下游純化中實(shí)驗(yàn)設(shè)計(jì)doe原理與實(shí)踐

Modalitysales2下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）

Concentration

WashVolume(CV)FlowRate

WashVolume(CV)FlowRate

Bias–Toohigh/lowRobustnesswithinResinId

SampleViscosity Hold

ActiveMonitoringof

BufferandSaltResinStability

Robustnessover thReliabilityofMonitors,ValvesandPumps

pH,Cond,SampleFlow SamplePressureSample

pHandVolume FlowRate

Conductivity

BufferandSaltTypepH,Conductivity

BufferandSaltTypeResinStabililtyAdditives Gradient

BufferandSaltTypeResin

3下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）DoEvsOneFactoratATimevsOFAT單因子OFATOneFactorataTime單因子FactorFactorY=a*X1+b*X2+單單因子法暗示系統(tǒng)響應(yīng)是關(guān)鍵因數(shù)的一個線性組5下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）Example–OFAT工??可工??可能操作空間在5-15ms/cm,pH6-借鑒過去的經(jīng)??時間&溫度會影響產(chǎn)每次改變一個因Cond06789pH0什么區(qū)域被真正探索到Wherewouldyourun?在哪兒進(jìn)行操作?Cond什么區(qū)域被真正探索到

6下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）Example87

87

TheOFATapproach(OneFactorAta 無法探索整個實(shí)驗(yàn)區(qū) 存在局部次優(yōu)化和不愉快的意外風(fēng)險7下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）Example讓我們來看看pH值和電導(dǎo)對回收率的真實(shí)9876

單因子法的缺點(diǎn)開始設(shè)置不同， 果也有可能不

8下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）DesignofExperiments9下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）DoEFactorABFactorAB?????

Twofactorsattwo

Factor

下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）DoE-”Designof

TraditionalParameter1Fixed,Parameter2changedorParameter2Fixed,Parameter1changedParameterParameterParameterDoEarrangedapproach:VaryallsimultaneouslyParameterParameterParameter下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）DoEStructuredapproachDOE方Traditional 得到的結(jié)果，采DoEStructuredapproachDOE方Traditional 下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）Qualityby為什么FDA要推行下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）什么是質(zhì)量源于設(shè)計(jì)定義：以完善的科學(xué)和質(zhì)量風(fēng)險管理為基礎(chǔ)，按照ICHHarmonisedTripartiteGuideline,PharmaceuticalDevelopmentQ8(R1),FDAGuidanceforIndustry,Q8(R1)PharmaceuticalDevelopment,June要點(diǎn)采用系統(tǒng)性的方預(yù)先定義目強(qiáng)調(diào)對產(chǎn)品和工藝的理解，以及對工藝的控基于充分的科學(xué)依據(jù)和質(zhì)量風(fēng)險管下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）QbD過程:需求向下決定控制策

CriticalQualityAttributes(CQA)

CriticalProcessParameters(CPP)

Design

Control下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）QbD過程:能力向上決定產(chǎn)品質(zhì)

ProductCriticalQualityAttributes(CQA)CriticalProcessParameters(CPP)Design

Controlstrategy控制策下游純化的實(shí)驗(yàn)設(shè)計(jì)（DoE）形Conformation

活

均一

濃表面電Chargevariants電荷不均Deamidation脫酰大

純ProductrelatedPrAleakageCellculture

序N-Disulfidebonds二硫鍵下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）Tool#1失效模式Preliminary ysis(PHA)程度(RiskPriorityNumber[RPN])失效的嚴(yán)重度x發(fā)生可Tool#2風(fēng) riskranking影響性（2-20）包括有效性，PK/PD-）程度(RiskScore)=影響性x不確定性（2-下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）FMEASSeverity失效SSeverity失效的嚴(yán)重度(1-10)OOccurrence頻度(1-10)D-Detection探測度(1-10)RPN風(fēng)險順序數(shù)SxOxRPN是一項(xiàng)設(shè)計(jì)風(fēng)險的指標(biāo)。當(dāng)RPN較高會出錯的可能性(概率)是什結(jié)果(嚴(yán)重性)是什ImportantthatasmanyaspossibleImportantthatasmanyaspossibleareincludedfrommultiplefunctions:R&D,ProcessdevelopmentManufacturing,Sourcing,QA,QCetcSOD嚴(yán)重度頻度探測度9-現(xiàn)行系統(tǒng)幾乎不可能檢測到失7-嚴(yán)重失效，TI>=1440min或出現(xiàn)不能發(fā)現(xiàn)，沒有任何可以在該失效模式發(fā)生前被探測到（設(shè)備廠商可以檢測到5-480min≤TI<1440min或影響到設(shè)難于發(fā)現(xiàn)，失效模式或原因被發(fā)3-min≤TI<480min，產(chǎn)品須篩選和便于發(fā)現(xiàn)，失效的原因或模式是可探測到1-要影響，修復(fù)時間TI<60min操作者可探測7737CarryBelowofCIPsolutionDOofFailureSofItem/ProcessExample:FMEA分?jǐn)?shù)RPN>50wasfurtherexamined*inprocesscharacterization*CaptoTMElutionCaptoTMElutionconditionsProductIntegrityand

SampleconditioningIntermediatewash:pHConductivity&additivesElutionconditionsProductIntegrityand

Bufferexchangestep

Bulkproduct--Protein-ProductIntegrityand

step

pHadjustmentProductIntegrityandactivity

ElutionconditionsProductIntegrityand

Bufferexchange

ElutionconditionsProductIntegrityandCaptoTMadhere步驟的魚骨

Concentration

WashVolume

WashVolume(CV)FlowRate

Bias–Toohigh/lowRobustnesswithinResinId

SampleHold

ActiveMonitoringof

BufferandSaltResinStability

Robustnessover

thValvesandPumps

pH,Cond,SampleFlow

Viscosity/PressurepHandConductivity

SampleAmountBufferandSaltpH,Conductivity

FlowRate BufferandSaltResinAdditives Gradient

BufferandSaltTypeResinStabililtyRPN 模RiskRiskPriority0

分值最高的前九項(xiàng)FMEACIPACIPACIPARPNscoresafterfirstmitigationHighestscorereducedfrom126to36RiskPriorityHighestscorereducedfrom126to36RiskPriority0 質(zhì)量源于設(shè)計(jì)(QbD)的工藝空產(chǎn)品質(zhì)量目

單個關(guān)鍵參數(shù)Acceptablerange設(shè)計(jì)范CharacterizationAcceptablerange設(shè)計(jì)范OperatingOperatingrange多個關(guān)鍵參數(shù)Characterizedspace特征空間Designspace設(shè)計(jì)空間controlspace控制空下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）質(zhì)量源于設(shè)計(jì)(QbD)的工藝空Screening篩WhichfactorsareWhataretheappropriaterangesforthesevitalfactors?Optimization優(yōu) ficationofthevitalfactors.Whataretheoptimalrangesforthesefactors?Robustness穩(wěn)健

特征空設(shè)計(jì)空操作空variationsintheinputparameters.下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）DoE&QbDr-Pro-Insulin重組胰島素B- SA-C-Mw~11pI~需要用8M復(fù)雜樣品 例如預(yù)先分析純化 下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）創(chuàng)制一種新的思維我們已經(jīng)發(fā)現(xiàn)陸地 我們接近目標(biāo)，準(zhǔn)備著陸 安全降落 DBCandfine

Furtheroptimization&Scale-UpPreDictor?PlatesAssistSoftware

?KTA?avant

下游純化中的實(shí)驗(yàn)設(shè) 原理與實(shí)B- SA-C- 1--

11-

下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）PreDictor高通量篩選5.3–0–5.3–0–1503.4–0–300ScreeningDifferentmediainsame50μl用于洗脫研下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）在PreDictor板中的結(jié)合研 pH間隔 3.4-5陽離子交換（和CaptoNaCl濃度

5.3-8.1陰離子交換（和CaptoResult:Nobindingofr-Pro-InsulintoAIEXandCaptoResult:Nobindingofr-Pro-InsulintoAIEXandCapto0-150mM陰離子交下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）4 Saltconcentration4 Saltconcentration

4

Saltconcentration

4

250Saltconcentration

SPSepharose Capto CaptoConclusion:Conclusion:CaptoMMCat150mMsaltis結(jié)論：選擇起始條件為CaptoMMC鹽濃度 DoE）原理與實(shí)在CaptoMMC上pHpH4-Bindingcapacity[flowthrough;adjusted]HighestbindingHighestbinding~16mg/mlmediaat~5.2&0-150mMNaClassumingthatr-Pro-Insulinconcentrationis~30%oftotalproteinconc.7pH-pH-54

Saltconcentration(mM)-

結(jié)論在pH~5.2&0-150mMNaCl時具有最高的動態(tài)下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）CaptoMMCPreDictor板上的洗脫實(shí)填料體積：50pH間隔:3.7-NaCl:150-下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）Elutionofr-Pro-InsulinfromCapto重組胰島素原在CaptoMMC上的洗脫條Yield[elution]7pH-pH-

54 Saltconcentration(mM)-下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）ConclusionsfromPreDictor用PreDictor板研究的結(jié)Bindingcapacity[flowthrough;adjusted]7657654pH- Saltconcentration(mM)-Yield[elution]ElutionconditionstobefurtheroptimizedpH>NaCl>400

7pHpH-54

600800化中的100

E）原理與實(shí)Saltconcentration(mM)-

OptimizationinColumn層析柱進(jìn)行優(yōu)ElutionElution 洗脫研 穩(wěn)健下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）CaptoMMC utionofr-Pro-CaptoMMC梯度洗脫純化重組胰島素Loadedvolume:10mlcrudesample/mlBindingconditons:50mMNaAcetate+150mMNaClpH5.2in8Murea.Restime5minElution:150-1000mMNaClandpHgradient5.2-7.5,20CVgradient0 下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）在?KTAavant25系統(tǒng)上進(jìn)行重組胰島素原的洗脫優(yōu) Tricorn5/50-CaptoRes 5min(0.2 AdjustedtopH5.2withHAc.2.5C.V.crudesamplewasloaded 50mMNaacetate+150mMNaClpH5.2*Step StudypH6.2-8and150-750mMNaClAcetate/Phosphate/FormatebufferPro(CIEX0-NaClpH2-No.ofexperiments:11(3centerpoint *Buffersin8Murea（緩沖液含8M的尿素 下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）結(jié)果：不同的pH&NaCl濃度下重組胰島素原的收

0- 700mM

Coefficientplot系數(shù)分布 pHxpH pHxsaltR2= Q2= 下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE） Capto AdjustpHofcrudesampleto5.2Load2.5xColumnVolumesStepelution:50mMPhosphatebufferpH8.0150mMNaCl8M下游純化中的實(shí)驗(yàn)設(shè)計(jì)（DoE）穩(wěn)健性實(shí)驗(yàn)：在?KTAavant25系統(tǒng)上用CaptoMMC進(jìn)行重組 Coefficientplot Coefficientplot 洗脫pH7.8-樣品S1&ColumnColumnSampleSampleColumnColumnS