JP5249489B2 - α1アンチトリプシンの精製法 - Google Patents
α1アンチトリプシンの精製法 Download PDFInfo
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- JP5249489B2 JP5249489B2 JP2004565589A JP2004565589A JP5249489B2 JP 5249489 B2 JP5249489 B2 JP 5249489B2 JP 2004565589 A JP2004565589 A JP 2004565589A JP 2004565589 A JP2004565589 A JP 2004565589A JP 5249489 B2 JP5249489 B2 JP 5249489B2
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Description
本発明は、タンパク質の分離および精製の方法に関する。より具体的には、本発明は、血漿分画のような複雑なタンパク混合物からのα1アンチトリプシン(AAT、α1プロテイナーゼ阻害剤、API、およびA1−PIとしても知られている)の分離と、医薬使用に適した組成物を提供するための、分離したAATのさらなる精製の方法に関する。
α1アンチトリプシン(AAT)は、プロテアーゼの糖ペプチド阻害剤であり、ヒトの血清および他の体液に見出されている。AATによるプロテアーゼ阻害は、組織タンパク分解の調節の必須要素であり、AAT欠損症は、いくつかの疾患の病理に関連している。例えば、α1アンチトリプシン欠損症を遺伝により受け継いだ個体は、ヒト白血球エラスターゼによる肺組織の非調節性の破壊の結果として、重篤な早期発症肺気腫に罹患する高いリスクを有する。外因性ヒトAATの投与は、エラスターゼを阻害することが示されていて、AAT欠損患者の生存の向上とその肺機能低下速度の抑制に関連づけられている(Crystalら,Am.J.Respir.Crit.Care Med.158:49−59(1998);概説については、R.MahadevaおよびD.Lomas,Thorax 53:501−505(1998)を参照のこと)。
本発明は、粗製AAT含有タンパク沈殿物よりAATを精製する方法を提供し、該方法は、本質的に以下の工程よりなる:(a)AATが溶けることを可能にする条件下でAAT含有タンパク混合物を緩衝液に懸濁する工程;(b)生じる懸濁液をジスルフィド還元剤と接触させて、還元懸濁液を産生する工程;(c)還元懸濁液を不溶性タンパク吸着材料と接触させる工程;および(d)不溶性材料を懸濁液より除去する工程。この方法は、先行技術の方法より低下したコストとより少ない時間で、クロマトグラフィー処理にそのまま適している濃縮AAT調製物を提供する。追加の精製工程は、以下にさらに記載するように、実施者の考え次第で実施してよい。
(a)α1アンチトリプシンは、SDSによれば、6%未満、好ましくは2%未満、そして最も好ましくは1%未満の汚染タンパク質を含有し、そして、
(b)0.1%未満のアルブミン;
(c)0.8%未満、そして好ましくは0.2%以下のα1酸性糖タンパク質;
(d)0.1%未満のα2マクログロブリン;
(e)0.1%未満のアポリポタンパク質A1;
(f)0.5%未満、そして好ましくは0.1%以下のアンチトロンビンIII;
(g)0.1%未満のセルロプラスミン;
(h)0.5%未満、そして好ましくは0.1%未満のハプトグロビン;
(i)0.2%未満、そして好ましくは0.1%未満のIgA;
(j)0.1%未満のIgG;
(k)0.1%未満のトランスフェリンを含有し;
(l)α1アンチトリプシンの比活性は、減衰係数としてE1% 1cm,280nm=5.3を使用するとき、1ミリグラムのタンパク質につき少なくとも0.99mgの機能性AATであり;
(m)本産物の8%未満、そして好ましくは、5%未満は、単量体AATより高い分子量であり;
(n)活性AATの抗原性AATに対する見かけの比は、終点比濁分析により測定するとき、1.08より大きい、好ましくは1.16より大きい、そして最も好ましくは1.23より大きい;
(o)広範囲の物理化学特性を代表するヒトおよびモデルウイルスを使用するスパイク試験において測定するとき、エンベロープウイルスは少なくとも11log10単位低減し、非エンベロープウイルスは、少なくとも6log10単位低減している;そして
(p)該産物は、凍結乾燥して、25℃以下で保存するとき、少なくとも2年間安定である。
以下に例示する本発明の特別な態様は、特別なCohn分画IVペーストを出発材料として利用するが、同様の血漿分画の使用も本発明の範囲内にあると考慮される。代わりの出発材料には、限定されないが、他のAAT含有Cohn分画(米国特許第4,697,003号を参照のこと)、Kistler−Nitschmann上清AまたはA+I由来の沈殿物(P.Kistler,H.S.Nitschmann,Vox Sang.,7:414−424(1962))、およびSchultzeら,により米国特許第3,301,842号に記載されるような血漿由来の硫酸アンモニウム沈殿物が含まれる。AAT産生組換え細胞または生物の培養物に由来するタンパク沈殿物、またはトランスジェニック哺乳動物の乳汁または血清に由来する沈殿物の使用も、本発明の範囲内にあると考慮される。
1.Cohn分画IV 1−4 の調製
ヒト血漿を−2〜2℃へ冷やし、そして6.9〜7.5のpHへ調整する。冷エタノールを加えて6〜10%の濃度とし、そして温度を−4〜0℃へ下げる。生じる沈殿物(「分画I」)を遠心分離または濾過により除去する。
分画IV1−4ペーストを懸濁緩衝液(例えば、100mM Tris,20mM NaCl,約7.5と約9.5の間、好ましくは約8と約9の間のpH)に懸濁させ、低温で少なくとも1時間撹拌する。使用する緩衝液の量は、1kgの血漿含有分画につき6〜10kgの緩衝液に及ぶ。
IEC平衡緩衝液で平衡化したアニオン交換樹脂を含有するクロマトグラフィーカラムへAAT最終濾液をそのまま適用する。IEC洗浄緩衝液でカラムを洗浄することによって汚染物をカラムより除去し、引き続き、IEC溶出緩衝液を使用してAATを溶出させる。
硫酸アンモニウムを加えて約1Mの最終濃度とすることによって、IECカラムからの溶出液をHICのために調製する。次いで、この溶液を濾過し、そしてHIC洗浄緩衝液に平衡化した疎水性相互作用クロマトグラフィーカラムへ適用する。洗浄緩衝液での初期溶出によりAAT含有流出液を得て、そして追加の洗浄緩衝液での溶出によってカラムに保持されたAATを除去する。流出液と洗液を合わせて限外濾過により濃縮し、そしてリン酸緩衝液へ透析濾過する。最終AAT濃度は、好ましくは、7%以下のタンパク質である。
ショ糖および酢酸カリウムの添加によりAAT濃縮物を低温殺菌のために安定化させて、そして約60℃で10〜11時間低温殺菌する。低温殺菌した溶液は、さらなる処理までは2〜8℃に保つ。
低温殺菌済みAAT溶液を最終製剤緩衝液で希釈する。次いで、この希釈した低温殺菌済みAAT溶液を2つの新しいYM−100(Amicon)らせん巻き限外濾過カートリッジに通して濾過する。このナノ濾過工程は、第二の主要なウイルス低減工程として役立つ。公称100,000ダルトンの分子量カットオフを有する膜によりウイルスが保持されるのに対し、ほぼ50kDの分子量を有するAATは素通りする。この第二フィルターの透過液とフィルター後洗液にAATを採取する。この最終濾液をバルク受け器に採取して、2〜8℃に保つ。
AAT含有最終濾液を濃縮し、そして15℃以下の温度で最終製剤緩衝液へ透析濾過して、最終バルク溶液を生成する。一連の滅菌、細菌保持フィルターの通過により、この溶液を浄化して滅菌する。この無菌バルク溶液を滅菌済みのガラス最終容器へ充填する。充填した容器を凍結乾燥させてから、真空で密封する。
9026リットルのヒト血漿より、Cohn血漿分画法により分画IV1−4沈殿物(667kg)を単離した。この材料をそれぞれほぼ75kgの9つのバッチへ分割した。プレスケーク(presscake)に対して6〜10(w/w)の緩衝液を使用して、各バッチをTris緩衝液に懸濁させた。この懸濁液を少なくとも15分間撹拌し、温度を2°〜8℃へ調整し、そして1N水酸化ナトリウムまたは1N塩酸を必要に応じて用いて、各懸濁液のpHを8.80〜8.95へ調整した。この懸濁液を15〜105分(平均45分)間撹拌し、そしてタンパク質(Bradfordアッセイ)と効力をモニターした。各バッチの比活性は、0.027〜0.045の範囲にあり、平均して0.037mgの機能性AAT/mgタンパク質であった。タンパク質全体のほぼ12%がアルブミンであり、ほぼ22%がトランスフェリンであった。
Claims (15)
- AAT含有タンパク質混合物よりAATを精製する方法であって:
(a)ジスルフィド還元剤とAAT含有タンパク質混合物を接触させて、2℃〜8℃で還元AAT含有混合物を産生する工程;
(b)還元AAT含有タンパク質混合物を不溶性タンパク質吸着材料と接触させる工程;および
(c)生じるAAT含有産物を単離する工程;
を含み、さらにアニオン交換クロマトグラフィー工程と疎水性相互作用クロマトグラフィー工程を含んでなり、かつ、塩析の工程を含まないことを特徴とする、前記方法。 - AAT含有タンパク質沈殿物よりAATを精製する方法であって:
(a)AATが溶けることを可能にする条件下でAAT含有タンパク質沈殿物を緩衝液に懸濁する工程;
(b)AAT含有懸濁液をジスルフィド還元剤と接触させて、2℃〜8℃で還元AAT含有懸濁液を産生する工程;
(c)還元AAT含有懸濁液を不溶性タンパク質吸着材料と接触させる工程;および
(d)生じるAAT含有産物を単離する工程;
を含み、さらにアニオン交換クロマトグラフィー工程と疎水性相互作用クロマトグラフィー工程を含んでなり、かつ、塩析の工程を含まないことを特徴とする、前記方法。 - ジスルフィド還元剤がジチオールである、請求項1または2に記載の方法。
- ジチオールがジチオスレイトールである、請求項3の方法。
- タンパク質吸着材料がシリカ吸着剤である、請求項1または2に記載の方法。
- タンパク質吸着材料がフュームド(fumed)シリカである、請求項5の方法。
- ウイルスの低減工程をさらに含んでなる、請求項1または2に記載の方法。
- ウイルスの低減工程が60〜70℃での低温殺菌を含む、請求項7の方法。
- 少なくとも40%(重量/重量)のショ糖を含有するAATの溶液に対して低温殺菌工程を行う、請求項8の方法。
- ウイルスの低減工程がウイルス粒子を除去するのに有効な濾過をさらに含む、請求項9の方法。
- 滅菌工程をさらに含んでなる、請求項1または2に記載の方法。
- 滅菌工程が細菌を除去するのに有効な濾過を含む、請求項11の方法。
- AAT含有タンパク質混合物がCohn分画IV沈殿物またはCohnII+III上清である、請求項1の方法。
- AAT含有タンパク質沈殿物がCohn分画IV沈殿物である、請求項2の方法。
- ジスルフィド還元剤を含む緩衝液を工程(a)に提供することによって工程(b)を実行する、請求項2の方法。
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BRPI0915948A2 (pt) * | 2008-07-18 | 2019-04-09 | Talecris Biotherapeutics Inc | método de preparar inibidor de alfa-1 proteinase |
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CN102180966B (zh) * | 2011-01-28 | 2013-04-03 | 哈尔滨派斯菲科生物制药股份有限公司 | 一种规模化生产人α1-抗胰蛋白酶的方法 |
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CN107163138B (zh) * | 2017-03-28 | 2021-02-09 | 深圳市卫光生物制品股份有限公司 | 一种人血浆蛋白α1-抗胰蛋白酶的分离纯化方法 |
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