Viral-vectored vaccines: a new approach inthe vaccine manufacturing processDr. Priyabrata PattnaikDirector – Asia Vaccine ...
Agenda 1   Vaccine Industry Trends 2   Market Trend of Viral based Biologics 3   Recombinant Virus as Vaccine Vectors 4   ...
Vaccine Industry Trends Vaccines segment is growing at 8%. ~ 400 vaccines are in commercial manufacture.    300 are vira...
Market Trend of Viral based Biologics       Biologics in Development     Vaccines   Recombinant   Mabs   Other            ...
Recombinant Viruses as Vaccine Vectors    Adenoviruses       Non-enveloped virus, 70-100 nm in diameter       Genome: li...
Adenovirus ControversyMerck & Co scraps Adenovirus based AIDS vaccine trial Adenovirus vector triggered immune response ...
Growth of R&D on Adenovirus Vaccine100000                                             20000                               ...
Oncolytic Vaccine Vector8               Source: J.-W. Choi et al. / Advanced Drug Delivery Reviews 64 (2012) 720–729
9
Generic Process of Vectored Vaccine Manufacturing10
Cell Culture for Adenovirus Typical cell culture: HEK293 Cell culture either in continuous or batch mode (w/ media excha...
Fast-Trap™ Lentivirus and AdenovirusPurification & Concentration Kit Kit contains necessary reagent Membrane-based, clos...
Adenovirus work before the controversy Collaboration with GenVec Inc.,  Gaithersburg, MD USA. Filter sizing for medium e...
Millistak+® Pod filter for lysate clarification                                                           Millistak+® fil...
Filter sizing for manufacturing scaleProcess Steps     Devices                       10 Liters                   100 Liter...
Nucleic Acid Removal in Adenovirus Process  General guidelines: 100 pg/dose[1] or <10 ng/dose[2]  Adenovirus-specific re...
Benzonase® for Nucleic Acid Removal fromAdenovirus Process                              rAd5 purification process        ...
Adenovirus Production Process                                              Liquid               Harvest                   ...
Fractogel® DEAE-650 M for purification of Adenovirus >92% purity and 70-80% yield                                         ...
Case studies20   Presentation title in footer | 00 Month 0000
Case study: ATOSUS Ark Therapeutics Oy Single Use Systems     Production of modified adenoviral products in GMP3 facility ...
Ark’s Ad5 Manufacturing Overview Adherent used for Phase III Cerepro supply                           Cell culture        ...
Process Development Concept     Cell Culture           10 L      10 L          Downstream Process                         ...
Scale-up of developed DSP of Adenoviral ATOSUS   Process from bench to pilot scale using commercially available,   off-the...
Spanning our clients’ Vaccine Processes     Services and Solutions                                                        ...
Acknowledgements Ark Therapeutics Minna Karhinen Robert Shaw Kassim Kolia David Venables Merck Millipore George Adams Nico...
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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.

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

  1. 1. Viral-vectored vaccines: a new approach inthe vaccine manufacturing processDr. Priyabrata PattnaikDirector – Asia Vaccine Initiative
  2. 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. 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. 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, Clinitrials.gov
  5. 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. 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. 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. 8. Oncolytic Vaccine Vector8 Source: J.-W. Choi et al. / Advanced Drug Delivery Reviews 64 (2012) 720–729
  9. 9. 9
  10. 10. Generic Process of Vectored Vaccine Manufacturing10
  11. 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. 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. 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. 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. 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. 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. 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. 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. 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. 20. Case studies20 Presentation title in footer | 00 Month 0000
  21. 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. 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. 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. 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. 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. 26. Acknowledgements Ark Therapeutics Minna Karhinen Robert Shaw Kassim Kolia David Venables Merck Millipore George Adams Nicolas Laroudie Paul Rickets26

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