Slide 1: A Comparative Analysis of Recombinant AAV9 Product Generated from Insect and Mammalian Bioproduction Processes
A Comparative Analysis of Recombinant AAV9 Product Generated from Insect and Mammalian Bioproduction Processes Phuong Nguyen Senior Scientist, Downstream Process Development, Neurogene Inc. ASGCT 2023
Slide 2: Disclosures
Disclosures Presenter: Phuong Nguyen Phuong Nguyen is Senior Scientist, Process Development at Neurogene Inc. Courtney Barlament is Associate Director, Upstream Process Development at Neurogene Inc. Natasha Patel is a Scientist, Process Development at Neurogene Inc. Adib Shafipour is an Associate Sc
Slide 3: Background
Background • Mammalian (HEK-AAV) and insect cell-based (Sf9-AAV) manufacturing systems are the two predominant AAV manufacturing platforms • Neurogene has established both manufacturing platforms and have cleared INDs with each process Sf9-AAV HEK-AAV • Neurogene has established both manufacturing p
Slide 4: Study Objective- Analytical Comparison of Mammalian and Insect Cell-based Manufacturing Systems
Study Objective- Analytical Comparison of Mammalian and Insect Cell-based Manufacturing Systems Two optimized, scalable platforms were utilized to generate AAV9 containing same transgene of medical importance (GMI) Insect cell-based (Sf9-AAV) Identical Cell lysis, transgene Analytical comparison cla
Slide 5: Process Overview of AAV9 Production Systems
Process Overview of AAV9 Production Systems Sf9-AAV HEK-AAV Rep2Cap9 rBV-Rep2Cap9 Upstream Sf9- pRep2Cap9 RVN Baculovirus expansion Co- Sf9- pHelper RVN Transfection HEK293 GMI infection pGMI Sf9- RVN rBV-GMI Downstream Same cell lysis, clarification, and purification processes AAV = adeno-associate
Slide 6: The Insect Cell-based System Yields Higher Productivity and Percent Full at Harvest
The Insect Cell-based System Yields Higher Productivity and Percent Full at Harvest Productivity and % full at harvest Volumetric yield 1E+13 1E+17 60 Lysate (vg/mL) Total vg (50L) 1E+12 40 % full 1E+16 1E+11 20 1E+10 0 1E+15 Sf9-AAV HEK-AAV Total yield from the same scale runs is ~10-fold higher us
Slide 7: Recoveries From Each Unit Operation are Similar Between the Two Processes
Recoveries From Each Unit Operation are Similar Between the Two Processes Sf9-AAV and HEK-AAV Step recoveries by ddPCR 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% Sf9-AAV 0% HEK-AAV Clarification TFF1 Affinity TFF2 AEX TFF3/BDS Overall Chromatography Chromatography TFF = tangential flow filtration; AEX
Slide 8: Both Processes Resulted in Similar AAV Particle Content by AUC
Both Processes Resulted in Similar AAV Particle Content by AUC Sf9-AAV HEK-AAV Full Full Partial Partial Empty Empty Particle Content (%) Sf9-AAV HEK-AAV Empty 10 6 Partial 8 7 Full 82 87 AUC = analytical ultracentrifugation 8
Slide 9: Similar Capsid Composition (Viral Protein Ratio) Observed in Both Products by CE-SDS
Similar Capsid Composition (Viral Protein Ratio) Observed in Both Products by CE-SDS Sf9-AAV HEK-AAV VP3 VP3 VP2 VP2 VP1 VP1 Species VP1 VP2 VP3 AAV Purity (%) Sf9-AAV 1.5 1.0 10 90 HEK-AAV 1.1 0.8 10 93 CE-SDS = capillary electrophoresis-sodium dodecyl sulfate 9
Slide 10: Overall Low Levels of PTM on the Capsid Surface, and the Difference between Products is within Assay Variability
Overall Low Levels of PTM on the Capsid Surface, and the Difference between Products is within Assay Variability Post-translational Modification 20 PTM (%) Sf9-AAV 10 HEK-AAV 0 Deamidation Oxidation Methylation Phosphorylation Acetylation AAV = adeno-associated virus; PTM = post-translational modifi
Slide 11: MiSeq Data Analysis Showed Similar Genome Integrity for Both Processes
MiSeq Data Analysis Showed Similar Genome Integrity for Both Processes Sf9-AAV HEK-AAV Regions (reads aligning to map %) (reads aligning to map %) NGN Construct (GMI) 86 91 Starting Plasmid Backbone 0.02 1.30 Baculo RepCap/Plasmid RepCap 0.18 0.48 Shuttle Vector 0.010 N/A Helper Plasmid N/A 0.21 Hos
Slide 12: Residual (Impurity) Analysis and Safety Testing Showed Comparable Profiles
Residual (Impurity) Analysis and Safety Testing Showed Comparable Profiles Assay Sf9-AAV HEK-AAV Endotoxin (EU/mL) < 0.05 < 0.05 Monomer = 97.6 Monomer = 99.2 SEC (%) HMWS = 2.4 HMWS = 0.8 Replication competent AAV (in 1E+11 vg) <10 rcAAV <10 rcAAV Residual Host Cell Protein (ng/mL) 8.1 <2.0 Residua
Slide 13: AAV Products from Both Processes Show Similar Activity Using a Functional (Enzymatic) Potency Assay
AAV Products from Both Processes Show Similar Activity Using a Functional (Enzymatic) Potency Assay Transform of galc enz 20221028 Plate1+Plate2 300000 ENG-201-002-DP activity (RFU) NG-076-BDS 200000 RFU Process Relative Potency (%) Enzymatic 100000 Sf9-AAV 100 HEK-AAV 87 Assay variability is +/- 25
Slide 14: Conclusions
Conclusions • We thoroughly characterized and compared the final products (containing the same GMI) generated using an Sf9 and a HEK process in order to address the question of which is a better process • Using developed processes, both methods yielded high quality vector with low amounts of impurit
Slide 15: Acknowledgements
Acknowledgements Process Development Analytical Development Courtney Barlament Xiaoyun Xu Natasha Patel Lexy Cattin-Roy Adib Shafipour Caitlin Tripp Shayan Kariminia Juliana Benito Rachel Stroup Rebecca Cram Ruda Cui Wei Liao Zheng (Judy) Chen … and the entire CMC team 15