JP7483679B2 - 改善されたタンパク質の回収 - Google Patents
改善されたタンパク質の回収 Download PDFInfo
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- JP7483679B2 JP7483679B2 JP2021507639A JP2021507639A JP7483679B2 JP 7483679 B2 JP7483679 B2 JP 7483679B2 JP 2021507639 A JP2021507639 A JP 2021507639A JP 2021507639 A JP2021507639 A JP 2021507639A JP 7483679 B2 JP7483679 B2 JP 7483679B2
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- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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Description
本願は、2018年8月14日に出願された米国仮出願第62/718,864号の優先権の利益を主張し、これは参照によりその全体が本明細書に組み込まれる。
本開示がより容易に理解できるように、特定の用語を最初に定義する。本願において使用される場合、本明細書で他に明示的に与えられている場合を除き、以下の各用語は以下に記載される意味を有するものとする。追加の定義が本願のあらゆる箇所に記載されている。
本開示は、濾過後の回収洗浄中において標的タンパク質の希釈を低減または防止する方法に関する。本開示のある態様は、(i)濾過が行われている間、標的タンパク質を含む試料を濾過アセンブリにフィード流速で通すこと、および(ii)回収洗浄中に緩衝液を濾過アセンブリに300リットル/平方メートル/時間(LMH)未満の洗浄流速で通すことにより、300LMHの洗浄流速で得られた標的タンパク質の希釈と比較して、標的タンパク質の希釈を低減または防止することに関する。別の態様において、本開示は、濾過後の回収洗浄中において標的タンパク質の濃度を改善または増加させる方法に関する。本開示のある態様は、(i)濾過が行われている間、標的タンパク質を含む試料を濾過アセンブリにフィード流速で通すこと、および(ii)回収洗浄中に洗浄緩衝液を濾過アセンブリに300リットル/平方メートル/時間(LMH)未満の洗浄流速で通すことにより、300LMHの洗浄流速で得られた標的タンパク質濃度と比較して、標的タンパク質濃度を改善することに関する。いくつかの実施態様において、標的タンパク質の所望の濃度は、約100mg/mL、約110mg/mL、約120mg/mL、約130mg/mL、約140mg/mL、約150mg/mL、約160mg/mL、約170mg/mL、約180mg/mL、約190mg/mL、または約200mg/mLよりも高い。
スケールダウンした設計の評価
潜在的な回収洗浄プロセスを評価するために,チューブサイズの変化を分析するスケールダウンしたモデルを設計した。このスケールダウンしたモデルは176cm2の膜面積を使用し、最初にサイズ16のチューブを用いて試験した。モデル化に使用した様々なチューブを表1に記載する。250g/Lの開始タンパク質濃度を使用し、様々な洗浄フィード流速を試験した。このデータを図1に示す。より高い洗浄フィード流速はより低い最終的なタンパク質濃度をもたらすため、図1に見られるようにより低い洗浄流速が望ましい。
表1.スケールに応じたチューブ
様々なモノクローナル抗体を用いた洗浄戦略の評価
3つのモノクローナル抗体(A、BおよびC)を設計された洗浄プロセスを用いて評価した。抗体Aの洗浄プロセスから得られたデータを表2に詳述する。これらのデータは、洗浄の流れ(LMH)、レイノルズ数(Re)、残余分の濃度、精製された原薬の濃度、収率、予想される収率、および洗浄量を含んでいる。スケールダウンした洗浄プロセスを評価した後、この手法をCM150タンジェンシャルフロー濾過(TFF)スケールでモノクローナル抗体Aについて検証した。すべての例において、流速を30リットル/M2/時間の流れに設定した。CM150システムで使用したチューブの断面積を考慮すると、これはCM150システムにおいて265のレイノルズ数に対応する。モノクローナル抗体BおよびCについて、追加のスケールダウンおよびCM150 TFFのデータを作成し、これらのデータをそれぞれ表2および表3に示した。
表2.モノクローナル抗体Aのデータ
Claims (24)
- (i)濾過が行われている間、標的タンパク質を含む試料を濾過アセンブリにフィード流速で通すこと、および(ii)回収洗浄中に緩衝液を濾過アセンブリに10~40リットル/平方メートル/時間(LMH)の洗浄流速で通すことを含む、濾過後の回収洗浄中において標的タンパク質の希釈を低減または防止する方法であって、ここで、回収洗浄における流動のレイノルズ数(「Re」)は1~50であり、Reは以下の式:
Re=Dυρ/μ
によって計算され、ここでDは流路の直径(m)または非円筒形流路形状の場合には相当直径であり、υは平均速度(m/s)(Q/Ac)であり、ρは密度(kg/m 3 )であり、μは粘度(Pa・s)である、方法。 - (i)濾過が行われている間、標的タンパク質を含む試料を濾過アセンブリにフィード流速で通すこと、および(ii)回収洗浄中に洗浄緩衝液を濾過アセンブリに10~40リットル/平方メートル/時間(LMH)の洗浄流速で通すことを含む、濾過後の回収洗浄中において標的タンパク質の濃度を増加させる方法であって、ここで、回収洗浄における流動のレイノルズ数(「Re」)は1~50であり、Reは以下の式:
Re=Dυρ/μ
によって計算され、ここでDは流路の直径(m)または非円筒形流路形状の場合には相当直径であり、υは平均速度(m/s)(Q/Ac)であり、ρは密度(kg/m 3 )であり、μは粘度(Pa・s)である、方法。 - 回収洗浄における流動のレイノルズ数(「Re」)が、
(i)
1~20である、請求項1または2に記載の方法。 - 回収洗浄における流動のレイノルズ数(「Re」)が、2~6である、請求項1または2に記載の方法。
- 回収洗浄における流動のレイノルズ数(「Re」)が、
1、2、3、3.8、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、または30である、
請求項1または2に記載の方法。 - 洗浄流速が、
(i)10LMH~30LMH、20LMH~40LMH、30LMH~40LMH、または20LMH~30LMHである;または
(ii)30LMHである、
請求項1~5のいずれか一項記載の方法。 - 回収洗浄後の標的タンパク質の濃度が、
(i)濾過の前に少なくとも100g/L、少なくとも110g/L、少なくとも120g/L、少なくとも130g/L、少なくとも140g/L、少なくとも150g/L、少なくとも160g/L、少なくとも170g/L、少なくとも180g/L、少なくとも190g/L、少なくとも200g/L、少なくとも210g/L、少なくとも220g/L、少なくとも230g/L、少なくとも240g/L、少なくとも250g/L、少なくとも260g/L、少なくとも270g/L、少なくとも280g/L、少なくとも290g/L、または少なくとも300g/Lである;または
(ii)100g/L~300g/L、110g/L~250g/L、120g/L~240g/L、150g/L~250g/L、130g/L~260g/L、140g/L~260g/L、160g/L~220g/L、または170g/L~230g/Lである;または、90g/L、100g/L、110g/L、120g/L、130g/L、140g/L、150g/L、160g/L、170g/L、180g/L、190g/L、200g/L、210g/L、220g/L、230g/L、240g/L、250g/L、260g/L、270g/L、280g/L、290g/L、または300g/Lである、
請求項1~6のいずれかに記載の方法。 - 回収洗浄後の標的タンパク質の収量が、(i)および(ii)からなる回収洗浄プロセスの前の標的タンパク質の収量と比較して少なくとも90%、少なくとも91%、少なくとも92%、少なくとも93%、少なくとも94%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、少なくとも99%、または少なくとも100%である;または
回収洗浄後の標的タンパク質の収量が、300LMHの流速による回収洗浄後に得られた標的タンパク質の収量と比較して少なくとも1%、少なくとも2%、少なくとも3%、少なくとも5%、少なくとも10%、少なくとも15%、または少なくとも20%増加している、請求項1~7のいずれかに記載の方法。 - フィード流速が、
(i)少なくとも200LMH、少なくとも210LMH、少なくとも220LMH、少なくとも230LMH、少なくとも240LMH、少なくとも250LMH、少なくとも260LMH、少なくとも270LMH、少なくとも280LMH、少なくとも290LMH、少なくとも300LMH、少なくとも310LMH、少なくとも320LMH、少なくとも330LMH、少なくとも340LMH、少なくとも350LMH、少なくとも360LMH、少なくとも370LMH、少なくとも380LMH、少なくとも390LMH、少なくとも400LMH、少なくとも410LMH、少なくとも420LMH、少なくとも430LMH、少なくとも440LMH、少なくとも450LMH、少なくとも460LMH、少なくとも470LMH、少なくとも480LMH、少なくとも490LMH、または少なくとも500LMHである;
(ii)200LMH~500LMH、200LMH~450LMH、250LMH~450LMH、450LMH、300LMH~450LMH、300LMH~400LMH、または300LMH~350LMHである;または
(iii)300LMHである、
請求項1~8のいずれかに記載の方法。 - 濾過緩衝液が方法工程(i)中において使用され、ここで濾過緩衝液が洗浄緩衝液と同じであるかまたはここで濾過緩衝液が洗浄緩衝液と異なっている、請求項1~9のいずれかに記載の方法。
- 洗浄緩衝液および/または濾過緩衝液が、
(i)有機酸または無機酸またはそれらの塩を含む;および/または
(ii)糖を含む、
請求項1~10のいずれかに記載の方法。 - (i)有機酸または無機酸は、クエン酸塩(例えば、クエン酸一ナトリウム-クエン酸二ナトリウム混合物、クエン酸-クエン酸三ナトリウム混合物、クエン酸-クエン酸一ナトリウム混合物など)、コハク酸塩(例えば、コハク酸-コハク酸一ナトリウム混合物、コハク酸-水酸化ナトリウム混合物、コハク酸-コハク酸二ナトリウム混合物など)、酒石酸塩(例えば、酒石酸-酒石酸ナトリウム混合物、酒石酸-酒石酸カリウム混合物、酒石酸-水酸化ナトリウム混合物など)、フマル酸塩(例えば、フマル酸-フマル酸一ナトリウム混合物、フマル酸-フマル酸二ナトリウム混合物、フマル酸一ナトリウム-フマル酸二ナトリウム混合物など)、グルコン酸塩(例えば、グルコン酸-グルコン酸ナトリウム混合物、グルコン酸-水酸化ナトリウム混合物、グルコン酸-グルコン酸カリウム混合物など)、シュウ酸塩(例えば、シュウ酸-シュウ酸ナトリウム混合物、シュウ酸-水酸化ナトリウム混合物、シュウ酸-シュウ酸カリウム混合物など)、乳酸塩(例えば、乳酸-乳酸ナトリウム混合物、乳酸-水酸化ナトリウム混合物、乳酸-乳酸カリウム混合物など)、酢酸塩(例えば、酢酸-酢酸ナトリウム混合物、酢酸-水酸化ナトリウム混合物など)、トリメチルアミン塩(例えば、Tris)、リン酸塩、またはヒスチジンである;および/または
(ii)糖は、スクロース、トレハロース、マンニトール、キシリトール、エリスリトール、ラクトース、グルコース、粉糖、またはプルランである、
請求項11に記載の方法。 - 洗浄緩衝液が20mMヒスチジンおよび250mMスクロースを含む、請求項1~12のいずれかに記載の方法。
- 洗浄緩衝液および/または濾過緩衝液が、4、4.5、5、5.5、6、6.5、7、7.5、8、8.5、9、または9.5のpHである、請求項1~13のいずれかに記載の方法。
- 試料が、純粋なタンパク質試料、清澄化されたバルクタンパク質試料、細胞培養物試料、およびそれらの任意の組合せからなる群から選択される、請求項1~14のいずれかに記載の方法。
- 細胞培養物試料が、哺乳類細胞を含む細胞培地から得られる、請求項15に記載の方法。
- 哺乳類細胞が、チャイニーズハムスター卵巣(CHO)細胞、HEK293細胞、マウス骨髄腫(NS0)、ベビーハムスター腎臓細胞(BHK)、サル腎臓線維芽細胞(COS-7)、メイディンダービーウシ腎臓細胞(MDBK)、またはそれらの任意の組合せから選択される、請求項16に記載の方法。
- 標的タンパク質が抗体または融合タンパク質;および/または酵素、ホルモン、サイトカイン、細胞表面受容体、プロテアーゼ、サイトカイン受容体、またはそれらの任意の組合せを含む、請求項1~17のいずれかに記載の方法。
- 標的タンパク質が抗体を含み、およびここで抗体が、
(i)IgM、IgA、IgE、IgD、およびIgGから選択されるアイソタイプである;および/または
(ii)抗GITR抗体、抗CXCR4抗体、抗CD73抗体、抗TIGIT抗体、抗OX40抗体、抗LAG3抗体、抗CSF1R抗体、または抗IL8抗体である、
請求項18記載の方法。 - IgG抗体は、IgG1、IgG2、IgG3、およびIgG4から選択される、請求項19に記載の方法。
- 標的タンパク質が融合タンパク質である、請求項18に記載の方法。
- 融合タンパク質が異種部分と融合している、請求項21に記載の方法。
- 異種部分が半減期延長部分である、請求項22に記載の方法。
- 半減期延長部分がFcを含む、請求項23に記載の方法。
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