• Document: Production and Purification of Virus like particle (VLP) based Vaccine. Priyabrata Pattnaik, PhD Director Worldwide Vaccine Initiative
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Production and Purification of Virus like particle (VLP) based Vaccine Priyabrata Pattnaik, PhD Director – Worldwide Vaccine Initiative Outline 1 VLPs as Hepatitis C vaccines 2 Baculovirus / insect cell expression platform 3 Challenges in VLP vaccine production and purification 4 VLP production in insect cell culture 5 Clarification of VLP 6 Concentration / Diafiltration of VLP 7 Chromatographic purification of VLP 8 Summary 2 Motivation  VLP vaccine candidates have 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 3 Why virus-like particles (VLPs)?  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) 4 Source: Roldão et al., Expert Reviews in Vaccines 9 (10), 1149-76 (2010) VLPs for hepatitis C vaccine development E1 and E2 glycoproteins from Hep C virus Hepatitis C  170 million people infected  Cirrhosis, liver cancer, death  Current therapies only partially effective, costly and poorly tolerated  No vaccine currently exists Capsid and structure VLP from retrovirus (murine leukemia virus) 5 Insect cell / baculovirus VLP production platform 60-80 nm Recombinant baculovirus (BV) is used to infect insect cells ~120 nm Key features Transient production High cell densities Regulatory acceptance VLP  Cervarix® (GSK) BV  Flublok® (Protein Sciences)  Several late-stage clinicals 6 Source: Mena et al., Expert Reviews in Vaccines 10 (1), 1063-81 (2011) Challenges in VLP vaccine production  Low production yields  Stability of enveloped VLPs  Difficulties in baculovirus (BV) removal lowers recovery  No established platform processes for purification BV VLP 7 Work carried out in collaboration with iBET iBET: Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal 8 Typical VLP-based vaccine process Insect cell / baculovirus VLP production platform Media and Inoculum Cell growth in Bioreactor Primary Bioburden UF/DF Preparation Virus Inoculation Clarification Reduction Sterile Polishing Baculovirus Purification UF/DF Filtration Chromatography Inactivation Chromatography 9 Typical VLP-based vaccine process Insect cell / baculovirus VLP production platform Media and Inoculum Cell growth in Bioreactor Primary Bioburden UF/DF Preparation Virus Inoculation Clarification Reduction Sterile Polishing Baculovirus Purification UF/DF Filtration Chromatography Inactivation Chromatography 10 Insect cell culture  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 11 Insect cell culture conditions improved based on experience with Mobius® bioreactor  Increased agitation rate  Increased cell density of inoculation  Replaced micro

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