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Photo credit Martin Web/Save the Children
Elizabeth and PatienceSave the Children clinic attendeeLiberia
Lentiviral Vector Manufacturing –Challenges and Solutions
Bo KaraCell Gene Therapy CMC
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Strategy: Break Through Innovation Therapies in Order to Treat More Common Diseases
Risk-benefit profile
EfficaciousCoGs driven
down
ADA SCID
Gene therapies to treat more common diseases with
multiple genetic factors, large unmet need
Knowledge gained on how to achieve long-term, durable responses using gene therapy
Target validated in monogenic diseaseUnmet need and validated target foster
innovation
Potential for Respiratory Indications, Colitis, Diabetes, Multiple Sclerosis
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 ultra rare diseases with
large unmet need
WAS
MLD
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Stem cell (e.g. Rare Diseases) or T-cell (e.g. Oncology): Platform Establishment
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T-CELL PROCESSING
VECTOR STEM CELL PROCESSING
PLASMID DNA’s
MCB’s/WCB’s
UpstreamProcess
DownstreamProcess
Bulk
Formulation,Fill, Freeze
293T MCB/WCB
Upstream(Transient)
Downstream
FormulationFill, Freeze
BM/MPB
FDG
EnrichmentDepletion
Transduction
Incubate/Wash
FormulationFill
Patient
Patient
LymphocyteApherisis
EnrichmentDepletion
Transduction
WashExpansion
WashHarvest
FormulationFill
Patient
Patient
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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 102 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
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Current – Transient adherent, some STR suspension, limited stable: moving towards industrial processes
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Production of Lentiviral Vectors, O-W Merten et al, Molecular Therapy — Methods & Clinical Development (2016) 3, 16017; doi:10.1038/mtm.2016.17
Lack of analytical standardisation - difficult to compare infective titres
Lentiviral Vector Manufacturing: Current State (largely)Majority of manufacturing small scale, high cost per ‘vector dose’
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Plasmid DNA Supply Chain
Downstream Processing
Chrm1Chrm2UFDF
LVV Product
HEK293TMCB
WCB
Seed Train
1’s to 10’s output
w.r.t. LVV ‘dose
Low USP Titres
Low DSP Recoveries
Vector Manufacturing – ScalabilityIndustrialisation of LVV Manufacturing
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Adherent + 1st Gen DSP
• 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
Suspension + 2nd Gen DSP• Exploit Biologics learning and
technologies
• 50-2000L harvests: scale to match vector market needs
• Closed processing – automation of unit operations
• 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
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Investing in Technology and Process Development
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• 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
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Bacterial Artificial Chromosome (BAC) encoding all vector
components
induce
HEK293T cells
Rapid Generation of Lentiviral Vector Producer Cell Lines Using a Single DNA Construct
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2L Suspension Culture – Stable Cell Line
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– Significant increase in functional titre (TU/ml) compared to transient suspension process
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Downstream Processing
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• Clarification• Membrane screening- improved
recoveries• Chromatography
• Use of matrices designed for LVV particles rather than proteins
• LVV pseudotype specific affinity capture-improved 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
Establish Process Platform Elements – then Select, Integrate, Confirm, Exploit
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Establishing Robust Lentiviral Manufacturing ProcessesControl Strategy – Risk Based Analysis
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• 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
In-process, Final Product Testing and Characterisation
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Analytical – Will Evolve as CQA’s Better Defined
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Ratio Infective to Non-infective impacts:• Transduction
efficiency• UF/DF• Sterile filtration• LVV aggregate
levels
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
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Questions?
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