004 TOYOPEARL填料在血浆蛋白中的应用ppt课件.ppt

血液制品层析工艺中TOYOPEARL填料 东曹 TOSOH 公司生命科学部门 1 2 PlasmaProductsReviewApplicationonPlasmaProtein Content 3 PlasmaProteinsofCurrentInterest 4 PlasmaProductsinChina 5 Plasma precipitation supernatant Cryoprecipitate FactorVIII FractionI Centrifugation4oC Ethanoltreatment Centrifugation supernatant precipitation Fibrinogen FactorIXAbtithrombinIII CohnColdEthanolFractionationProcess Continued 6 precipitation supernatant FractionIV FractionV Ethanoltreatment Centrifugation supernatant precipitation HaptoglobinAbtithrombinIII Albumin FractionII III supernatant precipitation precipitation Ethanoltreatment Centrifugation FractionII Globulin forintramuscular Globulin forintravenous Ethanoltreatment Ethanoltreatment Centrifugation CohnColdEthanolFractionationProcess 7 ProductionCapacityofPlasmaProteins Capacity MillionL Y CompanieswithyellowbarhaveappliedToyopearltotheirprocess 8 CSLBioplasma Australia CommonwealthSerumLaboratory CSL InternationalpharmaceuticalcompanybasedinAustraliaasCSLgroupsAllprocesseswereappliedbychromatographyforCohnethanolfractionationexceptFraction1in1994Thefourthlargestsupplierofplasmaproteins 2002 GlobalcompanywithmanufacturingplantinUSandEuropeaswell ConsultanttoChineseplasmaproteinsuppliers 9 WhyCSLBioplasmaAppliesChromatography CSLBioplasmadescribeschromatographyas HighlycosteffectiveprocessHighproductionefficiency IVIG 5 5g L Highquality Albumin 99 w v Achievableofautomatic closed processEasyextractionandseparationofby products i e otherplasmaproteins Advancedtechniqueinbio industry 10 ProductionProcessforPlasmaProteins SizeExclusionChromatographyHW 55FIon ExchangeChromatographyDEAE 650M QAE 550C SuperQ 650S MSP 650M SP 550CHydrophobicInteractionChromatographyButyl 650M C Phenyl 650S MAffinityChromatographyAF Formyl650M AF HeparinHC650M 11 ApplicationofPlasmaProducts 12 PurificationofAlbumin 13 PurificationofAlbuminbyAIEC ConditionColumn ToyoScreenSuperQ 650M 1mL Eluent A 50mMTris HCl B 50mMTris HCl 0 5MNaClGradient A B Linear30minFlowrate 1mL minDetection UV 280nm Sample HumanAlbumin Globulin1g LInjection 100uL 14 PurificationofAlbuminbyCIEC ConditionColumn ToyoScreenSP 650M 1mL Eluent A 50mMAceticbuffer pH5 0 B 50mMAceticbuffer 0 5MNaCl pH5 0 Gradient A B Linear30minFlowrate 1mL minSample HumanAlbumin Globulin1g LInjection 100uL 15 Anionexchanger Cationexchanger Albumin Globulin FractionV Loading Elution Loading Elution ConsiderationofAlbuminPurification 16 Chromatographyinplasmafractionation benefitandfuturetrendsT Burnouf J Chromatgr B664 1995 3 15 TOYOPEARLDEAE 650MisusedtoproduceahighlypurifiedFVIIIconcentrate PurificationofFactorVIII 17 StartingfromCohnfractionation chromatographyonTOYOPEARL DEAE 650MresultsinahighlypurifiedclottingfactorfortreatmentofhemophiliaA MethodislicensedfromOctapharmaandincludestwovirusinactivationsteps solvent detergentandheating Scaleishundredsofmillionunitsperyear TOYOPEARL DEAE 650Mchromatographyistheresinofchoice PurificationofFactorVIII Octanate 18 19 ConditionColumnsize 6 0mmID 4cmL 1 13ml Mobilephase Loading 0 4mg mlAT IIIin0 01MTris HClcontaining0 15MNaClpH7 5LinearVelocity 300cm minDetection UV 280nm ResinAT IIIbindingcapacity 10 leakage mg ml gel NewHeparin 650M3 9HeparinSepharose6FF2 4Heparin 650M1 9 Figure1Comparisonofbreakthroughcurveat300cm h ComparisonofdynamicAT IIIbindingcapacityat300cm h PurificationofAT III 20 Conditioncolumnsize 6 0mmID 4cmL 1 13ml Mobilephase Loading 0 4mg mlAT IIIin0 01MTris HClcontaining0 15MNaClpH7 5Detection UV 280nm Comparisonofdynamicbindingcapacity PurificationofAT III 21 AT III PurificationofAT IIIfromhumanplasmaonHeparin Toyopearl650MColumnsize 6 0mmI D X4 0cmSample Humanplasma18mlBuffers