JP7323512B2 - ラマン分光法を使用する細胞培養の自動制御 - Google Patents
ラマン分光法を使用する細胞培養の自動制御 Download PDFInfo
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Description
[0001]本出願は、いずれもその全体が参照によって本明細書に組み込まれる、2017年10月06日を出願日とする米国仮特許出願第62/569,076号および2017年10月06日を出願日とする米国仮特許出願第62/569,190号に基づき、その優先権を主張する。
[0022]本開示の完全かつ実施可能な開示は、添付の図面の参照を含む本明細書の残りの部分でより詳細に記載される。
[0026]本記述は例示的な実施形態の説明に過ぎず、本開示のより広い態様を限定するものとして意図されていないということは、当業者によって理解されるべきである。
[0072]異なるモノクローナル抗体(mAb)を産生する3種の異なるCHOK1SV GS-KO(商標)細胞株を本研究に使用した。2種の細胞株を較正モデル開発のために使用し、残りの細胞株をモデル認定のために使用した。全ての細胞株をプラットフォーム培地およびフィードで15日の期間にわたり培養した。較正モデル用の全ての細胞培養を、5リットルの作業容量を有する4つの撹拌槽型リアクター(STR)で実施した。各較正用細胞培養に対し、約3g/Lの標的残留グルコース濃度で作動する2つの制御を設けた。グルコースの操作範囲を拡大するために、2つのSTRを約1g/Lの追加の初期グルコースで操作し、1g/Lの標的残留グルコース濃度に維持した。さらに、5リットルの作業容量を有する4つのSTRにおける1ラウンドの異常な細胞培養からのデータを含めて、プラットフォームプロセスに関して予期された範囲から大きく外れた代謝産物および細胞濃度を調査した。モデル認定に関しては、5リットルの作業容量を有する2つのSTRを操作する一方で、10リットルの作業容量を有する1つのSTRを操作してモデル拡張性を評価した。
[0074]培養の間、オフライン試料(約20mL)を、代謝産物および細胞増殖の分析のために各培養物から1日2回収集した。グルコース、乳酸、グルタミン酸、およびアンモニウムのオフライン分析を、NOVA Bioprofile400(NOVA Biomedical)を使用して実施した。VCC(生存細胞濃度)およびTCC(総細胞濃度)のオフライン分析を、Vi-Cell XR(Beckman Coulter)を使用して実施した。培養4日目以降、1日2回、アリコート(約2mL)を産物濃度分析のために細胞培養上清から採取した。産物濃度を、プロテインA HPLCを介して分析した。
[0078]5リットルSTRにおいてプラットフォームプロセスを利用する12の細胞培養運転からのオフラインデータをそれぞれのラマンスペクトルと組み合わせて使用して、グルコース、グルタミン酸、乳酸、アンモニウム、生存細胞濃度(VCC)、総細胞濃度(TCC)、および産物濃度の較正モデルを生成した(表1)。全般に、モデルは全てのパラメータに関して低いRMSEE/RMSECV値とR2Y>0.90とを有した。さらに、アンモニウムを除く全てのモデルは、比較的少ない数の潜在変数(LV)を有した。アンモニウムモデルのLVの数が多いことは、このパラメータのモデルが過剰適合していることを指示し得る(R2cv=0.89)。モデルを構築する際は、プラットフォームプロセスの通常の操作条件から外れた範囲を包含するオフラインデータおよびスペクトルを収集した。これは、モデルが外挿することを要求される状況を引き起こすのを避けるために行われた。それぞれのオフライン参照方法と比較した、予測されたグルコース、グルタミン酸、乳酸、アンモニウム、VCC、TCC、および産物濃度の較正モデルのプロットは、全般に全てのモデルが測定した値と良好に相関することを指示する(図3)。
[0080]モデルを、較正モデルに含まれていない細胞株を使用して認定した。全ての認定用運転を、プラットフォームプロセスの通常の操作条件下で実施した。開発したモデルの潜在的な拡張性を調査するため、10リットル培養も実施した。3つ全ての認定用運転に関する、オフライン参照方法と比較した、結果として得た予測のプロファイルを図4に示す。予測された値のエラーバーは、各培養運転の±RMSEPである。オフライン参照方法のエラーバーは、販売業者によって明記された±精度である。全てのモデルは、比較的低いRMSEP値で所望のパラメータの変化をモニタリングすることができた(表2)。
[0090]本開示のシステムおよび方法に関するさらなる情報および例示も付加する。
[0092]ヒト間葉系幹細胞(MSC)を、マイクロキャリアを含む撹拌槽型バイオリアクターで培養した。培養期間は総計8日であった。最初の4日間は、細胞を2g/Lのグルコース濃度のMSC培地でインキュベートした。培養4日目から8日目までは、連続灌流を開始し、古い培地を4g/Lのグルコース濃度の新鮮なMSC培地と交換した。これらの8日の過程にわたり、試料をバイオリアクターから定期的に抜き出し、グルコース、乳酸、およびアンモニウム濃度を、新鮮な試料から、Nova BioProfile Flex測定器を使用して測定した。インラインラマンスペクトルを、特定のスペクトル範囲を走査する785nmのレーザーを備えたKaiser Optical System,Inc.製のRamanRXN2(商標)システムを使用して収集し、420~2465cm-1をモデル化に使用した。
[0094]3.5Lの培養物を含有する4つの5Lバイオリアクターを、モデル較正のために運転した。ラマン測定器は10秒の露光時間を使用し、各測定に関して75回の積算で収集した。測定を30分ごとに行った。総計47(N=47)の試料を4つの運転からNovaで測定した。
[0100]2~3Lの範囲の作業容量を有する7つの3Lバイオリアクターをモデル認定のために運転した。各バイオリアクター運転から平均7個の試料、総計50個の試料を収集した。少数の試料は乳酸およびアンモニウムの参照値が欠損していたが、これは、それらの特定の試料に関するNovaの技術的な問題、および一部の0の値によるものであった。認定用運転のためのMSCプロセスは大部分が較正に使用したプロセスと同一であったが、数少ない小さな例外を有した:異なるロットの培地を使用した、認定用運転はゲンタマイシンを使用して実施した、および認定用運転はプロセスの一部の間消泡剤の添加を含んだ。ゲンタマイシンの存在によって引き起こされたラマンスペクトルの差が存在し、これを計量化学によって補正した。
[0103]1つの運転を実施して、自動ラマン駆動制御を試験した。測定の頻度を増加させ、結果として10分ごとの測定とした。この運転を実行する前に、ラマンを介して測定される分子のいずれかに関して設定点を定義することができるように、ラマンシステムをバイオリアクターコントローラにつないだ。
[0114]一実施形態では、細胞としてT細胞または免疫細胞を挙げてもよい。例えば、細胞として、B細胞、ナチュラルキラー細胞、樹状細胞、腫瘍浸潤リンパ球、単球、または巨核球等を挙げることができる。
[0116]複数の実施形態では、細胞は肝細胞、例えばヒト肝細胞、動物肝細胞、または非実質細胞である。例えば、細胞は、接着型代謝試験用ヒト肝細胞、接着型誘導試験用ヒト肝細胞、接着型Qualyst Transporter Certified(商標)ヒト肝細胞、浮遊型試験用ヒト肝細胞(10名のドナーおよび20名のドナーのプールされた肝細胞を含む)、ヒト肝臓クッパー細胞、ヒト肝星細胞、イヌ肝細胞(単一のおよびプールされたビーグル肝細胞を含む)、マウス肝細胞(CD-1およびC57Bl/6肝細胞を含む)、ラット肝細胞(Sprague-Dawley、Wistar Han、およびWistar肝細胞を含む)、サル肝細胞(カニクイザルまたはアカゲザル肝細胞を含む)、ネコ肝細胞(ドメスティックショートヘア肝細胞を含む)、ならびにウサギ肝細胞(ニュージーランドホワイト肝細胞を含む)であり得る。肝細胞の例は、Triangle Research Labs,LLC、6 Davis Drive Research Triangle Park、North Carolina、USA 27709から市販されている。
[0121]複数の実施形態では、原核細胞はグラム陽性細胞、例えばBacillus、Streptomyces、Streptococcus、Staphylococcus、またはLactobacillusである。使用することができるBacillusは、例えば枯草菌(B.subtilis)、B.amyloliquefaciens、B.licheniformis、B.natto、またはB.megateriumである。複数の実施形態では、細胞は枯草菌、例えば枯草菌3NAおよび枯草菌168株である。Bacillusは、例えばBacillus Genetic Stock Center、Biological Sciences 556、484 West 12th Avenue、Columbus OH 43210-1214から取得可能である。
イ)、FX-06、AP-214、WAP-8294A(注射用)、ACP-HIP、SUN-11031、ペプチドYY[3-36](肥満、鼻腔内)、FGLL、アタシセプト、BR3-Fc、BN-003、BA-058、ヒト副甲状腺ホルモン1-34(点鼻、骨粗鬆症)、F-18-CCR1、AT-1100(セリアック病/糖尿病)、JPD-003、PTH(7-34)リポソームクリーム(Novasome)、デュラマイシン(点眼、ドライアイ)、CAB-2、CTCE-0214、糖PEG化エリスロポエチン、EPO-Fc、CNTO-528、AMG-114、JR-013、第XIII因子、アミノカンジン、PN-951、716155、SUN-E7001、TH-0318、BAY-73-7977、テベレリクス(即放性)、EP-51216、hGH(放出制御型、Biosphere)、OGP-I、シフビルチド、TV4710、ALG-889、Org-41259、rhCC10、F-991、チモペンチン(肺疾患)、r(m)CRP、肝選択的インスリン、スバリン、L19-IL-2融合タンパク質、エラフィン、NMK-150、ALTU-139、EN-122004、rhTPO、トロンボポエチン受容体アゴニスト(血小板減少性障害)、AL-108、AL-208、神経成長因子アンタゴニスト(疼痛)、SLV-317、CGX-1007、INNO-105、経口テリパラチド(eligen)、GEM-OS1、AC-162352、PRX-302、LFn-p24融合ワクチン(Therapore)、EP-1043、小児用肺炎球菌ワクチン、マラリアワクチン、B群髄膜炎菌ワクチン、新生児用B群レンサ球菌ワクチン、炭疽ワクチン、HCVワクチン(gpE1+gpE2+MF-59)、中耳炎療法、HCVワクチン(コア抗原+ISCOMATRIX)、hPTH(1-34)(経皮、ViaDerm)、768974、SYN-101、PGN-0052、アビスクミン(aviscumnine)、BIM-23190、結核ワクチン、マルチエピトープチロシナーゼペプチド、癌ワクチン、エンカスチム、APC-8024、GI-5005、ACC-001、TTS-CD3、血管標的TNF(固形腫瘍)、デスモプレシン(頬側放出制御型)、オネルセプト、およびTP-9201である。
Claims (15)
- 細胞培養物を増殖させるための方法であって、
バイオリアクター(10)中の細胞培養物をコヒーレント光源(52)に曝露して光を散乱させるステップ、
ラマン分光器(54)を使用して散乱光の強度を測定するステップ、
測定した散乱光強度に基づいて、コントローラ(60)を使用して細胞培養物における物質の濃度をパラメータとして決定し、決定されたパラメータの濃度に基づいて、コントローラ(60)が、バイオリアクター(10)へのパラメータ影響物質の流量(36、38、40)を選択的に増加または減少させて、パラメータを予め設定した限度内に維持するステップ
を含み、
該細胞培養物は、流体が連続的に除去および補充される灌流法で操作され、
該細胞培養物は、該パラメータの濃度を少なくとも4時間ごとに1回決定するためにコヒーレント光源に曝露され、そして
該コントローラが該バイオリアクターへのパラメータ影響物質の流量を選択的に増加または減少させるのに先立って、該パラメータの濃度が、最初の1から6日にわたって決定される、
方法。 - パラメータの濃度が、光強度データを参照データと比較することによって決定される、請求項1に記載の方法。
- コントローラ(60)が、決定されたパラメータの濃度に基づいてパラメータのその後の濃度を外挿する予測モデルを含み、算出したその後の濃度に基づいて、バイオリアクター(10)へのパラメータ影響物質の流量(36、38、40)を選択的に増加または減少させて、パラメータを予め設定した限度内に維持する、請求項1または2に記載の方法。
- パラメータが、グルコース、乳酸、グルタミン酸、アンモニウム、生存細胞濃度、総細胞濃度、または産物濃度を含む、請求項1から3のいずれか一項に記載の方法。
- 少なくとも2つの異なるパラメータの濃度が、測定した散乱光強度に基づいてコントローラ(60)によって細胞培養物において決定され、コントローラが、バイオリアクターへの1種または複数種のパラメータ影響物質の流量(36、38、40)を選択的に増加または減少させて、少なくとも2つのパラメータを予め設定した限度内に維持する、請求項1から4のいずれか一項に記載の方法。
- 少なくとも2つの異なるパラメータが、グルコース、乳酸、グルタミン酸、アンモニウム、生存細胞濃度、総細胞濃度、または産物濃度を含む群から選択される2つのパラメータを含む、請求項5に記載の方法。
- パラメータ影響物質が栄養培地を含む、請求項1から6のいずれか一項に記載の方法。
- 細胞培養物が哺乳動物細胞を含有する、請求項1から7のいずれか一項に記載の方法。
- 細胞培養物が幹細胞を含有する、請求項1から7のいずれか一項に記載の方法。
- 測定した散乱光強度の統計分析を実行するステップをさらに含み、統計分析が、標準正規変量を適用するステップ、一次微分を適用するステップ、およびパラメータと相関するスペクトル範囲を選択するステップを含み、標準正規変量を適用するステップの後にトレンド除去を行うステップを含む、請求項1から9のいずれか一項に記載の方法。
- 細胞培養物が、約6日間から約30日間増殖され、次いで回収される、請求項1から10のいずれか一項に記載の方法。
- パラメータの濃度が約12時間から約4日間で決定され、その後、コントローラ(60)がパラメータのその後の濃度を外挿する、請求項3に記載の方法。
- 細胞培養物が、コヒーレント光源(52)にインラインまたはアットラインで曝露される、請求項1から12のいずれか一項に記載の方法。
- コントローラ(60)が、パラメータ影響物質の流量(36)を選択的に増加または減少させるための制御モデルを用いてプログラムされ、制御モジュールが、異なる種類の細胞を含有する異なる細胞培養物内のパラメータを予め設定した限度内に維持することができる、請求項1から13のいずれか一項に記載の方法。
- 予測モデルが、参照試験中に少なくとも1つのパラメータの相関をなくすことによって創出される、請求項3に記載の方法。
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EP3692358A1 (en) | 2020-08-12 |
JP2020537126A (ja) | 2020-12-17 |
IL273549B2 (en) | 2023-10-01 |
KR20200068697A (ko) | 2020-06-15 |
US11609120B2 (en) | 2023-03-21 |
IL273549A (en) | 2020-05-31 |
US20190137338A1 (en) | 2019-05-09 |
US20230204421A1 (en) | 2023-06-29 |
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