Developing a single use adenovirus-vectored vaccine process through public-private collaboration
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This work highlights the importance of collaborations to accelerate vaccine process development and manufacturing under the constant pressure of emerging diseases and the growing need of global immunizations. We are collaborating with the Jenner Institute of the University of Oxford to advance the development of a rapid, scalable and GMP compliant process for simian adenoviruses used as vector for vaccines such as Rabies and emerging threats like Zika and Ebola. This webinar will describe the transition from a labor and time intensive process development to one utilizing a maximum of disposable technologies such as single use bioreactors and filtration technologies, using the rabies vaccine as a first candidate. We will highlight the challenges and their corresponding solutions that in the end created a template that can be used for different types of adenoviral vectors-based vaccines manufacturing. In this webinar, you will learn: - The challenges of creating a rapid and scalable process for Adenovirus vector manufacturing. - The solutions that overcame those challenges. - How public-private collaborations can accelerate vaccine process development.
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Developing a single use adenovirus-vectored vaccine process through public-private collaboration
- 1. Merck KGaA Darmstadt, Germany Anissa Boumlic-Courtade, PhD Associate Director EMEA Vaccines & Viral Therapies segment Process Solutions Life Sciences Through a public-private collaboration Developing a single use adenovirus based vaccine process
- 2. Developing a single use adenovirus-based vaccine Webinar The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada.
- 3. Agenda ▪ Introduction ▪ Viral vectors ▪ Jenner Institute-Oxford University project ▪ Overview of single use viral vector process at manufacturing scale ▪ Conclusions ▪ Acknowledgement Developing a single use adenovirus-based vaccine Webinar
- 4. Developing a single use adenovirus-based vaccine Webinar Vaccines save and improve lives And yet they’re still not accessible to everyone • Immunization saves up to 3 million children each year* • But in 2015, nearly 19,4 million children missed out on the basic vaccines they need to stay healthy* Shortages of certain vaccines because of unforcasted demand and/or long & aging manufacturing processes Pandemics and outbreaks are unevitable but the response is often slow A number of emerging pathogens are still without commercially available vaccines *source: unicef
- 5. Challenges & opportunities for vaccine development & manufacturing Developing a single use adenovirus-based vaccine Webinar Pathogen Funding Regulatory & laws Technology Market Manufacturing process Manufacturer- supplier One health Collaborations Cost
- 6. Different types of Vaccines Different technologies 6 Virus Live attenuated virus (Influenza) Inactivated virus (Influenza, Polio) Recombinant Virus-like particle (HPV Gardasil, Cervarix) Recombinant viral vector (Ebola) Synthetic nanoparticle Bacterial Bacterial sub-unit (Diphteria, Tetanos) Glyconconjugated polysaccharide (Pneumococcus, MenB, GBS) Recombinant protein Nucleic acid DNA (animal vaccines, Inovio Zika) RNA (Rabies, cancer) Pol- ysaccharide Z RSV Pre-fusion F protein RSV Pre-fusion F protein
- 7. Developing a single use adenovirus-based vaccine Webinar New technologies: Using platforms to accelerate vaccine development Example of Ebola 2014/15 pandemic Company Vector type Latest Phase MSD/New Links Profectus Biosciences VSV III -Filling GSK/Okairos J&J Jenner Institute, Oxford University Vaxart Jiangsu CDC Adenovirus III Bavarian Nordics Emergent Biosolutions MVA III Innovio DNA II Novavax Nanoparticle I Thomas Jefferson University Rabies virus I University Queensland/Lyon/QIMR VLP I Majority of platforms based on viral vector Strong reduction of time between Phase 1-3
- 8. Are genetically modified viruses used as tools for antigen expression, gene delivery or tumor targeting. Their therapeutic applications can therefore fall into: • Vaccine • Gene therapy • Cancer treatment Viral vectors Developing a single use adenovirus-based vaccine Webinar
- 9. Global Viral vector manufacturing High growth drivers Emerging diseases +20% CAGR* (billion USD) Genetic disorders Personalized medicines Availability of funding Highest growths in North America & Asia Effectiveness of vector based vaccines & gene therapy Source: Markets & Markets, 2018 Developing a single use adenovirus-based vaccine Webinar
- 10. Viral Vector Segmentation Clinical trials and market share Number of clinical trials registered on vectors globally Vector system Number of registered trials Share in the total number of trials (%) Adenovirus 547 22,2 Retrovirus 478 19,4 Naked/Plasmid DNA 442 17,9 Adeno-associated Virus 204 8,3 Lentivirus 196 8 Vaccinia Virus (MVA) 175 7,1 Poxvirus 107 4,3 Herpes Simplex Virus 93 3,8 Other Vectors 223 9 Source: the Journal of Gene Medicine
- 11. Objectives: Faster and cheaper template for efficient and safe vaccine or viral therapies This template would allow: Accelerating clinical phases Reducing time to market A faster response to outbreaks and pandemics Meeting the need for affordable medicines Viral vector manufacturing Developing a single use adenovirus-based vaccine Webinar
- 12. Developing a single use adenovirus-based vaccine Webinar Viral Vector Manufacturing Schematic viral vector process Raw materials Media prep Amplification & inoculation Cell culture Clarification (I & II) Bioburden removal Nucleic acid digestion ConcentrationBioburden removal AEX ChromConcentration & Diafiltration FormulationSterile filtration Final Filling Lysis SEC or AEX chrom Upstream Purification Formulation & Fill Finish Purification
- 13. Viral Vector Manufacturing Challenges Reaching sufficient viral titers PurityEnsure maximum virus recovery Purification cost & scalability Developing a single use adenovirus-based vaccine Webinar
- 14. Objectives Improve the manufacturing process for adenovirus based vaccines for Phase 1 materials. Develop a cost-effective and transferable manufacturing template that can be used to accelerate vaccine development and manufacturing worldwide. Collaboration with Oxford University
- 15. Objectives: • GMP process • Easy to operate • Single use • >50% efficiency • Phase 1 scale: >5x1013 VP (1000 doses) • Readily scalable to 5x1014 • Vaccine candidate: Rabies vaccine « ChAdOx2-RabGP » platform for vaccines Depth filtration Developing a single use adenovirus-based vaccine Webinar
- 16. Advantages 1 2 Induce a broad immune response against the target antigen. Simian adenovirus itself is not neutralized like human adenoviruses Can be engineered to be non-replicating in the vaccine recipient, lacking molecular mechanism for host genome integration 3 4 Efficiently infect a variety of mammalian cell types (including antigen-presenting cells) Amenable to scalable manufacturing processes such as the use of stirred tank bioreactors, high capacity filtration methods, and chromatographic purification procedures Why Simian Adenovirus Vector? Developing a single use adenovirus-based vaccine Webinar
- 17. Transition to a New Manufacturing Process Reduced handling, compressed process Developing a single use adenovirus-based vaccine Webinar Benzonase® Nuclease Clarification Millistak+® filters Concentration Pellicon® 2 cassettes Membrane chromatography NatriFlo® membrane Diafiltration/ Formulation Pellicon® 2 cassettes Final filtration Durapore® filters Centrifugation Ultra-centrifugationShake flasks Mobius® Bioreactor
- 18. Transition to new process Upstream Developing a single use adenovirus-based vaccine Webinar Mobius® 3L Bioreactor • 2X 3L vessels • HEK293/HEK293 T-Rex • HEK293 chemically defined media • Shake flask yield: 3-6 108 VP/mL HEK293 seed train Cell density at infection MOI Time of harvest Cell density at harvest Yield (qPCR) 1.5-2 x 106 3 42H 1.2 x 106 VP/mL ~5x1010 VP/mL
- 19. Transition to a new process DNA reduction with Benzonase® Nuclease Developing a single use adenovirus-based vaccine Webinar Rationale: - Regulatory requirements of 10ng/dose - Impact on viscosity - Impact on DSP performances Adding Benzonase® Nuclease at 60 U/mL of cell culture decreased the level of host cell DNA >1500 fold during 2h lysis in bioreactor and meet < 10ng/dose requirement Sample – Set point Host DNA ng/mL Bioreactor – pre-lysis 1850 Bioreactor – post lysis 1350 Benzonase® Nuclease addition 1650 30 min 8.6 1h 2.9 1h 30 min 1.4 2h 1.1 3h 30min 0.4 Method: - DNA reduction during in-bioreactor cell lysis - 60 U/mL of cell culture - Residual host DNA was assessed by qPCR targeting Alu repeats
- 20. Transition to new process Clarification using depth filtration Developing a single use adenovirus-based vaccine Webinar Rationale: - Replacement of the centrifugation unit operation - Fully disposable - Objective: >90% virus recovery Depth filter Name Media type Description Millistak+® filter C0HC Double layer, High capacity Cellulose Diatomaceous earth Nominal micron rating 0.2-2µ Millistak+® HC Pro filter C0SP Synthetic material Nominal micron rating 0.2-2µ Millistak+® filter CE50 Single layer Cellulose Nominal micron rating 0.6-1µ Clarisolve® filter CS20MS Polypropylene fibers Diatomaceous earth Nominal micron rating Clarisolve® filter CS60HX Polypropylene fibers Nominal micron rating Method: - 3 Millistak+® & 2 Clarisolve® depth filters were tested (different grades & charge)
- 21. Transition to new process Clarification using depth filtration Developing a single use adenovirus-based vaccine Webinar • Millistak+® HC Pro COSP filter achieves a single-step turbidity reduction 70 ~7 NTU, with 23 cm2 device (µPod® device) allows to process > 2.3L without reaching a true Pmax • Virus recovery: ~90% virus recovery (qPCR) C 0 H C C 0 S P C S 6 0 H X C S 2 0 M S C E 5 0 0 2 1 0 8 4 1 0 8 6 1 0 8 8 1 0 8 IU a s s a y o f F ilte r s tu d y p ro d u c ts F ilte r ty p e IU/ml N o F ilte r 0 .2 2 u m filte r
- 22. First tangential flow filtration step • 300 kDa Pellicon ® 2 Mini filter (0.1m2) with Biomax® membrane • 5L starting volume after clarification (Millistak+® HC Pro C0SP filter + 0.2µ filtration) • 10x volume concentration • 10 DV filtration against IEX-suitable buffer (containing 100 mM NaCl) • Permeate control Transition to new process Purification Developing a single use adenovirus-based vaccine Webinar ~80-88% product recovery 20-30-fold reduction of host cell protein contaminants
- 23. Permeate flow (flux) control to a fixed, robust setpoint throughout a process is achieved through the use of a permeate pump – the flux controlled TFF processes are also referred to as 2-pump TFF Transition to new process Recommendation for TFF: Permeate control Q Q Permeate control allows a stable low TMP and ensures a better virus recovery
- 24. 1 single step single use Anion exchange chromatography using Natriflo® HD-Q membrane Transition to new process Purification Developing a single use adenovirus-based vaccine Webinar • Quaternary amine grafted on macroporous Hydrogel coated PP membrane • Membrane deliver binding capacities that exceed resin- based columns with fast flow rates typical of membrane adsorbers • Highly versatile, and can be utilized in flow-through or bind-elute mode, with nearly any ion exchange Method Natriflo® HD-Q Recon Mini 0.2 ml Bind&Elute mode Loading: ~3.30 x 1011 VP Flow rate: 2m/min Equilibration & wash buffer: 50 mM NaPhosphate pH 6.5, 5% sucrose, 100 mM NaCl, 1 mM MgCl2, 0.1 % Tween®20 surfacant Elution buffer: 50 mM tris-HCl pH 8.0, 1M NaCl, 5% sucrose, 1 mM MgCl2, 0.1 % Tween® 20 surfacant
- 25. Gradient elution Natriflo® HD-Q membrane Transition to new process Purification Developing a single use adenovirus-based vaccine Webinar Natriflo® – HD Q membrane performance: 75-80% recovery Good purity (SDS Page)
- 26. New Adenovirus Process Performance summary Developing a single use adenovirus-based vaccine Webinar Benzonase® Nuclease Millistak+ HC pro C0SP filter Pellicon® 2 cassette NatriFlo® membrane Pellicon® 2 cassette Final filtration Durapore ® 0.2µ filter Mobius® Bioreactor Upstream Lysis & Nucleic acid digestion Clarification Concentration Chromatography Concentration & formulation Final filtration ~5E+10 VP/mL 60 U/ml 2H 37°C 1500 fold DNA reduction 2.3L: 23 cm2 90% recovery 5L: 0.1 m2 300KDa 80-90% recovery 1 step Bind & Elute 70-75% recovery 0.1 m2 Ongoing optimization Ongoing optimization 2 days 3 days
- 27. The new process at 3L scale is based on a single use flowpath • Bioreactor • Tubings & liners • Collection bags • Filters • Chromatography membrane Transition to New Process Integration of Single Use technologies Developing a single use adenovirus-based vaccine Webinar
- 28. Single Use Platforms for Viral Vector Bioreactor ConcentrationChromatographyConcentration & diafiltration Clarification Lysis & nucleic acid digestion Final formulation and filtration To filling Media & Buffer preparation Up to 2000 L scale Developing a single use adenovirus-based vaccine Webinar
- 29. D e v e l o p i n g a s i n g l e u s e a d e n o v i r u s - b a s e d v a c c i n e W e b i n a r Conclusions Accelerating vaccine development & manufacturing is possible GMP ready for various adenoviruses Full single use process Project met initial goals >2000 doses/4L culture 1 & 1/2 years project Collaboration between supplier/ manufacturer is key 5 days process time: « outbreak » ready
- 30. D e v e l o p i n g a s i n g l e u s e a d e n o v i r u s - b a s e d v a c c i n e W e b i n a r Acknowledgment Anissa Boumlic Ada Lam Anilkumar Kangokar Nicolas Laroudie Josselyn Haas Alex Xenopoulos Lenaig Savary Youness Cherradi Ranjeet Patil Sofiya Fedosyuk Tom Merritt Adam Richie Alexander Douglas
- 31. anissa.boumlic@emdmillipore.com Anissa Boumlic The vibrant M, Benzonase, Clarisolve, Durapore, Millistak+, Mobius, NatriFlo, Pellicon are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources. © 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.