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Production and Purification of Virus Like Particle (VLP) based Vaccine
This document summarizes a presentation on the production and purification of virus-like particle (VLP) based vaccines. It discusses using hepatitis C VLPs as a model system produced using an insect cell/baculovirus expression platform. Key points covered include: - Challenges in VLP vaccine production include low yields, stability issues, and difficulties removing baculovirus. - Hepatitis C VLPs containing E1 and E2 glycoproteins were successfully produced using Sf9 insect cells in a Mobius 3L disposable bioreactor, with comparable results to a glass bioreactor. - A depth filtration clarification process achieved around 70% DNA clearance while recovering approximately 70
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Production and Purification of Virus Like Particle (VLP) based Vaccine
- 1. Priyabrata Pattnaik, PhD Director– Worldwide Vaccine Initiative Bioprocess & Technology Conference, 27-30 October 2015, Singapore PRODUCTION ANDPRODUCTION ANDPRODUCTION ANDPRODUCTION AND PURIFICATION OF VIRUSPURIFICATION OF VIRUSPURIFICATION OF VIRUSPURIFICATION OF VIRUS LIKE PARTICLE (VLP)LIKE PARTICLE (VLP)LIKE PARTICLE (VLP)LIKE PARTICLE (VLP) BASED VACCINEBASED VACCINEBASED VACCINEBASED VACCINE
- 2. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore2 Presentation Outline VLP as Hepatitis C vaccines Baculovirus / insect cell expression platform Challenges in VLP vaccine production and purification VLP production in insect cell culture Clarification of VLP Concentration / Diafiltration of VLP Chromatographic purification of VLP Summary
- 3. VLP vaccine candidateshave become quite popular of late VLP-based processes are, however, currently quite diverse We undertook an effort to standardize the process We used hepatitis C VLP as a model This presentation will explain the approach taken and present the results obtained Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore3 Motivation
- 4. 4 Production andPurification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore Contain repetitive high-density displays of viral surface proteins that elicit strong T cell and B cell immune responses Non infectious because they do not contain genetic material, thus cannot replicate and are safer Their size (40-120 nm diameter) is optimal for uptake by dendritic cells Can be produced in a variety of cell culture systems Can self assemble in vivo Proven technology (Hepatitis B and Human Papilloma Virus vaccines) Why virus-like particles (VLPs)? Source: Roldão et al., Expert Reviews in Vaccines 9 (10), 1149-76 (2010)
- 5. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore5 Hepatitis C 170 million people infected Cirrhosis, liver cancer, death Current therapies only partially effective, costly and poorly tolerated No vaccine currently exists VLPs for hepatitis C vaccine development E1 and E2 glycoproteins from Hep C virus Capsid and structure VLP from retrovirus (murine leukemia virus)
- 6. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore6 Recombinant baculovirus (BV) is used to infect insect cells Key features Transient production High cell densities Regulatory acceptance Cervarix® (GSK) Flublok® (Protein Sciences) Several late-stage clinicals Insect cell / baculovirus VLP production platform ~120 nm VLP BV 60-80 nm
- 7. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore7 Low production yields Stability of enveloped VLPs Difficulties in baculovirus (BV) removal lowers recovery No established platform processes for purification Challenges in VLP vaccine production VLP BV
- 8. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore8 Work carried out in collaboration with iBET iBET: Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal
- 9. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore9 Typical VLP-based vaccine process Insect cell / baculovirus VLP production platform UF/DF Baculovirus Inactivation Purification Chromatography Media and Inoculum Preparation Cell growth in Bioreactor and Virus Inoculation Bioburden Reduction Primary Clarification Sterile Filtration Polishing Chromatography UF/DF
- 10. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore10 Typical VLP-based vaccine process Insect cell / baculovirus VLP production platform UF/DF Baculovirus Inactivation Purification Chromatography Media and Inoculum Preparation Cell growth in Bioreactor and Virus Inoculation Bioburden Reduction Primary Clarification Sterile Filtration Polishing Chromatography UF/DF
- 11. 11 Production andPurification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore Cell culture was carried out in stirred tank glass bioreactor and disposable bioreactor (Mobius® 3L bioreactor) Sf9 insect cells and Sf900II cell culture media were used in the process Mobius® 3L bioreactor was first operated at same conditions previously used for stirred tank glass bioreactors Cell aggregation Formation of foam Longer lag phase Lower viable cell concentration Insect cell culture
- 12. 12 Production andPurification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore Increased agitation rate Increased cell density of inoculation Replaced micro sparger with an open-pipe sparger Insect cell culture conditions improved based on experience with Mobius® bioreactor 0 20 40 60 80 100 0.0 0.5 1.0 1.5 2.0 0 24 48 72 96 120 144 Viability(%) ViableCellConcentration(106 cells/ml) Time (h) STR2 CR2 CR3 CR4 Viab STR2 Viab CR2 Viab CR3 Viab CR4
- 13. 13 Production andPurification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore Microscopic evaluation of cells Stirred glass bioreactor Mobius® 3L bioreactor Run CR2 Run CR3 Run CR4 45 hrs 70 hrs 95 hrs
- 14. 14 Production andPurification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore Western blot analysis of VLPs using three markers GAG- MLV STR1 CR2 CR3 CR4 HCV- E2STR1 CR2 CR3 CR4 Sucrose cushion purified VLPs VLPs pelleted by ultracentrifugation HCV- E1STR1 CR2 CR3 CR4 GAG-MLV (core protein) HCV-E1 (envelope protein) HCV-E2 (envelope protein) VLP HepC V
- 15. 15 Production andPurification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore Successful growth of Sf9 insect cells and infection with baculovirus for production of VLP vaccine using Mobius® 3L disposable bioreactor Comparable cell and VLP properties between disposable and glass bioreactors Reproducible performance of the disposable bioreactor was seen with identical results for three separate cell culture runs Successful use of Mobius® bioreactor for VLP production
- 16. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore16 Typical VLP-based vaccine process Insect cell / baculovirus VLP production platform UF/DF Baculovirus Inactivation Purification Chromatography Media and Inoculum Preparation Cell growth in Bioreactor and Virus Inoculation Bioburden Reduction Primary Clarification Sterile Filtration Polishing Chromatography UF/DF
- 17. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore17 Depth filtration Well suited for smaller vaccine batches Easier to scale Lower cost Disposable Gentle treatment Simpler process development Wide choice of depth filters Centrifugation Lab models used early on Well suited for large-scale production High capital expense Shear Clarification
- 18. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore18 Clarification: throughput data Disposable capsule filters Polygard® CN, nominal pore sizes of 10, 5, 0.6 and 0.3 µm Pleated, all-polypropylene depth filters Filter area: 17 cm2; Inlet flux: 988 LMH 0.0 0.5 1.0 1.5 2.0 0 50 100 150 200 250 300 350 Inletpressure(bar) Volume filtered (L/m2) Polygard CN filter train 5 0.3 µm 10 µm 5 µm 0 0.2 0.4 0.6 0.8 1 1.2 0 50 100 150 200 250 300 350 Inletpressure(bar) Volume filtered (L/m2) Polygard CN filter train 10 5 0.6 µm 10 µm 5 µm 0.6 µm
- 19. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore19 Clarification: recovery data Unlike centrifugation, depth filtration resulted in ~70% DNA clearance 0% 20% 40% 60% 80% 100% 10 μm → 5 μm → 0.6 μm 10 μm → 0.6 μm 5 μm → 0.6 μm 5 μm → 0.3 μm CFG → 0.6 μm CFG → 0.3 μm CFG VLP recovery HepC VLP clarification
- 20. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore20 Typical VLP-based vaccine process Insect cell / baculovirus VLP production platform UF/DF Baculovirus Inactivation Purification Chromatography Media and Inoculum Preparation Cell growth in Bioreactor and Virus Inoculation Bioburden Reduction Primary Clarification Sterile Filtration Polishing Chromatography UF/DF
- 21. 21 Production andPurification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore Pellicon® cassettes Two different ultrafiltration membranes 300 kD composite regenerated cellulose (Ultracel® membrane, “CRC”) 100 kD polyethersulfone (Biomax® membrane, “PES”) Similar process conditions employed 4-5x concentration factor Loading: 72 L/m2; Feed flux: 480 LMH; TMP: 1 bar; Pfeed: 0.6-0.9 bar; Pretent: 1.1-1.4 bar Concentration of clarified VLP harvest
- 22. 22 Production andPurification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore Concentration of clarified VLP harvest – results 90% 35% 85% 80% 28% 91% 58% 90% 96% 38% 0% 25% 50% 75% 100% HCV-VLP recovery % BV removal % Total Protein removal % DNA removal % HCP removal% Pellicon® (PES 100 kD) Pellicon® (CRC 300 kD) Both membranes were fully retentive of the VLP
- 23. 23 Production andPurification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore Clarification Filter-only clarification train can be used without compromising recovery yield of VLPs. Filter cascade composed of a Polygard® CN 5 µm filter followed by a 0.3 µm depth filter showed the highest recovery of HCV-VLP, improving on centrifugation/2° depth filtration Moderate DNA removal with depth filtration was seen UF/DF Pellicon® cassette with 300 kD regenerated cellulose membrane offered the best combination of recovery and purification Polygard® CN depth filters and Pellicon® cassettes with Ultracel® membrane offered best results
- 24. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore24 Typical VLP-based vaccine process Insect cell / baculovirus VLP production platform UF/DF Baculovirus Inactivation Purification Chromatography Media and Inoculum Preparation Cell growth in Bioreactor and Virus Inoculation Bioburden Reduction Primary Clarification Sterile Filtration Polishing Chromatography UF/DF
- 25. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore25 Purification strategy Anion exchange chromatography (AEX) resins used Identify purification goal Ensure analytics are available Batch adsorption Resin in multiwell plates Vary pH, conductivity Measure recovery, purity Chrom bind/elute Prepacked columns Confirm batch adsorption Chrom breakthrough Prepacked columns Capacity measurements Scale up Iterations… Iterations…
- 26. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore26 Batch adsorption experiments (bind-elute) Fractogel® and two anion exchnage prototypes approach target of 2 BV LRV Yield increases with increasing ligand density for prototypes DMAE TMAE Q92 Q17 Q54 Q48 0 1 2 3 0 20 40 60 80 BVLRV %yield ligand density (µmol/g) Load at 150 mM, elute at 400+700 mM Fractogel® – red AEX prototypes – blue Fractogel® – dotted line DMAE TMAE Q92 Q17 Q54 Q48 0 1 2 3 0 20 40 60 80 BVLRV % VLP yield Load at 150 mM, elute at 400+700 mM
- 27. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore27 Batch adsorption experiments (flow-through) Inadequate performance in pure flow-through mode; Similar trends with ligand density Adopted strategy: collect the flow-through fraction, then wash/elute the resin to recover more material DMAE TMAE Q92 Q17 Q54 Q48 0 1 2 3 0 20 40 60 80 BVLRV %yield ligand density (µmol/g) Flow-through at 300 mM DMAE TMAE Q92 Q17 Q54 Q48 0 1 2 3 0 20 40 60 80 BVLRV % VLP yield Flow-through at 300 mM Fractogel® – red AEX prototypes – blue Fractogel® – dotted line
- 28. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore28 Column experiments Breakthrough curves for dynamic binding capacity 10% dynamic binding capacity ranges at 900-1300 ng VLP / mL of packed resin The prototype resins has about 30% higher DBC compared to Fractogel® 0 500 1000 1500 2000 Q17 Q48 Q54 Q92 TMAE DMAE DBC(ngVLP/mLofresin) 10% 50% Prototype AEX Fractogel® Q17 Q48 Q54 Q92 TMAE DMAE 0 20 40 60 80 800 1000 1200 1400 %yield 10% DBC (ng VLP / mL of resin)
- 29. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore29 DOE of flow-through conditions: Fractogel® TMAE Inputs: load NaCl (100/200/300 mM) and flow rate (100/200/400 cm/hr) Responses: % VLP recovery and BV LRV Higher flow rate OR Higher load conductivity Recovery LRV Flow rate (mL/min) NaCl(mM) Higher recovery AND Lower BV LRV
- 30. 30 Production andPurification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore Successfully purified VLPs using Fractogel® TMAE commercial resins and AEX prototype resins Yield of >60% with ~2 LRV baculovirus can be achieved with a flow-through/wash purification strategy for both resins Options to increase recovery or purification depending on product value by varying process conditions Successful purification of VLPs using Fractogel® and prototype AEX chromatographic resins
- 31. 31 Production andPurification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore Optimum performance achieved Traditional lab process New scalable process Purity Baculovirus clearance 94% 97.6% DNA clearance DNA 99.9% HCP clearance HCP 82% Recovery by P30 ELISA VLP recovery VLP < 10% ~ 65%
- 32. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore32 Typical VLP-based vaccine process Insect cell / baculovirus VLP production platform UF/DF Baculovirus Inactivation Media and Inoculum Preparation Bioburden Reduction Sterile Filtration Polishing Chromatography Mobius® bioreactor Polygard®-CN 5.0 0.3 µm filters Fractogel® AEX resins Pellicon® Ultrafiltration cassettes Ultracel® 300 kD membrane
- 33. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore33 Typical VLP-based vaccine process Insect cell / baculovirus VLP production platform Mobius® Bioreactor Polygard®-CN 5.0 0.3 µm filters Pellicon® Ultrafiltration cassettes Ultracel® 300 kD membrane Fractogel® AEX resins
- 34. 34 Production andPurification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore Successfully used Mobius® 3L disposable bioreactor for production of VLP-based vaccine in insect cell culture system Optimized downstream processing using Polygard® CΝ 5.0 0.3 µm depth filters followed by UF/DF using Pellicon® cassette with Ultracel® 300 kD membrane Purified VLP by using Fractogel® commercial resins and anion exchange prototype resins Integrated all the above components to achieve recovery and impurity clearance in line with requirements Summary
- 35. Production and Purificationof Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore35 EMD/Merck Millipore Alex Xenopoulos Elina Gousseinov Shannon Ryan Beth Goodridge Achim Schwaemmle Andreas Stein Annika Aldinger Sylvain Ribaud Lenaig Savary Cristina Peixoto Ricardo Silva Rute Castro Ana Sofia Coroadinha Paula Alves Manuel Carrondo Team and acknowledgments
- 36. THANK YOUTHANK YOUTHANKYOUTHANK YOU 36 Priyabrata Pattnaik, PhD priyabrata.pattnaik@merckgroup.com @pattnaik_p https://sg.linkedin.com/in/priyabratapattnaik https://plus.google.com/109816383630328905377