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Sophie Vazulka

Sophie Vazulka

University of Natural Resources and Life Sciences, Austria

Title: Production of Antibody Fragments with Plasmid-based and Genome Integrated T7 E. coli Expression Systems – Evaluation of Systems Performance in Microtiter Fed-batch like Cultivations

Biography

Biography: Sophie Vazulka

Abstract

Although E. coli is the most prominent bacterial production host for recombinant proteins, some proteins with high economic potential can still hardly be produced at remunerative levels. We selected four different fabs (fragment antigen binding) (BIBH1, BIWA4, CIMZIA and FabX) with identical constant domains representing such challenging proteins. Fab yield can be affected by miss-folding, aggregation or unbalanced expression, translation and translocation levels of sub-units making it still challenging to efficiently design expression systems and production processes. For translocation to the periplasm a post-translational (OmpA) and a co-translational (DsbA) leader sequence were used. E. coli BL21(DE3) and E. coli HMS174 (DE3) were transformed either via pET vectors or genome integration. The resulting 32 clones were cultivated under fed-batch like conditions in the BioLector. Cell growth was not affected by leader/fab combinations but the yield of correctly folded fab ranged from 0 to 12.5 mg/g CDM. Higher expression rates caused higher amounts of free light chain and K12 strain reached higher yields. Except of CIMZIA with DsbA leader, genome integrated versions showed higher fab yields, reduced levels of free light chain and basal expression than plasmid-based systems. Independent of the expression system used, highest yields were obtained with CIMZIA, followed by BIWA4, BIBH1 and FabX. Leader sequence cleavage efficiency for DsbA was significantly lower than for OmpA, both showed lowest with CIMZIA. Summarizing, we showed that the selected set of host/gene dosage/leader/fab combinations resulted in a broad range of variation in terms of fab yields and processing and will be studied in detail during bench-scale fermentations.