lentiviral vector manufacturing – challenges and solutions · pdf filecontrol strategy...

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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

AMC, Cardiff, Oct 2017 2

Stem cell (e.g. Rare Diseases) or T-cell (e.g. Oncology): Platform Establishment

AMC, Cardiff, Oct 2017

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

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 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


Current – Transient adherent, some STR suspension, limited stable: moving towards industrial processes


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’


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


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

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

8

Bacterial Artificial Chromosome (BAC) encoding all vector

components

induce

HEK293T cells

Rapid Generation of Lentiviral Vector Producer Cell Lines Using a Single DNA Construct


2L Suspension Culture – Stable Cell Line

10

– Significant increase in functional titre (TU/ml) compared to transient suspension process


Downstream Processing


• 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


Establishing Robust Lentiviral Manufacturing ProcessesControl 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

In-process, Final Product Testing and Characterisation


Analytical – Will Evolve as CQA’s Better Defined


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


Questions?


lentiviral vector manufacturing – challenges and solutions · pdf filecontrol strategy...

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