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Volume 8
Journal of Biotechnology & Biomaterials
ISSN: 2155-952X Euro Biotechnology 2018
October 11-12, 2018
October 11-12, 2018 | Moscow, Russia
21st European
Biotechnology Congress
conferenceseries.com
Page 18
Production of antibody fragments with plasmid-based and genome integratedT7 E. coli expression
systems: Evaluation of systems performance in microtiter fed-batch like cultivations
Monika Cserjan1, Mathias Fink1, Sophie Vazulka1, Johanna Jarmer2 and Gerald Striedner1
1University of Natural Resources and Life Sciences, Austria
2Boehringer Ingelheim RCV GmbH & Co KG, Austria
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 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 the dsbA leader, genome
integrated versions showed higher Fab yields, reduced levels of free light chain and basal expression than plasmid-based
systems. Independent from used expression system, 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 detailed in bench-scale
fermentations.
Biography
Monika Cserjan has completed her PhD at the University of Natural Resources and Life Sciences, Vienna in 1998. She is Senior Scientist in the Christian
Doppler Laboratory for production of next-level biopharmaceuticals in E. coli at the Department of Biotechnology (Fermentation Technology Group), Vienna
and Project Leader at the Austrian Centre of Industrial Biotechnology (ACIB).
Monika Cserjan et al., J Biotechnol Biomater 2018, Volume 8
DOI: 10.4172/2155-952X-C5-100