JP2013522252A - 生物学的活性型組換えヒトg−csfを得るための方法 - Google Patents
生物学的活性型組換えヒトg−csfを得るための方法 Download PDFInfo
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- JP2013522252A JP2013522252A JP2012557447A JP2012557447A JP2013522252A JP 2013522252 A JP2013522252 A JP 2013522252A JP 2012557447 A JP2012557447 A JP 2012557447A JP 2012557447 A JP2012557447 A JP 2012557447A JP 2013522252 A JP2013522252 A JP 2013522252A
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Abstract
Description
(a)封入体中に含まれるG−CSFを、変性剤および還元剤を含有する可溶化緩衝液で可溶化するステップ;
(b)可溶化物を、還元型グルタチオンおよび酸化型グルタチオンを含有するリフォールディング緩衝液で10℃を超える温度にて希釈することによりG−CSFをリフォールディングするステップ;および
(c)逆相(RP)クロマトグラフィーを含む少なくとも1つクロマトグラフィーステップによりリフォールディングされたG−CSFを精製するステップ
を含む、生物学的に活性型のヒトG−CSFを封入体から得るための方法に関する。
(i)限外ろ過/ダイアフィルトレーションステップ;
(ii)カチオン交換(CEX)クロマトグラフィーステップ:
(iii)精密ろ過ステップ;
(iv)逆相(RP)クロマトグラフィーステップ;および
(v)限外ろ過/ダイアフィルトレーションステップ
を含む。
G−CSFを、pBR322由来の発現ベクターである03−221C−pHIP中で発現させる。大腸菌宿主株BNN93を、産生用にこのプラスミドで形質転換した。BNN93は大腸菌K12誘導体である。G−CSF合成を、30℃から42℃への温度シフトにより誘発されるラムダプロモーターにより調節し、その結果、封入体中で一次不溶性G−CSFが過剰発現される。
1,000mlのカナマイシンを含まない複合培地で希釈したワーキングセルバンク(WCB)の1つのバイアルの内容物を用いて、予備発酵のための「希釈したワーキングセルバンク」を生成させる。希釈は、クリーンルームグレードBで層流フード(LAF)グレードA中で実施する。前培養物を、20Lの予備発酵槽中に直接無菌的に移す。培養は、30±1℃で開始し、OD6000.3〜0.6に達するまで250rpmで6〜12時間撹拌する。カナマイシンを含まない複合培地であらかじめ満たした1000Lの発酵槽中に、完全発酵ブロスを移す。培養物を、30±1℃にて≧200rpmで撹拌しながらインキュベートして、5〜7のOD600に達するまでpO2≧30%およびpH7.0±0.2を9〜11時間保証する。
G−CSFの発現は、温度を42℃まで上昇させ、6.75〜7.25時間、≧200rpmで撹拌しながらさらにインキュベートして、pO2≧30%を確実にすることによって誘発する。細菌細胞を室温でディスクセパレーターにより150L/hにて採取し、その結果、約100Lの濃縮された細胞懸濁液を得る。
濃縮された細胞懸濁液を200Lに希釈し、高圧ホモジナイザーを用いて20mMのリン酸ナトリウム、pH8.0中に300L/hで3回引き続いて通過させることにより、500バールで均質化する。放出された封入体を続いて17,000rpmにて50L/hでの円筒篭型遠心分離により採取する。18mMのリン酸ナトリウム、2%(w/v)のTriton X−100、5mMのEDTA、pH8.0中で10分間再懸濁させた後、封入体を17,000rpmにて50L/hで再度遠心分離する。結果として得られたペレットを18mMのリン酸ナトリウムpH8.0で10分間洗浄し、続いて17,000rpmにて50L/hでさらに遠心分離する。ペレットを注射用水(WFI)中に再懸濁させ、3つの等しいアリコートに分注する。封入体を次いで<−15℃で保存する(ホールドステップ)。
1,000リットルの発酵から得られる全封入体量の3分の1を解凍した後、封入体を25mMのTris、6MのGuHCl、1mMのEDTA、pH8.0中で5.5〜6.5時間、撹拌しながらインキュベートすることにより可溶化し、続いて1.5μmのフィルターを用いたろ過、例えば深層ろ過(清澄化ろ過)を行う。G−CSFを次いで、25mMのTris、0.8Mのアルギニン、1mMのグルタチオン(還元型)1mMのグルタチオン(酸化型)、10mMのEDTA、pH8.0中に20±2℃で希釈することによりリフォールディングする。これに続いて、ポリソルベート80を0.01%(w/v)の終濃度になるまで添加し、酢酸でpH4.0に調節する。リフォールディング後、限外ろ過およびダイアフィルトレーションステップを実施し(MWCO 10kDa、8.4m2、約8×緩衝液交換)、この場合、緩衝液を10mMの酢酸ナトリウム、0.01%(w/v)のポリソルベート80、pH4.0により交換する。最後に、10μmおよび1.2μmのフィルターカスケードを通して溶液をろ過する。ろ過後、溶液を22±2℃で保存する(ホールドステップ)。
クロマトグラフィー精製ステップは、細胞培養化合物、細菌細胞不純物、プロセス関連および生成物関連不純物を除去し、製剤処方の調製のためまたはその後のペグ化のために一貫したG−CSF含有量を保証するために設計される(以下の実施例を参照のこと)。本発明のプロセスの概要を図1に示し、下記表1に記載する。カチオン交換およびRP−HPLCクロマトグラフィーの実施のさらに詳細な説明を実施例7により表2および3で提供する。
ダイアフィルトレーションした濃縮物をカチオン交換(CM Sepharose(登録商標)FF)カラム上にロードする。ロード後、カラムを5カラム容積(CV)の平衡緩衝液(20mMの酢酸、0.004%(w/v)のポリソルベート80、pH5.5)で洗浄し、結合したG−CSFをCMカラムから、直線的塩化ナトリウム勾配を用いて、20mMの酢酸、pH5.5、0.004%(w/v)のポリソルベート80および約50〜80mg/mlのソルビトールを含有するバッグ中に溶出させる。溶出プロファイルをUV検出(280nm、260nmおよび215nm)によりモニタリングし、伝導率を測定することにより勾配を制御する。溶出されたG−CSFを、伝導率および吸光度プロファイルによって同定する。G−CSFプールフラクションの濃度を、UV280吸光度により測定し、≦1.6mg/mLまで希釈する。
ダイアフィルトレーションろ液を同じ緩衝液で1.0±0.1mg/mlのタンパク質濃度まで希釈し、次いで5±3℃で保存(ホールドステップ)するために、1つの使い捨てのバッグ(プレフィルターは、ろ過前にLAF中で最終容器に連結され、最終フィルターは、50Lの一次包装バッグにあらかじめ組み立てられ、照射により滅菌され、ろ過および充填後に密封することにより無菌的に遮断される)中に直接二重ろ過した(カスケード中の2つの0.22μmフィルター)。
Claims (26)
- 生物学的に活性型のヒトG−CSFを封入体から得るための方法であって、以下のステップ:
(a)封入体中に含まれるG−CSFを、変性剤および還元剤を含有する可溶化緩衝液で可溶化するステップ;
(b)可溶化物を、還元型グルタチオンおよび酸化型グルタチオンを含有するリフォールディング緩衝液で、10℃を超える温度にて希釈することにより、G−CSFをリフォールディングするステップ;および
(c)逆相(RP)クロマトグラフィーを含む少なくとも1つクロマトグラフィーステップによりリフォールディングされたG−CSFを精製するステップ
を含む、方法。 - 変性剤がグアニジン−HClであり、好ましくはグアニジン−HClの濃度が4.0〜8.0mol/lである、請求項1記載の方法。
- 還元剤がDTTである、請求項1または2記載の方法。
- 可溶化緩衝液中の還元剤の濃度が1〜100mmol/l、好ましくは1〜10mmol/lである、請求項1〜3のいずれか1項に記載の方法。
- 封入体1グラムあたり10〜100mlの可溶化緩衝液を使用する、請求項1〜4のいずれか1項に記載の方法。
- 可溶化緩衝液および/またはリフォールディング緩衝液が、キレート剤、好ましくはEDTA二ナトリウムをさらに含有する、請求項1〜5のいずれか1項に記載の方法。
- 可溶化時間が、1〜10、好ましくは4〜8、最も好ましくは6±0.5時間である、請求項1〜6のいずれか1項に記載の方法。
- リフォールディング緩衝液が、アルギニン−HClをさらに含有する、請求項1〜7のいずれか1項に記載の方法。
- 還元型グルタチオンおよび酸化型グルタチオンの濃度が、それぞれ0.2〜10mmol/lである、請求項1〜8のいずれか1項に記載の方法。
- 可溶化物を1:20の比にてリフォールディング緩衝液で希釈する、請求項1〜9のいずれか1項に記載の方法。
- リフォールディングを20±2℃で少なくとも3時間実施する、請求項1〜10のいずれか1項に記載の方法。
- 可溶化物を、リフォールディング緩衝液で希釈する前にろ過に供する、請求項1〜11のいずれか1項に記載の方法。
- 逆相(RP)クロマトグラフィーステップがRP高圧液体クロマトグラフィー(RP−HPLC)である、請求項1〜12のいずれか1項に記載の方法。
- Jupiter C4、Source 15 RPCまたはSource 30 RPCクロマトグラフ用樹脂を使用する、請求項1〜13のいずれか1項に記載の方法。
- Jupiter C4クロマトグラフ用樹脂を逆相高圧液体クロマトグラフィー(RP−HPLC)で使用する、請求項14記載の方法。
- RPクロマトグラフィーステップに先行してイオン交換クロマトグラフィーを行う、請求項1〜15のいずれか1項に記載の方法。
- イオン交換クロマトグラフィーがカチオン交換(CEX)クロマトグラフィーである、請求項16記載の方法。
- ステップ(c)が:
(i)限外ろ過/ダイアフィルトレーションステップ;
(ii)CEXクロマトグラフィーステップ;
(iii)精密ろ過ステップ;
(iv)RPクロマトグラフィーステップ;および
(v)限外ろ過/ダイアフィルトレーションステップ
を含む、請求項1〜17のいずれか1項に記載の方法。 - 水溶性ポリマーをG−CSFに共有結合させることをさらに含む、請求項1〜18のいずれか1項に記載の方法。
- 水溶性ポリマーがポリエチレングリコール(PEG)である、請求項19記載の方法。
- ポリマーの分子量が約10〜30kDa、好ましくは20kDaである、請求項19または20記載の方法。
- 生物学的に活性型のG−CSFを得るための、前記請求項のいずれか1項で特徴付けられる分取RPクロマトグラフィーステップの使用。
- 請求項1〜21のいずれかに記載の方法または請求項22記載の使用により得られるG−CSFの調製物。
- 組換えG−CSFならびに薬学的に許容される添加剤、例えば緩衝液、塩および安定剤からなる医薬組成物の製造法であって、請求項1〜21のいずれか1項に記載のG−CSFを得るための方法または請求項22記載の使用を含む、方法。
- 精製された生物学的に活性型のG−CSFを、10mMの酢酸(pH4.0)、0.0025%のポリソルベート80および50g/lのソルビトール中に配合する、請求項24記載の方法。
- 請求項23のG−CSF調製物を含むか、または請求項24もしくは25記載の方法により得られる医薬組成物。
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JP2018505210A (ja) * | 2014-12-22 | 2018-02-22 | ユーシービー バイオファルマ エスピーアールエル | タンパク質製造方法 |
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WO2011108482A1 (ja) * | 2010-03-05 | 2011-09-09 | Dic株式会社 | 賦型シート及びその製造方法 |
HUP1200172A2 (en) * | 2012-03-19 | 2013-10-28 | Richter Gedeon Nyrt | Methods for refolding g-csf from inclusion bodies |
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WO2014155365A2 (en) * | 2013-03-29 | 2014-10-02 | Dr.Reddy's Laboratories Limited | Purification method |
EP2978770B1 (en) | 2013-03-29 | 2019-11-06 | Dr. Reddy's Laboratories Limited | Refolding of proteins |
CN103694339B (zh) * | 2013-12-04 | 2017-10-13 | 珠海联邦制药股份有限公司 | 一种甘精胰岛素前体的复性方法 |
US10457716B2 (en) | 2014-08-06 | 2019-10-29 | University Of Notre Dame Du Lac | Protein folding and methods of using same |
GB201506870D0 (en) * | 2015-04-22 | 2015-06-03 | Ucb Biopharma Sprl | Method |
CN110382524B (zh) * | 2017-01-16 | 2024-02-06 | 南沙生物科技(香港)公司 | 用于在酵母中生产重组il-11的***和方法 |
JP7335664B1 (ja) | 2023-04-05 | 2023-08-30 | アポロ興産株式会社 | 包装材の製造方法 |
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AU2011229491A1 (en) | 2012-09-06 |
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CN102892780B (zh) | 2015-07-29 |
WO2011113601A1 (en) | 2011-09-22 |
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US8703123B2 (en) | 2014-04-22 |
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ZA201206208B (en) | 2013-05-29 |
IL221808B (en) | 2018-06-28 |
US20120328560A1 (en) | 2012-12-27 |
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