Abstract
Prokaryotic production systems have been widely used to manufacture
recombinant therapeutic proteins. Economically, the prokaryotic
production - especially of small therapeutic molecules - is advantageous
compared to eukaryotic production strategies. However, due to the
potential endotoxin and host cell protein contamination, the
requirements for the purification process are disproportionately higher
and therefore more expensive and elaborate to circumvent. For this
reason, the goal of this work was to develop and establish a rapid,
simple, inexpensive and `up-scalable' production and purification
process, using the therapeutic relevant protein anti-EGFR scFv hu225 as
model molecule. Configuring high cell density cultivation of Escherichia
coli - using the rha-BAD expression system as production platform - a
specific product concentration up to 20 mg(scFv)/g(CDW) was obtained. By
combining freeze-and-thaw, osmotic shock and pH induced host cell
protein precipitation, almost 70\% of the product was extracted from the
biomass. In a novel approach a mixed mode chromatography was implemented
as a capturing and desalting step, which allowed the direct application
of further ion exchange chromatography steps for purification up to
pharmaceutical grade. Thereby, 50\% of the produced scFv could be
purified within 10 h while maintaining the biological activity. (C) 2014
Elsevier B.V. All rights reserved.
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