, polylysine production method, polylysine composition, and pharmaceutical production method for removing endotoxin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33502197A JP4606524B2 (en) | 1997-11-19 | 1997-11-19 | Polylysine, polylysine production method, polylysine composition, and pharmaceutical production method for removing endotoxin |
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| Publication Number | Publication Date |
|---|---|
| JPH11152330A JPH11152330A (en) | 1999-06-08 |
| JP4606524B2 true JP4606524B2 (en) | 2011-01-05 |
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| JP33502197A Expired - Fee Related JP4606524B2 (en) | 1997-11-19 | 1997-11-19 | Polylysine, polylysine production method, polylysine composition, and pharmaceutical production method for removing endotoxin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| HUP9902217A3 (en) * | 1999-06-29 | 2002-07-29 | Szegoe Peter | Polycation based bioconjugates and process for producing thereof |
| JP4996791B2 (en) * | 2001-03-14 | 2012-08-08 | Ｊｎｃ株式会社 | Endotoxin adsorbent and method for removing endotoxin using the same |
| JP2004292357A (en) * | 2003-03-27 | 2004-10-21 | Kumamoto Technology & Industry Foundation | Agent for adsorption and removal of intraoral endotoxin |
| JP4989039B2 (en) * | 2005-04-27 | 2012-08-01 | Ｊｎｃ株式会社 | Nucleic acid complex and method for introducing nucleic acid into cells using the same |
| JP5207505B2 (en) * | 2006-09-04 | 2013-06-12 | 国立大学法人大阪大学 | Thermosensitive polylysine |
| GB201106742D0 (en) * | 2011-04-20 | 2011-06-01 | Spheritech Ltd | Cross-linked poly-e-lysine |
| GB2562004B (en) * | 2012-04-20 | 2019-10-23 | Spheritech Ltd | Cross-linked poly-e-lysine particles |
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