Monday, July 30, 2007
P30

High Throughput Strain Evaluation for Therapeutic Protein Expression using Pfenex Expression Technology™

Hongfan Jin1, Diane Retallack1, J. Carrie Schneider1, Russell Coleman2, Ying Shao1, Huizhu Liu1, Kathryn Woodard1, and Lawrence Chew1. (1) Core Biotechnology R&D, The Dow Chemical Company, 5501 Oberlin Dr, San Diego, CA 92121, (2) Biopharma, The Dow Chemical Company, 5501 Oberlin Dr, San Diego, CA 92121

Multiple factors can affect product yield and quality, for example: protease degradation, protein folding/ inclusion body formation, efficient secretion, and correct disulfide bond formation. Finding the best expression host quickly is critical for speed to the clinic. Microbial expression systems are particularly amenable to rapid host strain development because of the rapid doubling time of these organisms, available genome information, reliable molecular biology tools and their ready scalability for manufacturing. The Pfenex Expression Technology™ platform, a robust Pseudomonas fluorescens-based system for the expression of recombinant proteins has been developed for the expression of therapeutic proteins. We have assembled useful host strains to enable proper protein folding, secretion and/or disulfide bond formation.  These, together with a set of compatible plasmids with a variety of transcription and translation regulators as well as a collection of periplasmic secretion signals, enable construction of hundreds of combinations to be tested in parallel to identify the optimal expression strain. High throughput (HTP) transformation and expression of recombinant therapeutic proteins can be readily performed in P. fluorescens to identify optimal expression plasmid/host strain combinations. As an example, human gamma-interferon was expressed in 90 P. fluorescens host strains and analyzed in a HTP format using 96-well SDS-CGE.  Within two weeks, several host strains were identified with greatly improved soluble expression of gamma-interferon as compared to the P. fluorescens wild type strain. We have observed that growth and expression results obtained through HTP expression analyses are typically reproducible at the 20L fermentation scale.

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