title: A Simplified and Efficient Process for Insulin Production in Pichia pastoris.
abstract: A significant barrier to insulin is affordability. In this manuscript we describe improvements to key steps in the insulin production process in Pichia pastoris that reduce cost and time. The strategy for recovery and processing of human insulin precursor has been streamlined to two steps from bioreactor to the transpeptidation reaction. In the first step the insulin precursor secreted during the methanol induction phase is recovered directly from the culture broth using Tangential Flow Filtration with a Prostak™ module eliminating the laborious and time-consuming multi-step clarification, including centrifugation. In the second step the protein is applied at very high loadings on a cation exchange resin and eluted in a mixture of water and ethanol to obtain a concentrated insulin precursor, suitable for use directly in the transpeptidation reaction. Overall the yield from insulin precursor to human insulin was 51% and consisted of three purification chromatography steps. In addition we describe a method for recovery of the excess of H-Thr(tBu)-OtBu from the transpeptidation reaction mixture, one of the more costly reagents in the process, along with its successful reuse.
authors.affiliation: ICGEB, Trieste, Italy.
authors.forename: Sulena
authors.initials: S
authors.lastname: Polez
authors.affiliation: Biomanufacturing Sciences Network, Process Solutions, Merck SpA, Vimodrone (Milan), Italy.
authors.forename: Domenico
authors.initials: D
authors.lastname: Origi
authors.affiliation: ICGEB, Trieste, Italy.
authors.forename: Sotir
authors.initials: S
authors.lastname: Zahariev
authors.affiliation: ICGEB, Trieste, Italy.
authors.forename: Corrado
authors.initials: C
authors.lastname: Guarnaccia
authors.affiliation: ICGEB, Trieste, Italy.
authors.forename: Sergio G
authors.initials: SG
authors.lastname: Tisminetzky
authors.affiliation: ICGEB, Trieste, Italy.
authors.forename: Nataša
authors.initials: N
authors.lastname: Skoko
authors.affiliation: ICGEB, Trieste, Italy.
authors.forename: Marco
authors.identifier: http://orcid.org/0000-0002-0417-1013
authors.initials: M
authors.lastname: Baralle
mesh_terms: D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D002845:Chromatography / Q000379:methods; D005285:Fermentation; D006801:Humans; D007328:Insulin / Q000096:biosynthesis* / Q000302:isolation & purification; D010843:Pichia / Q000378:metabolism*; D059748:Proteolysis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D007328:Insulin; D011994:Recombinant Proteins