production and purification of viral vectors for gene and cell therapy applications
TRANSCRIPT
Priyabrata Pattnaik, PhDDirector & Head of Biologics OperationsAsia Pacific
Production and purification of Viral vectors for gene and cell therapy applications
The cell & gene therapy market is poised for rapid growthwith projections reaching ~$10B in 5 years
Source: Seed Planning; METI; Kuick Research; Med Market Diligence;Transparency market research
Global market size & CAGRUSD B
Products in the pipeline
7474
345475
Stem celltherapy
106
17
14
Cellular therapy, other
12315
Gene therapy
14416
Phase I
Phase II
Phase III
109
1
12
8
3
2020
+36% p.a.
20182012
Kuick research (India)See Planning/METI (Japan)
Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 20162
The cell & gene therapy market can be split into 3 broad segments...
Description and examples Major players
Cell & gene therapy
Non-geneti-cally modified cell therapies
• Purified populations of allogeneic cells are expanded and injected topically
• E.g., mesenchymal stem cells
Viral gene therapies
• Drug product is virus particle, injected into patient topically (or systemically)
• E.g., Spark’s night-blindness therapy
• Autologous patient cells are extracted, genetically modified using viral transduction, then reinfused into the patient
• E.g., CAR T-cell therapy
Genetically modified cell therapies
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3 broad segments
Gene Therapy Stem-cell Therapy Cell Immunotherapy
Gene Therapy
Stem-cell Therapy
Cell Immunotherapy
Nucleic acid Lenti/Adeno virus-based delivery Somatic or germline
Stem cells banking Replace or regenerate organs and tissue Broad use of bone marrow in ALL
Autogenetic CAR-T1. Most common2. Immune cells separation3. Lentivirus-based delivery4. Chimeric Antigen Receptors (CARs)
Allogeneic modified T cells1. Industrialized trend2. Genetic modification of immune cells3. Allogeneic T cell generation4. Lentivirus-based delivery or genome edit (CRISPR)
CAR-TVirusStem cells
T-cells
Virus
CAR-T4
Methods for producing genetically modified T-cells for immunotherapy
Source: Bure et al. (2016) Automation of CAR-T Cell Adoptive Immunotherapy Bioprocessing. Bioprocess International, 14(4)s: 22-31.
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Overall Process Map of CAR-T
T-cell separation
Virus generationGMP-grade lentivirus Recovery rate= 70% 2.5*109 virus per trial 5*106 titer/ml 500ml bulk
T-cell modification MOI=5 1.7*109 virus per trial
Car-T Cell Amplification
Reintroduction 5*106 CAR-T/kg E.g.: 70 kg weighting 3.5*108 CAR-T per trial
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Viral Vectors For Gene TherapyIntroduction
Gene Therapy – Main Applications:• Treat Heridatery Single-Gene Defects.• Cancer• Cardiovascular Diseases• Ocular Diseases• Infectious Diseases• Other Diseases Where Gene Transfer Will Have an Impact
Main Features for Virus-Based Vectors:• Lacks Viral Genes Involved in Replication.• Expression Cassette is Cloned into the Vector.• Helper Function is Provided in trans• Co-Transfection of Vector Genome and Packaging Construct Produce Recombinant Vecotor
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~60% of the vaccines undergoing clinical trials are viral based. ~ 640 viral vaccines ~ 200 viral vectors ~ 60 virus like particles
Another ~240 gene therapy products in development that utilize the same technology.
Example of vectors:
Adenovirus (70-100 nm)
Lentivirus (80-100 nm)
HSV (300 nm)
Viral Vectors – Introduction
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Features AAV LentivirusGenome ssDNA ssRNA(+)Virus Coat Non-Enveloped EnvelopedDiameter 18 – 26nm 80 – 130nmPackaging size 4.5 kb 8 kbInfection Range Dividing & Non-Dividing Cells Mostly DividingPost Infection Mostly Non-Integrating* Integrates into Host GenomeGene Expression Long lasting (?) ProlongedMain Advantage Non-pathogenic; Non-inflamatory Persistent Gene TransferMain Disadvantage Small packaging capacity Oncogenesis may occur
Characteristics of AAV & Lentivirus
^ AAV (mainly serotype 2) may integrate into Chromosome 19
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AAV Serotype Tissue Tropism
AAV1 Muscle, Heart, Ocular, CNSAAV2 CNS, Kidney, Muscle, Testes
Various in vitro applicationsAAV4 Lung, AAV5 Lung, CNS, Ocular, PancreasAAV6 Lung, HeartAAV7 Muscle, LiverAAV8 Liver, Muscle, Ocular, CNS, HeartAAV9 Lung, Liver, Muscle, Heart, CNS, Kidney, TestesAAVrh10 Pleura, CNSOther Various
AAV Serotypes and Tissue Tropism
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Transient Transfection Mediated by PEI or Calcium Phosphate. Co-transfection of AAV production cells with 3 plasmids:
• Plasmid with AAV ITR and the gene of interest• Plasmid with AAV rep/cap• Plasmid providing the helper genes isolated from adenovirus.
Wild Type Adenovirus Infection Into Cell Lines with AAV rep/cap Genes and AAV Vector.
Infection using two HSV viruses harboring the gene of interest and the rep/cap genes to produce AAV.
Infecting sf9 cells with two baculoviruses harboring the gene of interest and the rep/cap genes to produce AAV.
Production Modes for Recombinant AAV
Sufficient Production of virus vector copies
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Adenovirus vectors manufacturingKey requirements and needs
Antigens attributes have to meet pre-set Critical Quality Attributes
Reproducibility & consistency
Biosafety
Sufficient recovery to achieve acceptable economics
Ex: nucleic acid content, product identity & safety, etc.
Titer
Regulatory
Recovery
Purity & Quality
Process Schematics of AAV vector production and Purification
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UF/DF BenzonaseTreatment
Primary Purification
Chromatography
Media and Inoculums Preparation
Cell growth in BioreactorHEK 293 Cells
Virus InoculationTransfection
VirusHarvest (Lysis)
PrimaryClarification
UF/DFSterile
Filtration
SecondaryClarification
SecondaryPurification
ChromatographyFill & Finish
Thaw and Expand
Cells and Seeds
Optional BenzonaseTreatment
Process Schematics of AAV vector production and Purification
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UF/DF BenzonaseTreatment
Primary Purification
Chromatography
Media and Inoculums Preparation
Cell growth in BioreactorHEK 293 Cells
Virus InoculationTransfection
VirusHarvest
PrimaryClarification
UF/DFSterile
Filtration
SecondaryClarification
SecondaryPurification
ChromatographyFill & Finish
Thaw and Expand
Cells and Seeds
Optional BenzonaseTreatment
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Amplification & culture of HEK-293
Bioreactors
T-Flasks*
*Source: Wang and Rivière (2015) Manufacture of tumor-and virus-specific T lymphocytes for adoptive cell therapies, Cancer Gene Therapy (2015) 22, 85–94.
Segura et al., Biotechnology & Bioengineering, 2005, 90 : 391-404
Virus Total Protein DNA
MCBInnoculationShake/spin
Sf-9
HEK 293EB66® Cells
BHK21
Sf-9
HEK 293EB66® Cells
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Minimize Your Process Development Efforts Leverage Merck’s Experience with Various Cell Lines in Mobius® Bioreactors
CHOHybridoma
SP 2/0
Vero
h-MSCT cells
HepaRG®
Adherent
Suspension
Public references at the bench (2 L) … and production scale (50 to 2000 L)
Vero
Power/Volume
Agitation speed
MDCK
S2 Drosophilia
mAb / Rec-protein
Virus Production (Suspension)
CellTherapy
Virus production (Adherent)
h-MSC
HepaRG®
CHO
Hybridoma
*application data available
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Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016
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• Standard media: EX-CELLTM 293 Serum-Free Medium for HEK 293 Cells in suspension
• Custom media on demand
• Supplements
Cell culture Media preparation
Durapore® or Express® 0.22 or
0.1 µmSterilizing
Or Viresolve®
Barrier (available soon)
Lynx sterile connectors
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Bioreactor Biosafety: Preventing contamination
Control
Protect
Maximum control of animal derived materials In-coming test of critical materials Move to animal free raw materials processes. Recombinant versions of Trypsin, insulin, albumin etc
(Cellprime® range).
• As a minimum: sterile filtration of cell culture media (Express SHC for example)
• Implementation of a Mycoplasma or a Virusbarrier: filtration of cell culture media for example using Viresolve® barrier
• Sterile Sampling with NovaSeptum• Aervent filters for the aeration (0.22µ)• Sterile connectors (Lynx)
Process Schematics of AAV vector production and Purification
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UF/DF BenzonaseTreatment
Primary Purification
Chromatography
Media and Inoculums Preparation
Cell growth in BioreactorHEK 293 Cells
Virus InoculationTransfection
VirusHarvest (Lysis AAV)
PrimaryClarification
UF/DFSterile
Filtration
SecondaryClarification
SecondaryPurification
ChromatographyFill & Finish
Thaw and Expand
Cells and Seeds
Optional BenzonaseTreatment
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Adherent cells may be lysed in situ or detached from the growth substrates
Lysis can happen by freeze-thaw lysis, mechanical homogenization or chemically via the use of surfactants.
Large volume suspension cultures may be treated with surfactants, e.g. Triton X-100, or homogenized with a mechanical device.
Nuclease treatment is incorporated following lysis to reduce viscosity and facilitate subsequent filtration and chromatography steps.
Cell lysis to release virus particles
Purification
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Clarification Centrifugation
Centrifugation allows effective removal of microcarriers, but not so much for cell/debris separation; the viral yield recovery is typically low (≤30-50%)
Post-centrifugation clarification using double layer PES filter offer low throughput due to insufficient clarity of the post-centrifuge viral supernatant and cause additional losses of viral yield
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Clarification strategiesCell density & size matters
Low Concentration
High Concentration
Small particlesColloids
Charged Depth Filters
TFF Filters
Surface Filters
Non-charged Depth Filters
Filter capacities depend on cell density and degree of lysis and particle size distribution
Large/hard particles
Milligard, Polysep, Polygard CN
Polygard CR, Clarisolve 60HX
Prostak, Pellicon 2
Millistak+
Clarisolve 20/40 MS
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Case study: Clarification of AAV8 • Adeno-associated virus harvest clarification. Previous process
using a competitive depth filter.
• Clarisolve 20 MS was selected for primary clarification
• No pre-treatment required
• Adeno-associated virus harvest clarification. Previous processusing a competitive depth filter.
• Clarisolve 60 HX was tested for primary clarification.
Increase of 4 x throughput vs previous depth filtration filter = reduction of footprint
• No pre-treatment required
Depth filtration based clarification – primaryAAV case studies
20MS: Polypropylene & Millistak® media 60HX: Polypropylene media construction with no (+)-charged resin binder and no DE offer inert surface, hence no virus binding issues
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Millistak+ D0HC can be also used for the clarification of virus lysates despite of (+) charges (cellulose fibers with Diatomaceous earths).
Can be an option to test side by side with Clarisolve(similar format, same holder for large scale) & NFF filters
Example: Capacity range from 30 – 300 L/m2 for Adenovirus from Per.C6 cell harvest
Depth filtration based clarification Millistak+ for primary clarification
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NFF based clarification of AAVFiltration train example
Cell/Virus
Harvest
Polysep II
1.2/0.5 µm
Durapore0.45µm
Pellicon 2 Biomax 100-300kD Polygard CR
5µm
OR
Clarigard3 µm
Clarification Bioburdenreduction
Concentration
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Clarification systems with Single Use flow pathsSemi-automated systems
For POD formats (Millistak+ & Clarisolve®
For NFF filter capsules
Process Schematics of AAV vector production and Purification
Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201627
UF/DF BenzonaseTreatment
Primary Purification
Chromatography
Media and Inoculums Preparation
Cell growth in BioreactorHEK 293 Cells
Virus InoculationTransfection
VirusHarvest
PrimaryClarification
UF/DFSterile
Filtration
SecondaryClarification
SecondaryPurification
ChromatographyFill & Finish
Thaw and Expand
Cells and Seeds
Optional BenzonaseTreatment
Solution: Genetically engineered endonuclease that cleaves all forms of DNA and RNA.
Presence of Mg2+ (1-2 mM) is required for enzyme activity. One unit of Benzonase® degrades approximately 37µg DNA in 30 min to as
low as 3-8 base pairs (<6 kDa). Benzonase® can be detected with dedicated ELISA kit. Sensitivity 0.2 ng/ml
Nucleic Acid Digestion Benzonase® Endonuclease
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Regulatory: <10 ng nucleic
acid/dose
CharacteristicsOrigin Serratia marcescens
Expression E. Coli K-12 mutant
Molecular mass ~ 30 KD
Isoelectric point 6.85
Functional in pH range 6-10
Temperature 0-42°C
Bioprocess International, February 2014
Parameters Biomax 300kD
Feed flow (l/min/m²) 6
TMP (bar) <0.3
Initial flux (LMH) 30
Final flux (LMH) 30
Average flux (LMH) 30
Volumetric Concentration Factor 10
Diafiltration volume 2
Purification: First StepTarget: 25-fold conc + DF, vector recovery of 90%
Success Criteria Good yield & Retention Higher purity and
Contaminant Removal- hcDNA- HCP- Spent Benzonase
SolutionPermeate controlPellicon 2 Biomax 300 kD, V Screen for Lentivirus100 kD for AAV
Lentivirus & AAV: Typical TFF process parameters
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TFF-MF (0.1-0.65 µm) and Open UF (300-1000 kDa) optimization
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Process Schematics of AAV vector production and Purification
Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201631
UF/DF BenzonaseTreatment
Primary Purification
Chromatography
Media and Inoculums Preparation
Cell growth in BioreactorHEK 293 Cells
Virus InoculationTransfection
VirusHarvest
PrimaryClarification
UF/DFSterile
Filtration
SecondaryClarification
SecondaryPurification
ChromatographyFill & Finish
Thaw and Expand
Cells and Seeds
Optional BenzonaseTreatment
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Purification of AAV Using cation exchange Fractogel EMD SO3- as primary step
“The recovery from the Fractogel SO3 − column was almost 100% based on the infective viral particle recovery. In our laboratory the Fractogel SO3 − has consistently shown a recovery between 80 and 100%, which is dependent upon the extraction procedures and the variability in the infective viral particle assay”
+ Benzonase®
Fractogel® EMD
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Purification of AAV Ion exchange schemes
(A) Two-step purification protocol involving a strong cation exchange chromatography resin (Fractogel SO3-) followed by a strong anion exchange resin (Fractogel TMAE)
(B) Capture of the AAV vector by anion exchange chromatography using a strong anion exchange resin (Fractogel TMAE) with subsequent polishing by gel filtration chromatography.
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Best method of separation is ultra centrifugation, but has challenges of scalabaility.
Ion exchange chromatography with dual shallow gradient (pH & conductivity) can be used.
pI of empty particles could be higher than that of packaged virions.
AAV empty capsid separation
SOURCE: Okada et al., (Sep. 2009) Scalable Purification of Adeno-Associated Virus Serotype 1 (AAV1) and AAV8 Vectors, Using Dual Ion-Exchange Adsorptive Membranes. Human Gene Therapy, 20:1013–1021
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TFF & chrom systems with Single Use flow pathsFully automated, recipe driven
Chromatography TFF (UF/DF)Controls Automated with recipe control CCP6
Operating pressure
Up to 60 psi
Operable fluid temp
4oC-30oC 4oC-45oC
Area Column diameter: 60 – 350 mm
System 1: 0.5-2.5 m2
System 2: 2.5-5 m2
SensorsPre & post columnpH, conductivity, UV, temperature, pressure
Added sensors for conductivity, UV
Flow Rate System 1: 0.1 – 2.2 L/minSystem 2: 1.6 - 8L/min
System 1: up to 18 L/minSystem 2: up to 28 L/min
OtherLinear & step gradient mix: Mixing range 10-90%Accuracy :+/-2 %
Mixing tank 50L to 100/200LOptional jacketed
Process Schematics of AAV vector production and Purification
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UF/DF BenzonaseTreatment
Primary Purification
Chromatography
Media and Inoculums Preparation
Cell growth in BioreactorHEK 293 Cells
Virus InoculationTransfection
VirusHarvest
PrimaryClarification
UF/DFSterile
Filtration
SecondaryClarification
SecondaryPurification
ChromatographyFill & Finish
Thaw and Expand
Cells and Seeds
Optional BenzonaseTreatment
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Screening for excipients that prevent AAV2 vector aggregation
SOURCE: Wright et al., 2005. Identification of Factors that Contribute to Recombinant AAV2 Particle Aggregation and Methods to Prevent Its Occurrence during Vector Purification and Formulation. Mol. Ther., 12(1): 171-178.
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Formulation Storage & Transport
Range of formulation buffers and excipients to ensure long-term stability
• Buffers (ex: Tris, HEPES, PBS)
• Salts (freeze-dry)
• Stabilizers (ex: Polysorbate)
• Polyols: Manitol, sorbitol, PEG
• Sucrose, Trehalose (coming soon)
Emprove® dossier
Single Use Mixing & transport technologies
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Bioburden monitoring is essential (<10 cfu/ml) Durapore 0.22µm filter can be used as final filter
and had to undergo retrospective validation Process need to operate at low pressure to avoid
product loss during filtration Lot release will depend on sterility testing of final
dosage form & filter IT test
Sterile filtration and storage
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Wide range of buffers in solid or liquid forms• Biological buffers (organic, ex amino-acids)
• Purfication & formulation buffers (ex: NaOH, NaCl, PBS, etc..)
• Cleaning in place (ex: NaOH, HCl)
• Emprove® dossier c
Buffers
Liquid buffers: dual sourcing strategy • Berlin, Germany (batch size: up to 2000 L)
• Irvine, Scotland (batch size: up to 10000 L)
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Final FillingComponent choice
CriteriaGamma compatibility
>40kGy
Statement of animal origin
USP<88> Class VIpost-gamma >40kGy
USP<85> Endotoxin,post-gamma >40kGy
USP<788> Particulates,post-gamma >40kGy
USP<661> Physicochemical,post-gamma >40kGy
Shelf life >2.5 years, post-gamma>40kGy
Total Bioburdenpre gamma
Bacteriastatis/Fungistasis,Post-gamma >40kGy
Configurable Assembly
Component Library
All availableComponents
Solutions from Merck for AAV vector production and Purification
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Pellicon 2Biomax 100 kD
BenzonaseELISA Kit IIMobius Mix
Fractogel TMAE
Express HPF > SHR Mobius BioreactorNovAseptum SamplingAervent (vent filter)
Mobius Mix
Polygard CR 5µmClarisolve 60HX
Pellicon 2Biomax 300 kD
Durapore0.22µm
Polysep II 2.0/1.2
Fractogel DMAE
Mobius IntegratedFill FinishSolutions
Thaw and Expand
Cells and Seeds
Optional BenzonaseTreatment
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Benzonase® Treatment Clarification Intermediate TFF
Tangential Flow Filtration
Last StepDrug
Substance
First stepCell thawing
Cell culture (perfusion) – 5 weeks Virus infection – 2 days
3-6
weeks
USPweek 1 to 5
DSPweek 5
DSPweek 6
Final Filtration
2 stage Ion Exchange Chromatography
Virus vector– Single Use Process manufacturing overview
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Quality-control assays for clinical-grade AAV vectors
SOURCE: Arie van Oorschot (UniQure) Setting up a market scale manufacturing platform from scratch. Cell Culture World Conference. 23 February 2016
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Schematic of the manufacturing steps associated testing regimes for a cell therapy production process
Source: Alison Armstrong (2016) Advances in Assay Technologies for CAR T-Cell Therapies. BioPahrm International, 28(2): 32-37.
Quality, Science and Services You Can Trust
Merck Life Science (SAFC and BioReliance) provide the greatest array of process development, manufacturing and testing support services for our clients.
Viral Based Gene Therapy products (Adenovirus, AAV, Retrovirus, Lentivirus, others)
Cell Banking Viral Banking Bulk Drug Substance Bulk Drug Product Custom Assay Development BioSafety Testing
Viral Vector Production at SAFC Carlsbad Facility
Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201646
47 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016
Merck offers wide range of technology, tools
and services for Production and purification of
Viral vectors for gene and cell therapy
Thank You
Priyabrata Pattnaik, [email protected]
@pattnaik_p
https://sg.linkedin.com/in/priyabratapattnaik
https://plus.google.com/109816383630328905377