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Viral-vectored vaccines: a new approach in the vaccine manufacturing process


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Presentation in Vaccine World Summit, Pune, India. 3-7 March 2013.

Presentation in Vaccine World Summit, Pune, India. 3-7 March 2013.

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  • 1. Viral-vectored vaccines: a new approach inthe vaccine manufacturing processDr. Priyabrata PattnaikDirector – Asia Vaccine Initiative
  • 2. Agenda 1 Vaccine Industry Trends 2 Market Trend of Viral based Biologics 3 Recombinant Virus as Vaccine Vectors 4 Growth of R&D on Adenovirus Vaccine 5 Evaluation and feasibility study for Adenovirus vaccine 6 Case study: Ark Therapeutic’s ATOSUS 7 Process Development Concept and Scale-up of Adenovirus Process 8 Conclusion and Acknowledgement
  • 3. Vaccine Industry Trends Vaccines segment is growing at 8%. ~ 400 vaccines are in commercial manufacture. 300 are viral based. ~1400 vaccines are in development ~ Half of the vaccines undergoing clinical trials are viral based.  ~ 640 viral vaccines  ~ 200 viral vectors  ~ 60 vaccine like particles Another ~240 gene therapy products in development that utilize the same technology.3
  • 4. Market Trend of Viral based Biologics Biologics in Development Vaccines Recombinant Mabs Other 25% Vaccine Clinical Trials Viral Vaccine Trials Vaccine Trials 600 500 400 300 200 100 0 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 YTD 4 Source: Expression Systems Analysis, G Adams 2011,
  • 5. Recombinant Viruses as Vaccine Vectors Adenoviruses  Non-enveloped virus, 70-100 nm in diameter  Genome: linear double-stranded DNA  Efficiently transduces non-active and dividing cells  Large (>30 kb) transgene capacity  Easily produced in high titers  CAR receptor dependent transduction  Immunogenic  Existing humoral response to certain serotypes5
  • 6. Adenovirus ControversyMerck & Co scraps Adenovirus based AIDS vaccine trial Adenovirus vector triggered immune response Body flooded with CD4 cells CD4 is also target for HIV Since then……  Mol. Biol. of adenovirus improved  Adenovirus engineered to increase target specificity  Adenovirus induced immunology is better understood6
  • 7. Growth of R&D on Adenovirus Vaccine100000 20000 15000 50000 10000 5000 0 Vaccine Vectored Adenovirus 0 Vaccine Vaccine Number of citation in Google Scholar in 2012 Citation on Adenovirus vaccine in Google Scholar 7
  • 8. Oncolytic Vaccine Vector8 Source: J.-W. Choi et al. / Advanced Drug Delivery Reviews 64 (2012) 720–729
  • 9. 9
  • 10. Generic Process of Vectored Vaccine Manufacturing10
  • 11. Cell Culture for Adenovirus Typical cell culture: HEK293 Cell culture either in continuous or batch mode (w/ media exchange) Accumulating lactic acid is detrimental to virus production Virus yield drops significantly when the media pH < 7 Typical cell concentration during virus infection is 2 - 10 x106 cells/ml Adenovirus titre during harvest generally ranges from 109 to 1011 pfu/ml.11 Cell culture growth plot, Courtesy: Dr. David Venables, Ark Therapeutics
  • 12. Fast-Trap™ Lentivirus and AdenovirusPurification & Concentration Kit Kit contains necessary reagent Membrane-based, closed vacuum-driven device High recoveries of purified viable viral particles in under two hours Source: Merck Millipore Literature No. AN1066EN00 Rev. B
  • 13. Adenovirus work before the controversy Collaboration with GenVec Inc., Gaithersburg, MD USA. Filter sizing for medium exchange, lysate clarification, post- clarification filtration, Ultrafiltration/ diafiltration, and post-hold sterile filtration prior to column chromatography13
  • 14. Millistak+® Pod filter for lysate clarification  Millistak+® filter C0HC and DE30 provided good capacity (>150L/m2)  Millistak+® filterC0HC and DE30 provided desired turbidity reduction  Millistak+® filter B1HC filter plugged prematurely  Millistak+® filter CE30 filter led to turbidity breakthrough14 Work in collaboration with GenVec Inc., Gaithersburg, MD, USA. Reference: Bioprocessing Journal, Fall 2006, pp 67-74.
  • 15. Filter sizing for manufacturing scaleProcess Steps Devices 10 Liters 100 Liters 1,000 LitersMedium ProstakTM 0.33 m2 membrane 3.4 m2 membrane 34.0 m2 membraneExchange 1 x 4 Stak Module 2 x 20 Stak Module 20 x 20 Stak ModuleLysate Option 1/Step1:Clarification Millistak+® DE30 or 0.093 m2 area 0.7 m2 area 7.0 m2 area Millistak+® C0HC 0.060 m2 area 0.47 m2 area 4.7 m2 area Option 2/Step 1: Clarigard® (3 µm) 1 x 10” device 4 x 30” 34 x 30” Option 2/Step 2: Polysep IITM (1/0.5 µm) 1 x 2” Opticap® XL 1 x 10” Opticap® XL 4 x 30” Opticap® XLPost- 0.45 µm Durapore® 0.025 m2 membrane 0.25 m2 membrane 2.5 m2 membraneclarificationfiltrationConcentration/ Pellicon® 2 Module with 0.3 m2 membrane 2.5 m2 membrane 25 m2 membraneDiafiltration Biomax® membrane, (3X) (10X) 500kD, C screenPost-Hold/ Pre- 0.22 µm Durapore® 5 Liters 50Liters 500 LitersColumn Millipak® 20 Millipak® 200 1 x 20” CapsuleFiltration 100 cm2 membrane 1000 cm2 membrane 1.38 m2 membrane Work in collaboration with GenVec Inc., Gaithersburg, MD, USA. Reference: Bioprocessing Journal, Fall 2006, pp 67-74.
  • 16. Nucleic Acid Removal in Adenovirus Process  General guidelines: 100 pg/dose[1] or <10 ng/dose[2]  Adenovirus-specific regulatory guidance: 10 ng would only be acceptable provided that the DNA was digested to less than 100-200 base pairs in length[3]  Adenoviruses are typically produced at about 104-105 viral particles (vp)/cell[4]  Mammalian cells have a genome of about 10 pg[5]  7 logs of DNA clearance would be required in order to attain levels below 100 pg/dose for a high (1012 vp) dose of adenovirus.[1] Acceptability of cell substrates for the production of biologicals. Report of a WHO Study Group. In WHO Technical Report Series; World Health Organization: Geneva, 1987.[2] Grachev et al., WHO requirements for the use of animal cells as in vitro substrates for the production of biologicals. Biologicals 1998, 26 (3), 175-193.[3] Bauer et al., Testing of Adenoviral Vector Gene Transfer Products: FDA Expectations. In Adenoviral Vectors for Gene Therapy; Curiel, D. T., Douglas, J. T., Eds.; AcademicPress: New York, 2002; pp 615-654.[4] Nadeau and Kamen. Production of adenovirus vector for gene therapy. Biotechnol. Adv. 2003, 20 (7-8), 475-89.[5] Kraiselbuld et al., Presence of aherpes simplex virus DNA fragment in a L cell clone obtained after infection with irradiated herpes simplex virus 1. J. Mol. Biol. 1975, 97, 533-542.0
  • 17. Benzonase® for Nucleic Acid Removal fromAdenovirus Process  rAd5 purification process  Host cell DNA to below a detectable limit  Combination of ultrafiltration and anion exchange chromatography Source: Konz et al., Biotechnol. Prog. 2005, 21, 466-472 (Merck & Co)
  • 18. Adenovirus Production Process Liquid Harvest Cell lysis Adenovirus production Solid Benzonase® Size Exclusion treatment/ Filtration Chromatography Centrifugation (adenovirus) Retentate Anion Exchange Chromatography on Fractogel® DEAE Purified media Adenovirus Ultrafiltration/ Concentration18 Kamen and Henry, Development and optimization of an adenovirus production process, J Gene Med 6, S184–S192, 2004
  • 19. Fractogel® DEAE-650 M for purification of Adenovirus >92% purity and 70-80% yield Sample preparation: Viruses propagated in HEK293 cellsA260 Lysis by 3 cycles of freeze/thaw of washed cells after centrifugation; 0.5 M NaOH Benzonase® (final conc. 100 U/ml) was 1M virus added for 30 min at RT. 0.35 M Chromatography: 0.25 M -Viral lysate loaded onto a 3 x 6 cm Fractogel® DEAE (M) column -Equilibrated with 50 mM TRIS/HCl, 0.1 M 100 mM NaCl, 2 mM MgCl2, 2% sucrose; pH 8. time - Sequential washing with 0.1 & 0.25 M NaCl. Elution: Bound virus was eluted at 0.35 M NaCl.19 Source: Puresyn, Inc.; Malvern, PA
  • 20. Case studies20 Presentation title in footer | 00 Month 0000
  • 21. Case study: ATOSUS Ark Therapeutics Oy Single Use Systems Production of modified adenoviral products in GMP3 facility (ATOSUS)  High cell densities achieved, up to Thawing and expansion of MCB 30E+6 cells/ml. processing or WCB Upstream Medium preparation  High upstream infections achieved, Infection of expanded cells with cell yields > 60,000 vp/cell. MVSS or WVSS ’Bulk Harvest’  High downstream purified yields achieved, >2x1015vp/10L batch Cell lysis and clarification of CVL ’Clarified Harvest’  HPLC analysis validated for Crude Downstream processing Viral Lysate, to allow tracking of vps Buffer conditioning by crossflow ultrafiltration ’TFF 1 Product’ Buffer-, storage- &  Light scattering analysis to monitor CIP solutions Capture and polishing with preparations aggregation/precipitation – correlates chromatography ’Chromo 1 Product’ with HPLC  Potency, infectivity, hcDNA, HCP ’Chromo 2 Product’ Concentration and diafiltration by crossflow ultrafiltration achieved within product specifications ’Purified Bulk’ Final Formulation  Single use processing - plug and Buffer preparation Fill & Finish play for other AdV projects with ’Drug Substance’ ’Drug Product’ tweaks21
  • 22. Ark’s Ad5 Manufacturing Overview Adherent used for Phase III Cerepro supply Cell culture ~ 5-6 weeks Viral Infection – Harvest ~ 2 days Lysis and Purification ~3 days Sterile filtration and filling ~1 day22
  • 23. Process Development Concept Cell Culture 10 L 10 L Downstream Process 10 L 10 L Process Development DSP 10 L 10 L 10 L 10 L Develop 100L CC Robust 10L GMP Production 100 L 10 L 100 L 100 L Scale Up DSP x10 100 L 100 L Robust 100L GMP Production Develop 300L CC 300 L 100 L 300 L 300 L Scale Up DSP x 3 300 L 300 L Robust 300L GMP Production  Stepwise Progression of Existing Process  Each Unit Operation Developed and Scaled Independently  Stepwise Scale Up reduces unknowns and risk of failure  Time given for Engineering Runs and Requirements for cGMP23
  • 24. Scale-up of developed DSP of Adenoviral ATOSUS Process from bench to pilot scale using commercially available, off-the-shelf systems and single use assemblies First step Cell thawing 6 weeksUSPweek 1 to 5 Cell culture (perfusion) – 5 weeks Virus infection – 2 daysDSP Lysis & Benzonase® Treatment Clarification Intermediate TFFweek 5 Last Step Drug SubstanceDSP 2 stage Ion Exchange Chromatography Tangential Flow Filtration Final Filtrationweek 6
  • 25. Spanning our clients’ Vaccine Processes Services and Solutions Services and Solutions Ingredients/CIP Enzymes Chromatography Buffers EMPROVE® bio Supplements Clarification Filtration EMPROVE® bio Lipids / PEGs EMPROVE® bio Filtration Virus Clearance Parenterals Media Production Clarification eprova Single-use production / sampling Services and Solutions25
  • 26. Acknowledgements Ark Therapeutics Minna Karhinen Robert Shaw Kassim Kolia David Venables Merck Millipore George Adams Nicolas Laroudie Paul Rickets26