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 integration into the host genome. The resulting 32 clones were cultivated in microtiter format, under fed-batch like, C-limited conditions (BioLector). Cell growth was not affected by different leader sequence/Fab combinations, but the yield of correctly folded, soluble Fab ranged from 0 to 7.4 mg/g CDM. In most cases the K12 strain reached higher yields then the B strain counterparts. Except of two systems, genome integrated systems showed higher Fab yields, reduced levels of free light chain, inclusion body formation and basal expression than the plasmid-based counterparts. 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 lower than for OmpA, both 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, which provides a platform for more detailed characterization in bench-scale fermentations. For characterization studies, the genome integrated systems showed to be suitable, due to good yields, elimination of plasmid-mediated variation and reduced metabolic load, compared to plasmid-based counterparts.

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

2019 Spring National Meeting

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