Lentiviral Vector Manufacturing Challenges and Solutions Bo Kara Cell Gene Therapy CMC Elizabeth and Patience Save the Children clinic attendee Liberia Photo credit Martin Web/Save the Children
Strategy: Break Through Innovation Therapies in Order to Treat More Common Diseases Gene therapies to treat ultra rare diseases with large unmet need Expand into in vivo therapies and new cell types based on experience with lentiviral platform: e.g. B-Thal, NY-ESO-1 TCR, other Gene therapies to treat more common diseases with multiple genetic factors, large unmet need. Potential for Respiratory Indications, Colitis, Diabetes, Multiple Sclerosis MLD ADA SCID. Knowledge gained on how to achieve long-term, durable responses using gene therapy. Target validated in monogenic disease Unmet need and validated target foster innovation WAS Risk-benefit profile Efficacious CoGs driven down AMC, Cardiff, Oct 2017 2
Stem cell (e.g. Rare Diseases) or T-cell (e.g. Oncology): Platform Establishment PLASMID DNA s MCB s/wcb s VECTOR 293T MCB/WCB STEM CELL PROCESSING Patient T-CELL PROCESSING Patient Upstream Process Upstream (Transient) BM/MPB FDG Lymphocyte Apherisis Enrichment Depletion Downstream Process Downstream Enrichment Depletion Transduction Transduction Wash Expansion Bulk Formulation Fill, Freeze Incubate/Wash Formulation Fill Wash Harvest Formulation Fill Formulation, Fill, Freeze Patient Patient AMC, Cardiff, Oct 2017 3
LVV Product Properties Challenge for Process Development & Manufacturing LVV: ssrna, ~7-11kb (homodimer) Enveloped Strong net negative charge: useful handle but 60-120nm diameter ~2500 x 10 2 kda (vs. mab ~150kDa) Shear sensitivity Temperature sensitivity (infective) Freeze-thaw sensitivity Salt sensitivity (v. narrow range) Cell production non-infective and infective particles formed Heterogeneity Infectivity infective to non-infective Impact on process options Transient production: potential for batch to batch variation AMC, Cardiff, Oct 2017 4
Current Transient adherent, some STR suspension, limited stable: moving towards industrial processes Lack of analytical standardisation - difficult to compare infective titres Production of Lentiviral Vectors, O-W Merten et al, Molecular Therapy Methods & Clinical Development (2016) 3, 16017; doi:10.1038/mtm.2016.17 AMC, Cardiff, Oct 2017 5
Lentiviral Vector Manufacturing: Current State (largely) Majority of manufacturing small scale, high cost per vector dose Plasmid DNA Supply Chain Seed Train Low USP Titres HEK293T MCB WCB 1 s to 10 s output w.r.t. LVV dose LVV Product UF DF Chrm2 Chrm1 Low DSP Recoveries Downstream Processing AMC, Cardiff, Oct 2017 6
Vector Manufacturing Scalability Industrialisation of LVV Manufacturing Adherent + 1 st Gen DSP Suspension + 2 nd Gen DSP Exploit Biologics learning and technologies 50-2000L harvests: scale to match vector market needs Closed processing automation of unit operations 1L - <100L total harvest Manual unit ops Batch to batch variation Scale-up limited Low DSP recovery 1 s to 10 s w.r.t. lentivirus dose per batch Cost per Transforming Unit v.high Robust and consistent performance Improved DSP recovery/quality 100 s to 1000 s w.r.t. lentivirus dose per batch Cost per Transforming Unit reduced AMC, Cardiff, Oct 2017 7
Investing in Technology and Process Development Suspension exploit volumetric productivity increases Optimise: Transfection Potential for stable cell line systems Batch medium Fed-batch: feed strategy Improved DSP: Step recoveries Potential for Affinity capture Improved recovery infectious vs. total particles Improved understanding of vector CQA s Improved analytics AMC, Cardiff, Oct 2017 8
Rapid Generation of Lentiviral Vector Producer Cell Lines Using a Single DNA Construct Bacterial Artificial Chromosome (BAC) encoding all vector components HEK293T cells induce AMC, Cardiff, Oct 2017 9
2L Suspension Culture Stable Cell Line Significant increase in functional titre (TU/ml) compared to transient suspension process AMC, Cardiff, Oct 2017 10
Downstream Processing Clarification Membrane screening- improved recoveries Chromatography Use of matrices designed for LVV particles rather than proteins LVV pseudotype specific affinity captureimproved and selective recoveries UF/DF, Filtration Membrane screening improved recoveries Formulation Development/screening Improved stability Reduced aggregation Small molecule Protein, e.g. hgh mab, e.g. IgG1 Lentiviral vector AMC, Cardiff, Oct 2017 11
Establish Process Platform Elements then Select, Integrate, Confirm, Exploit AMC, Cardiff, Oct 2017 12
Establishing Robust Lentiviral Manufacturing Processes Control Strategy Risk Based Analysis Identify LVV Critical Process Parameters (CPPs), vs LVV CQA s Process risk assessment Analytical risk assessment View on LVV assay(s) robustness Design and implement process controls CPPs maintained within defined acceptable ranges Risk assess - impact of raw and starting materials on the CQA s Define relationships: raw and starting material attributes vs. product attributes during development Control raw and starting materials Material release specifications and/or material storage conditions Ensure product CQAs maintained within the defined acceptable ranges Align material control strategy with the microbial control strategy (LVV required as sterile product) Including risks associated with viral and TSE contamination AMC, Cardiff, Oct 2017 13
In-process, Final Product Testing and Characterisation AMC, Cardiff, Oct 2017 14
Analytical Will Evolve as CQA s Better Defined Ratio Infective to Non-infective impacts: Transduction efficiency UF/DF Sterile filtration LVV aggregate levels AMC, Cardiff, Oct 2017 15
Finally Roadmap Continued innovation Vector and cell line(s): Increase specific productivity: understand Infective LVV synthetic pathway flux analysis Understand vector CQA s Improve infective to non-infective ratio Upstream Processing Adherent to suspension SUB s to 2000L Transient to stable inducible systems - modulation of expression Downstream processing Improved clarification bespoke LVV filtration Given pseudotyping affinity capture Formulation In-process stability enhancement Higher concentrations w/o aggregation AMC, Cardiff, Oct 2017 16
Questions? AMC, Cardiff, Oct 2017 17