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![M. Hebben
CONFIDENTIAL
POROS®CaptureSelect™ AAV9
• Affinity resin developed by Thermo Fisher Scien@fic
• Ligand based on a single-domain [VHH] an@body fragment
• VHH affinity ligands are produced in yeast (S. cerevisiae) in an
animal origin free produc@on process
– ISO9001 cer@fied manufacturing facility (Netherlands)
12-15kDa
• Small size:12-15 kDa
fragment: ~1/10th mAb
• Tunable specificity/affinity
CaptureSelect™ technology
Caution: For manufacturing, processing, or repacking.](https://image.slidesharecdn.com/bils2015mhebben-160418133722/85/BILS-2015-Genethon-M-Hebben-17-320.jpg)
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BILS 2015 Genethon M. Hebben
The document discusses the development and manufacturing challenges of AAV9 gene therapy vectors, highlighting the history and evolution of gene therapy from the 1980s to the present. It details GeneThon's operations, including its GMP manufacturing capabilities and the effectiveness of AAV9 in treating various diseases, as well as the purification processes necessary for large-scale production. The conclusion emphasizes that manufacturing AAV9 vectors at a large scale is feasible with a high yield process, suitable for clinical applications.
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BILS 2015 Genethon M. Hebben
- 1. M. Hebben CONFIDENTIAL Debottlenecking DownstreamProcess of AAV9 Gene Therapy Vectors using Customized Chromatography Resin BioInnovation Leaders Summit London, Feb 11th 2015 Matthias Hebben, Ph.D. Bioprocess Development 11/02/2015
- 2. M. Hebben CONFIDENTIAL Genethon ata Glance • Genethon Bioprod: GMP manufacturing site granted in 2013 by ANSM • Produc@on of clinical batches • Vector plaCorms: AAV and len@viral vectors. • Created in 1990 by AFM (French Associa@on of Muscular Dystrophies) • An integrated, non profit organiza@on of 220 people • Transla@onal research and clinical development in the area of gene therapy 11/02/2015
- 3. M. Hebben CONFIDENTIAL In vivoand ex vivo gene therapy Muscular Dystrophies Myotubular myopathy Eye diseases Liver-mediated gene therapy in vivo delivery Recombinant AAV vectors are directly injected into the target organ Hematopoietic stem cells taken from the patient are transduced with an HIV- derived lentivector and reinfused Primary immunodeficiencies, Hemoglobinopathies ex vivo delivery 11/02/2015
- 4. M. Hebben CONFIDENTIAL Gene TherapyEvolution since 1980 1980 1990 2000 2010 2020 2030 Proof of concept Clinical Trials Commercial Phase 11/02/2015
- 5. M. Hebben CONFIDENTIAL STRONG FLOWOF ACQUISITIONS AND LICENSE AGREEMENTS WITH BIG HEALTHCARE PLAYERS: MORE THAN USD 470 M FUND RAISED IN 2012 AND 2013: (Chatham Therapeutics) An incredible progression in 2014! 11/02/2015
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- 7. M. Hebben CONFIDENTIAL Vector scAAV9-SMN1 11/02/2015 Transgenecassette DNA AAV vector
- 8. M. Hebben CONFIDENTIAL AAV9 asa Gene Therapy Vector 11/02/2015 • Unique features of AAV9: – Topism for muscles and heart – Tropism for CNS – Capability to cross BBB Foust et al. Nat. Biotechnol. 2009
- 9. M. Hebben CONFIDENTIAL AAV9 asa Gene Therapy Vector Disease Vector Administra=on route Authors SMA scAAV9-SMN IV Dominguez et al. 2010 MPSIIIA AAV9-SGSH IC/ICV Haurigot et al. 2013 MPSIIIB AAV9-NAGLU IV Fu et al. 2011 Pompe AAV9-GAA intrapleural Falk et al. 2013 ALS AAV9-ADAR2 IV Yamashita et al. 2013 11/02/2015 • AAV9 suitable for gene therapy of: – Neuromuscular disorders – Lysosomal storage diseases affec@ng the brain • 1 clinical trial ini@ated in US: scAAV9-SMN1 for GT of SMA (AveXis) • Large scale manufacturing in GMP condi@ons is needed to support future clinical trials
- 10. M. Hebben CONFIDENTIAL AAV9 UpstreamProcess: Transfection Transfection of suspension cells PEI Scale: 200L disposable bioreactors Transfection of adherent cells Calcium Phosphate Scale: 24 CF10 (25L) Triple plasmid transfection 11/02/2015 HEK293 cells +
- 11. M. Hebben CONFIDENTIAL Baculovirus genome recombination in E.coli Generation of infectious Baculovirus in Sf9 cells Production 2x 200L in disposable bioreactors Bac-Rep/cap rep polh p10 pA pA cap promoter- Transgene Bac-rAAV-ITR ITR ITR rBac-rAAVrBac-Rep/Cap Co infection Sf9 cells AAV9 Upstream Process: Baculovirus 11/02/2015
- 12. M. Hebben CONFIDENTIAL AAV UpstreamProcesses Transfec=on of HEK293 cells Adherent cells (cell factories) or suspension cells (s@rred tanks) Versa@le process Fast implementa@on Expensive (plasmid cost) Main impuri@es: DNA/Transfec@on agent Baculovirus / Sf9 cells Suspension cells (s@rred tanks) Versa@le process Slow implementa@on (8 months) Cost decreases with number of lots Main impuri@es: Baculovirus DNA and proteins 11/02/2015
- 13. M. Hebben CONFIDENTIAL Purification ofAAV9 Vectors • No industrial friendly method available to purify AAV9 at large scale • Immuno affinity AVB Sepharose (GE Healthcare): – Originally developped for AAV1 – Cross binding of AAV2, AAV5, AAV6, AAV8, AAV10 – No binding of AAV9 capsids • Need to develop a purifica@on process from scratch 11/02/2015
- 14. M. Hebben CONFIDENTIAL AAV9 DownstreamProcess using IEX Clarification CEX AEX AEX Negative Mode TFF 11/02/2015 SF
- 15. M. Hebben CONFIDENTIAL AAV9 DownstreamProcess using IEX 11/02/2015 0 20 40 60 80 100 120 Clarifica@on CEC AEC 1 AEC 2 TFF % vector recovery Average yield (n=6) AAV9 5e9VG 1e10VG
- 16. M. Hebben CONFIDENTIAL AAV9 DownstreamProcess using IEX • Low yield (approximately 20%) – Implies a large batch size to generate the required amount of vector – Implies a high concentra@on factor to achieve target @ters in clinical doses – At small scale, the highest @ter obtained was 1-2e12 VG/mL • Scalability issues – Afemps to scale up revealed technical piCalls: very narrow pH/ temperature opera@ng range affec@ng purity and vector stability • Process not viable? 11/02/2015
- 17. M. Hebben CONFIDENTIAL POROS®CaptureSelect™ AAV9 • Affinity resin developed by Thermo Fisher Scien@fic • Ligand based on a single-domain [VHH] an@body fragment • VHH affinity ligands are produced in yeast (S. cerevisiae) in an animal origin free produc@on process – ISO9001 cer@fied manufacturing facility (Netherlands) 12-15kDa • Small size:12-15 kDa fragment: ~1/10th mAb • Tunable specificity/affinity CaptureSelect™ technology Caution: For manufacturing, processing, or repacking.
- 18. M. Hebben CONFIDENTIAL POROS®CaptureSelect™ AAV9 • Resin based on POROS® beads – Polystyrene-Divinylbenzene Backbone • Rigid, Incompressible, Easy-to-Handle, – Perfusive Media • Large Throughpores Unlock Bead Interior; High Surface Area – 50 micron Par@cle Size • Provides Superior Resolu@on Independent of Scale and Flow Rate • Resin is highly selec@ve for Adeno-Associated Virus Serotype 9 (AAV9) • CaptureSelect™ AAV9 ligand leakage ELISA kit available Caution: For manufacturing, processing, or repacking.
- 19. M. Hebben CONFIDENTIAL Resin Selectivity 11/02/2015 • No residual proteins detected • Same purity profile as 3 steps of IEX chromatography
- 20. M. Hebben CONFIDENTIAL Resin Capacity • The higher the flow rate, the higher the capacity! • Capacity = 1e12 VG/ml of resin at 450 cm/h using Baculovirus-expressed AAV9 • Considering 30% of full par@cles à binding of 3e12 capsids per mL of resin • High capacity results in small volume of resin • Small resin volume results in high concentra@on factor 11/02/2015 0.0E+00 5.0E+11 1.0E+12 1.5E+12 2.0E+12 0 100 200 300 400 500 600 700 AAV9 =ter (VG/mL) Flow rate (cm/h) Capacity of POROS AAV9 Eluate Flow Through
- 21. M. Hebben CONFIDENTIAL AAV9 PurificationProcess Clarification Immuno Affinity Chromatography Concentration and Formulation Sterile Filtration 11/02/2015 Fill & Finish 200L 50mL
- 22. M. Hebben CONFIDENTIAL Process Yield 11/02/2015 0 20 40 60 80 100 120 POROS AAV9 TFF STERILE FILTRATION % vector recovery Vector recovery at key steps Batch #1 Batch #2 Batch #3 • ≥ 80% vector recovery at each step • Overall process yield = 50 to 65% • Reproducibility at 10L and 50L scales
- 23. M. Hebben CONFIDENTIAL Process Efficiencyto Remove DNA 11/02/2015 -5 -4 -3 -2 -1 0 DNA reduc=on (log) Baculovirus DNA removal Batch #1 Batch #2 Batch #3 Total process allows reduction of 4 log of residual DNA
- 24. M. Hebben CONFIDENTIAL Conclusions • Manufacturing of AAV9 vectors feasible at large scale – Process efficiency confirmed at 50L scale – Scale up to 200L scale in progress • Immunoaffinity chromatography allows a simple, efficient and high yield process – Generic produc@on plaCorm for AAV9 vectors • Full process generates final product around 1e13 VG/mL 11/02/2015
- 25. M. Hebben CONFIDENTIAL Thanks Généthon – Fulvio Mavilio, CSO • Bioprocess Development – Mohammed Risi – Ludivine Dejoint – Laurent Bortolussi – Nicolas Marceau – Christophe Lecomte – Francis Boussicault – Delphine Dufour – Laurence Guianvarc’h • Analy@cs – Chris@ne Le Bec – Bruno Dalle • R&D – Ofo Merten – Samia Mar@n – Federico Mingozzi 11/02/2015