title: Evaluation of different primary recovery methods for E. coli-derived recombinant human growth hormone and compatibility with further down-stream purification.
abstract: Due to advances in fermentation technology, it is now possible to obtain fermentation broth with over 30% solids. The high solid content makes the clarification step difficult, especially at large scale. The primary protein recovery step is challenging due to the heterogeneous solution of soluble and insoluble material. In this study, we compare different primary recovery routes and the compatibility with the initial capture chromatography step. The primary recovery routes studied are standard clarification by centrifugation and extraction in aqueous two-phase systems. The compatibility of the feed streams from the different primary recovery steps with the first chromatography step is addressed. An anion-exchange column was used as the first capture column in the purification process. The aqueous two-phase system was composed of a random copolymer of ethylene oxide and propylene oxide (EOPO) in combination with a waxy starch. The target protein in this study was human growth hormone (hGH) produced in recombinant Escherichia coli. The purity of hGH in the top phase after aqueous two-phase extraction was found to be significantly higher than in clarified homogenate supernatant and increased as the EOPO polymer concentration in the aqueous two-phase system increased. Stability of the supernatant and EOPO top phases and hGH were determined by turbidity measurements and LC-MS assay. All of the feed-streams from the primary recovery steps were compatible with the anion-exchange chromatography step; however, the capacity of the resin was strongly dependent on the purity of the load. Different process aspects, e.g., resin capacity, viscosity, purification, and yield of hGH and scalability are compared.
authors.affiliation: Genentech, Inc., 1 DNA Way, South San Francisco, CA, USA.
authors.forename: Josefine
authors.initials: J
authors.lastname: Persson
authors.forename: Dana C
authors.initials: DC
authors.lastname: Andersen
authors.forename: Philip M
authors.initials: PM
authors.lastname: Lester
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D019382:Human Growth Hormone / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007218:Industrial Microbiology / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D019382:Human Growth Hormone