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Lysis-free extraction of biopharmaceuticals from the periplasm of Clean Genome E. coli

Blattner, Frederick R
Scarab Genomics, LLC
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Thirty percent of recombinant therapeutic proteins are made in E. coli and include important vaccinecomponents such as carrier proteins. Further, burgeoning new immunotherapies that use proteins such assingle chain antibodies and virus-like particles are enjoying increasing success. To meet the production needsof these expanding areas, Scarab Genomics proposes to extend the versatility of its Clean Genome® E.coliproduction system to include a method by which recombinant therapeutic proteins delivered into the cellperiplasm can be extracted directly into the culture medium for easy purification. Combined with Scarab?sextended fermentation platform, which enables high-density growth of Clean Genome E. coli and the constantproduction of target protein, a simplified recombinant protein extraction method will expand Scarab?s platformand facilitate continuous production, as mandated by the FDA. The development of an extraction method willreduce production time and effort, greatly simplify purification of therapeutic proteins such as the carrier proteinCRM197 and dramatically reduce total production costs. Preliminary experiments using the carrier proteinsCRM197, Haemophilus protein D and Pseudomonas exoprotein A indicate that subtle changes to theproduction host strain combined with a proprietary extraction buffer can induce up to 90% recovery ofrecombinant periplasmic protein. Further, recombinant therapeutic proteins released into the medium werestable for more than two weeks further emphasizing the versatility of the system. In Phase 1, fermentation andextraction conditions for the test carrier protein CRM197 will be improved and reagents needed to optimizeexpression and extraction will be developed. The first aim of Phase 2 proposes to determine whether deletionsof key transpeptidase enzymes that are important in the stability of the periplasm, and which have been shownto enhance the release of recombinant proteins from the periplasm, can improve extraction. Similarly, theinfluence of peptidoglycan-modifying enzymes expressed from the production plasmid will be examined in thecontext of the extraction method. The second aim will examine the expression and extraction of four targetproteins in extended fermentation to test the extraction system at production scale. In addition to the carrierproteins Haemophilus protein D and Pseudomonas exoprotein A, a single chain antibody and a single-chainvariable fragment will be examined. The antibody fragments will also be subjected to purification to confirm thatextraction of recombinants into culture medium simplifies purification, as was evident for carrier proteins. Thethird aim proposes to devise a method for removing cells and cell debris from the extracted solution with thegoal of providing continuous chromatographic systems with clarified material for a truly continuous process.
Funding Source
Nat'l. Inst. of Allergy and Infectious Diseases
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Escherichia coli
Bacterial Pathogens