Production and purification of Viral vectors for gene and cell therapy applications
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The cell and gene therapy market is growing rapidly and is projected to reach $10 billion in 5 years. There are three main segments: gene therapy, stem cell therapy, and cell immunotherapy. Gene therapy uses viral vectors like lentivirus or adenovirus to deliver nucleic acids. The production of viral vectors like AAV involves growing HEK 293 cells in bioreactors, transfecting them with plasmids, harvesting and purifying the virus through clarification, filtration, and chromatography. CAR-T cell therapy is also discussed as an example of cell immunotherapy, which uses lentivirus to modify patient T-cells that are then reintroduced to the patient.
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Production and purification of Viral vectors for gene and cell therapy applications
- 1. Priyabrata Pattnaik, PhD Director & Head of Biologics Operations Asia Pacific Production and purification of Viral vectors for gene and cell therapy applications
- 2. The cell & gene therapy market is poised for rapid growth with projections reaching ~$10B in 5 years Source: Seed Planning; METI; Kuick Research; Med Market Diligence;Transparency market research Global market size & CAGR USD B Products in the pipeline 74 74 34 54 75 Stem cell therapy 106 17 14 Cellular therapy, other 123 15 Gene therapy 144 16 Phase I Phase II Phase III 10 9 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
- 3. 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 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 20163
- 4. Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016 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-T 1. Most common 2. Immune cells separation 3. Lentivirus-based delivery 4. Chimeric Antigen Receptors (CARs) Allogeneic modified T cells 1. Industrialized trend 2. Genetic modification of immune cells 3. Allogeneic T cell generation 4. Lentivirus-based delivery or genome edit (CRISPR) CAR-TVirus Stem cells T-cells Virus CAR-T 4
- 5. 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. Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 20165
- 6. Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016 Overall Process Map of CAR-T T-cell separation Virus generation GMP-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 6
- 7. 7 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016 Viral Vectors For Gene Therapy Introduction 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
- 8. 8 ~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 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016
- 9. 9 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016 Features AAV Lentivirus Genome ssDNA ssRNA(+) Virus Coat Non-Enveloped Enveloped Diameter 18 – 26nm 80 – 130nm Packaging size 4.5 kb 8 kb Infection Range Dividing & Non-Dividing Cells Mostly Dividing Post Infection Mostly Non-Integrating* Integrates into Host Genome Gene Expression Long lasting (?) Prolonged Main Advantage Non-pathogenic; Non-inflamatory Persistent Gene Transfer Main Disadvantage Small packaging capacity Oncogenesis may occur Characteristics of AAV & Lentivirus ^ AAV (mainly serotype 2) may integrate into Chromosome 19
- 10. 10 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016 AAV Serotype Tissue Tropism AAV1 Muscle, Heart, Ocular, CNS AAV2 CNS, Kidney, Muscle, Testes Various in vitro applications AAV4 Lung, AAV5 Lung, CNS, Ocular, Pancreas AAV6 Lung, Heart AAV7 Muscle, Liver AAV8 Liver, Muscle, Ocular, CNS, Heart AAV9 Lung, Liver, Muscle, Heart, CNS, Kidney, Testes AAVrh10 Pleura, CNS Other Various AAV Serotypes and Tissue Tropism
- 11. 11 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016 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
- 12. Sufficient Production of virus vector copies Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201612 Adenovirus vectors manufacturing Key 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
- 13. Process Schematics of AAV vector production and Purification Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201613 UF/DF Benzonase Treatment Primary Purification Chromatography Media and Inoculums Preparation Cell growth in Bioreactor HEK 293 Cells Virus Inoculation Transfection Virus Harvest (Lysis) Primary Clarification UF/DF Sterile Filtration Secondary Clarification Secondary Purification ChromatographyFill & Finish Thaw and Expand Cells and Seeds Optional Benzonase Treatment
- 14. Process Schematics of AAV vector production and Purification Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201614 UF/DF Benzonase Treatment Primary Purification Chromatography Media and Inoculums Preparation Cell growth in Bioreactor HEK 293 Cells Virus Inoculation Transfection Virus Harvest Primary Clarification UF/DF Sterile Filtration Secondary Clarification Secondary Purification ChromatographyFill & Finish Thaw and Expand Cells and Seeds Optional Benzonase Treatment
- 15. Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201615 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 MCB Innoculation Shake/spin
- 16. Sf-9 HEK 293 EB66® Cells BHK21 Sf-9 HEK 293 EB66® Cells 16 Minimize Your Process Development Efforts Leverage Merck’s Experience with Various Cell Lines in Mobius® Bioreactors CHO Hybridoma SP 2/0 Vero h-MSC T 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) Cell Therapy Virus production (Adherent) h-MSC HepaRG® CHO Hybridoma *application data available * * * * * * * * * * * * * * * * * * * * * Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016
- 17. 17 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016 • 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 µm Sterilizing Or Viresolve® Barrier (available soon) Lynx sterile connectors
- 18. 18 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016 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 Virus barrier: filtration of cell culture media for example using Viresolve® barrier • Sterile Sampling with NovaSeptum • Aervent filters for the aeration (0.22µ) • Sterile connectors (Lynx)
- 19. Process Schematics of AAV vector production and Purification Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201619 UF/DF Benzonase Treatment Primary Purification Chromatography Media and Inoculums Preparation Cell growth in Bioreactor HEK 293 Cells Virus Inoculation Transfection Virus Harvest (Lysis AAV) Primary Clarification UF/DF Sterile Filtration Secondary Clarification Secondary Purification ChromatographyFill & Finish Thaw and Expand Cells and Seeds Optional Benzonase Treatment
- 20. 20 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016 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
- 21. Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201621 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
- 22. Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201622 Clarification strategies Cell density & size matters Low Concentration High Concentration Small particles Colloids 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
- 23. 23 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016 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 process using 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 – primary AAV 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
- 24. 24 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016 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
- 25. 25 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016 NFF based clarification of AAV Filtration train example Cell/Virus Harvest Polysep II 1.2/0.5 µm Durapore 0.45µm Pellicon 2 Biomax 100-300kDPolygard CR 5µm OR Clarigard 3 µm Clarification Bioburden reduction Concentration
- 26. Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201626 Clarification systems with Single Use flow paths Semi-automated systems For POD formats (Millistak+ & Clarisolve® For NFF filter capsules
- 27. Process Schematics of AAV vector production and Purification Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201627 UF/DF Benzonase Treatment Primary Purification Chromatography Media and Inoculums Preparation Cell growth in Bioreactor HEK 293 Cells Virus Inoculation Transfection Virus Harvest Primary Clarification UF/DF Sterile Filtration Secondary Clarification Secondary Purification ChromatographyFill & Finish Thaw and Expand Cells and Seeds Optional Benzonase Treatment
- 28. 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 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201628 Regulatory: <10 ng nucleic acid/dose Characteristics Origin 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
- 29. 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 Step Target: 25-fold conc + DF, vector recovery of 90% Success Criteria Good yield & Retention Higher purity and Contaminant Removal - hcDNA - HCP - Spent Benzonase Solution Permeate control Pellicon 2 Biomax 300 kD, V Screen for Lentivirus 100 kD for AAV Lentivirus & AAV: Typical TFF process parameters 29 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201629
- 30. TFF-MF (0.1-0.65 µm) and Open UF (300-1000 kDa) optimization Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201630
- 31. Process Schematics of AAV vector production and Purification Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201631 UF/DF Benzonase Treatment Primary Purification Chromatography Media and Inoculums Preparation Cell growth in Bioreactor HEK 293 Cells Virus Inoculation Transfection Virus Harvest Primary Clarification UF/DF Sterile Filtration Secondary Clarification Secondary Purification ChromatographyFill & Finish Thaw and Expand Cells and Seeds Optional Benzonase Treatment
- 32. Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201632 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
- 33. Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201633 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.
- 34. 34 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016 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
- 35. Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201635 TFF & chrom systems with Single Use flow paths Fully automated, recipe driven Chromatography TFF (UF/DF) Controls Automated with recipe control CCP6 Operating pressure Up to 60 psi Operable fluid temp 4o C-30o C 4o C-45o C Area Column diameter: 60 – 350 mm System 1: 0.5-2.5 m2 System 2: 2.5-5 m2 Sensors Pre & post column pH, conductivity, UV, temperature, pressure Added sensors for conductivity, UV Flow Rate System 1: 0.1 – 2.2 L/min System 2: 1.6 - 8L/min System 1: up to 18 L/min System 2: up to 28 L/min Other Linear & step gradient mix: Mixing range 10-90% Accuracy :+/-2 % Mixing tank 50L to 100/200L Optional jacketed
- 36. Process Schematics of AAV vector production and Purification Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201636 UF/DF Benzonase Treatment Primary Purification Chromatography Media and Inoculums Preparation Cell growth in Bioreactor HEK 293 Cells Virus Inoculation Transfection Virus Harvest Primary Clarification UF/DF Sterile Filtration Secondary Clarification Secondary Purification ChromatographyFill & Finish Thaw and Expand Cells and Seeds Optional Benzonase Treatment
- 37. Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201637 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.
- 38. Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201638 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
- 39. 39 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016 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
- 40. 40 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016 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)
- 41. 41 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016 Final Filling Component choice Criteria Gamma compatibility >40kGy Statement of animal origin USP<88> Class VI post-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 Bioburden pre gamma Bacteriastatis/Fungistasis, Post-gamma >40kGy Configurable Assembly Component Library All available Components
- 42. Solutions from Merck for AAV vector production and Purification Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201642 Pellicon 2 Biomax 100 kD Benzonase ELISA Kit II Mobius Mix Fractogel TMAE Express HPF > SHR Mobius Bioreactor NovAseptum Sampling Aervent (vent filter) Mobius Mix Polygard CR 5µm Clarisolve 60HX Pellicon 2 Biomax 300 kD Durapore 0.22µm Polysep II 2.0/1.2 Fractogel DMAE Mobius Integrated Fill Finish Solutions Thaw and Expand Cells and Seeds Optional Benzonase Treatment
- 43. Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201643 Benzonase® Treatment Clarification Intermediate TFF Tangential Flow Filtration Last Step Drug Substance First step Cell thawing Cell culture (perfusion) – 5 weeks Virus infection – 2 days 3-6weeks USP week 1 to 5 DSP week 5 DSP week 6 Final Filtration 2 stage Ion Exchange Chromatography Virus vector– Single Use Process manufacturing overview
- 44. Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 201644 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
- 45. 45 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016 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
- 46. 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. 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
- 48. Thank You Priyabrata Pattnaik, PhD priyabrata.pattnaik@merckgroup.com @pattnaik_p https://sg.linkedin.com/in/priyabratapattnaik https://plus.google.com/109816383630328905377