title: A simple Pichia pastoris fermentation and downstream processing strategy for making recombinant pandemic Swine Origin Influenza a virus Hemagglutinin protein.
abstract: The present Influenza vaccine manufacturing process has posed a clear impediment to initiation of rapid mass vaccination against spreading pandemic influenza. New vaccine strategies are therefore needed that can  accelerate the vaccine production. Pichia offers several advantages for rapid and economical bulk production of recombinant proteins and, hence, can be attractive alternative for producing an effective influenza HA based subunit vaccine. The recombinant Pichia harboring the transgene was subjected to fed-batch fermentation at 10 L scale. A simple fermentation and downstream processing strategy is developed for high-yield secretory expression of the recombinant Hemagglutinin protein of pandemic Swine Origin Influenza A virus using Pichia pastoris via fed-batch fermentation. Expression and purification were optimized and the expressed recombinant Hemagglutinin protein was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot and MALDI-TOF analysis. In this paper, we describe a fed-batch fermentation protocol for the secreted production of Swine Influenza A Hemagglutinin protein in the P. pastoris GS115 strain. We have shown that there is a clear relationship between product yield and specific growth rate. The fed-batch fermentation and downstream processing methods optimized in the present study have immense practical application for high-level production of the recombinant H1N1 HA protein in a cost effective way using P. pastoris.
authors.affiliation: Bioprocess and Scale Up Facility, Defence Research and Development Establishment, Ministry of Defence, Govt. of India, Gwalior 474 002, MP, India. athmabiotech@gmail.com
authors.forename: T N
authors.initials: TN
authors.lastname: Athmaram
authors.forename: Anil Kumar
authors.initials: AK
authors.lastname: Singh
authors.forename: Shweta
authors.initials: S
authors.lastname: Saraswat
authors.forename: Saurabh
authors.initials: S
authors.lastname: Srivastava
authors.forename: Princi
authors.initials: P
authors.lastname: Misra
authors.forename: M
authors.initials: M
authors.lastname: Kameswara Rao
authors.forename: N
authors.initials: N
authors.lastname: Gopalan
authors.forename: P V L
authors.initials: PV
authors.lastname: Rao
mesh_terms: D000818:Animals; D018533:Biomass; D019149:Bioreactors; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000032:analysis; D005285:Fermentation; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis* / Q000235:genetics; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics* / Q000378:metabolism; D009976:Orthomyxoviridae Infections / Q000453:epidemiology / Q000821:virology; D058873:Pandemics; D010843:Pichia / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013552:Swine / Q000821:virology*
chemical_list: D004731:Endotoxins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011994:Recombinant Proteins