Yung-Chih Hu, Alan

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Engineering Conferences InternationalECI Digital ArchivesVaccine Technology IV ProceedingsSpring 5-24-2012Purification of cell-based influenza H5N1 virusesby liquid chromatography technologiesAlan Yung-Chih HuNational Institute of Infectious Diseases and Vaccinology (NIIDV), NHRIFollow this and additional works at: http://dc.engconfintl.org/vaccine_ivPart of the Biomedical Engineering and Bioengineering CommonsThis Conference Proceeding is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion inVaccine Technology IV by an authorized administrator of ECI Digital Archives. For more information, please contact franco@bepress.com.Recommended CitationAlan Yung-Chih Hu, "Purification of cell-based influenza H5N1 viruses by liquid chromatography technologies" in "VaccineTechnology IV", B. Buckland, University College London, UK; J. Aunins, Janis Biologics, LLC; P. Alves , ITQB/IBET; K. Jansen,Wyeth Vaccine Research Eds, ECI Symposium Series, (2013). http://dc.engconfintl.org/vaccine_iv/46Purification of Cell-Based Influenza H5N1 Viruses by Liquid Chromatography TechnologiesVaccine Technology IV, May 24th 2012 , Albufeira, Portugal National Health Research InstitutesAlan Yung-Chih Hu, Ph.D.Assistant InvestigatorNational Institute of Infectious Diseases and Vaccinology (NIIDV), NHRINational Health Research Institutes (NHRI)Since 19962National Institute of Infectious Diseases and Vaccinology, NHRI2Moved to this campus in 2004National Institute of Cancer Research Institute of Cellular and System Medicine Division of Aging-Related Musculoskeletal Diseases Division of Cardiovascular and Metabolism Medicine Division of Regenerative Medicine Institute of Population Sciences Center for Health Policy Research & Development Division of Biostatistics and Bioinformatics Research Programs3National Institute of Infectious Diseases and Vaccinology, NHRI3Division of Gerontology Research Division of Mental Health & Substance Abuse Research Institute of Biotechnology & Pharmaceutical Research National Institute of Infectious Diseases and Vaccinology Division of Environmental Health & Occupational Medicine Division of Medical Engineering Research Division of Molecular & Genomic Medicine Center for Nanomedicine Research Institution Objectives and manpowerImprove the health and well-being of TaiwaneseEnhance the quality of biomedical research and medical careDevelop medical and 4National Institute of Infectious Diseases and Vaccinology, NHRI4pharmaceutical technologyTrain and cultivate biomedical researchersPromote health policy research and implementation Total: over 1600 As of May 2012Platform TechnologyDevelopmentcGMP AreaNIIDV Organization & StructureEffective, May, 2011National Institute of Infectious Diseases and Vaccinology ( NIIDV )Vaccine  R&D CenterDivision of Infectious DiseasesPathogenicSurveillance Clinic Policy13+58 12+125 ( 91 )5National Institute of Infectious Diseases and Vaccinology, NHRI5BCG ProductionViral Vaccine ProductionAntivenins ProductionGenetic Engineering PlatformFormulation and Deliver PlatformAnimal model developmentBioprocess Development PlatformPurification/Bioanalytical PlatformQuality AssuranceRegulatory AffairsBacterial Vaccine ProductionRegulatory  & Quality Management Innovation  ManagementStrategic PlanningManagement  •Budget Control •Procurements •Project ManagementQuality ControlFacility EngineeringWarehouse and ShippingCentral ServiceProjectManagement ProductionManagementFacilityManagement•Tech Transfer •IP Management•Service ContractVaccinomics PlatformHigh-through put sequencing to identify novel and unknown pathogens and new strategyMolecular mechanisms of drug resistance, molecular pathogenesisViral pathogenesis (EV, Influenza, HIV)Viral tumorigenesis (HBV, HCV, EBV)TSAR/TSARYLocal important virus (influenza, EV, HIV)mechanismsepidemiology trials advocacyNIIDV programs• Antibiotic resistance, microbial genomics and infection control Advocacy• Tuberculosis research and novel BCG/ TB vaccine development• Emerging virus infections and vaccine 6National Institute of Infectious Diseases and Vaccinology, NHRIdevelopment• Tumor viruses and therapeutic development• Novel vaccine technology and bioproduct development6Target Status 2012 FundingCategory 1:Production contracts from Taiwan CDCBCG Vaccine Taking over Taiwan CDC orphan vaccine productionDOH/CDCAntivenin downstream productionCategory 2:Emergency manufactureFlu Vaccine Enhance national Bio-security and capability to produce vaccine under emergency and /or pandemic diseaseDOH Regular Funding/ IndustryEnterovirus : EV71 (B4/C4)Category 3:Developing  Meningoccoccal Group B VaccineComplete product development up to phase 2 clinical trial and then MOEA/ Industry/ DOHPipelines of NHRI Vaccine Center7National Institute of Infectious Diseases and Vaccinology, NHRI7vaccine with commercial valuetechnology and product transferMeningococcal group B using lipoprotein technology is going to enter into Phase I in Q4/2011HPV Therapeutic VaccineDengue VaccineNovel Adjuvant DevelopmentRSV/PIV3 Combined VaccineCategory 4:Novel vaccine and technology developmentRecombinant BCG/TB VaccineVaccine platform technology development and novel vaccine R&D to assist, reduce the cost and timelines and enhance the product pipelines in Taiwan vaccine industryBasic R&D by NSC/NHRI/PP projectUniversal pneumococcal VaccineBioprocess DevelopmentVaccine R&D : NHRI cGMP Pilot Plant8National Institute of Infectious Diseases and Vaccinology, NHRI8viralBacteriaBCGCentralservicePilot Plant layout9National Institute of Infectious Diseases and Vaccinology, NHRI9Filling&packingwarehouseP3 animal facilityBiologicals10National Institute of Infectious Diseases and Vaccinology, NHRI10Source from: N Engl J Med; pp. 2540~2543, 2008Our choicePandemic manufacturing strategy for H5N1 preparednessNHRI cell-base technology200,000 doses/ 3monthsFrontline workersAdimmune 11National Institute of Infectious Diseases and Vaccinology, NHRI11egg-base technology(30 million doses/year)The nation(23 million people)Adimmune / MedigenCell-ase technology(capacity ??)Roller-bottle process flowBatch0101421Cell Production Bank850 RB x 151700RB x 100 (20L)Virus InfectionHarvest and Clarification6375 T-flask x 5850 RB x 3 75 T-flask x 123Bioreactor scale-up12National Institute of Infectious Diseases and Vaccinology, NHRI12Batch2728355663Concentration & Host DNA RemovalPurificationDiafiltrationInactivationFormulationImmunizationQC assay242563 daysProcess Development in VRDC2.2L 7.5L 30 L 150 L13National Institute of Infectious Diseases and Vaccinology, NHRI13(a) Confluent microcarriers (b) Cytopathic effect   (c) Purfied Virus particle .(a) (b) (c)Various scale of microcarrier cell culture bioreactors in Vaccine center.Single-use bioreactorsWave bioreactorCesco 200LTideCell14National Institute of Infectious Diseases and Vaccinology, NHRI14Hyclone 50L/200L SUB Sartorius 70L Cultibag STRDownstream Purification using Continuous ultracentrifuge15National Institute of Infectious Diseases and Vaccinology, NHRI15BezonaseConcentrationBezonaseHA yieldresidual Proteinresidual DNADepth filtrationFlowchart of downstream purification schemeFilter selectioninactivationinactivation16National Institute of Infectious Diseases and Vaccinology, NHRIultracentrifugeSECSepharose 4FF DiafiltrationDiafiltrationSECSepharose 4FFAEXDiafiltration16AEXinactivationDepth filter selection17National Institute of Infectious Diseases and Vaccinology, NHRI17*The use of DE: diatomaceous earth (硅藻土) had significant effect on DNA  and protein removal(DE) (DE) (DE) (DE) (PP) (DE+PP)Depth filter test on large scale18National Institute of Infectious Diseases and Vaccinology, NHRI18DNA removal by different anion resins0100200300400500600050010001500200025000 2 4 6 8 10 12 14 16 18 ng/mlHA/50ulViralQHA dsDNA01002003004005006007000500100015002000250030003500400045000 2 4 6 8 10 12 14 16 18 ng/mlHA/50ulCVQXLHA dsDNA19National Institute of Infectious Diseases and Vaccinology, NHRI19CV01002003004005006007008000200400600800100012000 2 4 6 8 10 12 14 16 18 20 ng/mlHA/50ulCVPOROS HQ50 HA dsDNA0501001502002503003504000102030405060700 2 4 6 8 10 12 14 16 18 20 22 ng/mlHA/50ulCVPOROS HQ20 HA dsDNAOptimization of size exclusion Liquid chromatographyHA units (/50 µl)HA units (/50 µl)20National Institute of Infectious Diseases and Vaccinology, NHRI20HA units (/50 HA units (/50 µl)HA units (/50 HA units (/50 µl)1      2    3      4      5       6               1     2    3     4     5     62201601201009080706050401701258162534321National Institute of Infectious Diseases and Vaccinology, NHRI1: After Concentration2: After Gel filtration3: After Anion-exchange302520151032251714Loading: 8µl /well0.65 µm214.0 µm4: After Concentration5: After Gel filtration6: After Anion-exchangeProcess scale-up in downstream purification10002000300040005000600020040060080010001200HAunit/50ulUV280HA unitXK 16/40 UV280 Hiscale 50/40 UV 280XK 16/40 HA Hiscale 50/40 HA51205121024XK 16/40HiScale 50/4022National Institute of Infectious Diseases and Vaccinology, NHRI2210x scale-up-10000-20000 20 40 60 80 100min-2000020004000600080001000012000-2000200400600800100012000 20 40 60 80 100ng/mlUV280minds DNA concentrationXK 16/40 UV280 Hiscale 50/40 UV 280XK 16/40 dsDNA Hiscale 50/40 dsDNAComparison of purification using different schemesTC001-1 ultracentrifuge Bulk (0.65)TC001-1 NB Bulk (0.65)TC001-1 BBulk (0.65)TC001-2 NBbulk (4um)TC001-2 Bbulk (4um)SRD (ug/ml) 76.1 32.32 33.18 24.67 29.81BCA (µg/ml) NA 68.06 69.21 45.56 41.19$ 282 $ 26523National Institute of Infectious Diseases and Vaccinology, NHRIµg/dose NA 30.9 31.5 28.5 20.6residue DNA(ng/ml)8.334 10.926 14.006 3.094 2.6residue DNA (ng/dose)1.6 5.0 6.4 1.9 1.3Predicted Doses(based on 4L harvet)1015 1465 1504 1118 135123Before 0.2 µmTideCell + Plus_MDCKMC + OptiproDNA variation from different culturing system24National Institute of Infectious Diseases and Vaccinology, NHRI240 5000 10000 15000 20000 25000 30000 35000 40000Roller bottle + Plus_MDCKMC + Plus_MDCKng/mlNBS 5-33Gel filtration NBS 33 run of 5L SEC 5040001:10_UV1_280nm  NBS 33 run of 5L SEC 5040001:10_UV2_260nm  NBS 33 run of 5L SEC 5040001:10_Cond NBS 33 run of 5L SEC 5040001:10_Fractions  NBS 33 run of 5L SEC 5040001:10_Inject  NBS 33 run of 5L SEC 5040001:10_Protein (ug per ml) NBS 33 run of 5L SEC 5040001:10_dsDNA by Qbit (ng per ml)  NBS 33 run of 5L SEC 5040001:10_HA unit per 50ul   0200040006000Act  0 100 200 300 400 500 600 700 ml1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 WasteOpti-pro25National Institute of Infectious Diseases and Vaccinology, NHRISub 3 column test 13 SEC 5040001:10_UV1_280nm  column test 13 SEC 5040001:10_UV2_260nm  column test 13 SEC 5040001:10_Cond column test 13 SEC 5040001:10_Fractions  column test 13 SEC 5040001:10_HA unit per 50ul  column test 13 SEC 5040001:10_dsDNA by Q bit column test 13 SEC 5040001:10_Protein by coomassie plus   0200040006000Act  0 100 200 300 400 500 600 700 ml1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 WastePlus_MDCKSummary• The use of diatomaceous earth dad significant effect on DNA  and protein removal, but mechanism is not clear how DE absorbed the impurities• Low DNA content can be achieved by SEC and AEX columns, thus no addition of DNase is needed• The use of LC chromatography showed a similar results that 26National Institute of Infectious Diseases and Vaccinology, NHRIcompared to ultracentrifuge technology• Variations from Upstream harvest could affect heavily on downstream purification• New method need to be implemented for improving viral yield26Acknowledgements• Department of Health• National Science Council• Taiwan CDC27National Institute of Infectious Diseases and Vaccinology, NHRI27Bioprocess team• Center for Drug Evaluation• Science Advisory Board

Purification of cell-based influenza H5N1 viruses by liquid chromatography technologies

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Purification of cell-based influenza H5N1 viruses by liquid chromatography technologies

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