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.
CELL-DENSITY FERMENTATION; THERAPEUTIC PROTEINS; E. COLI; EXCHANGE
CHROMATOGRAPHY; MONOCLONAL-ANTIBODIES; ENDOTOXIN REMOVAL; EXPRESSION
VECTOR; DRUG DEVELOPMENT; FV FRAGMENTS; PURIFICATION
number-of-cited-references
56
funding-text
We would like to thank Elke Gerlach<SUP>1</SUP> for providing us with
the A431 cells and for her help in the early morning experiments. We
also thank the fermentation engineers of the IBVT, Andreas
Freund<SUP>2</SUP> and Salaheddine Laghrami<SUP>2</SUP>, for their great
support during the HCDC. We would like to show our gratitude to M. Kinan
Hayani and Niki Francesco Macri for their proof reading. This work was
supported by the Bundesministerium fur Bildung und Forschung (BMBF),
Germany.
%0 Journal Article
%1 ISI:000345970300023
%A Lindner, Robert
%A Moosmann, Anna
%A Dietrich, Alexander
%A Boettinger, Heiner
%A Kontermann, Roland
%A Siemann-Herzberg, Martin
%C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS
%D 2014
%I ELSEVIER SCIENCE BV
%J JOURNAL OF BIOTECHNOLOGY
%K Ion Mixed Periplasmic chain chromatography; disruption} exchange fragment mode myown variable; {Single
%N A
%P 136-145
%R 10.1016/j.jbiotec.2014.10.003
%T Process development of periplasmatically produced single chain fragment
variable against epidermal growth factor receptor in Escherichia coli
%U https://doi.org/10.1016/j.jbiotec.2014.10.003
%V 192
%X 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.
@article{ISI:000345970300023,
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.}},
added-at = {2018-01-25T13:38:08.000+0100},
address = {{PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}},
affiliation = {{Siemann-Herzberg, M (Reprint Author), Univ Stuttgart, Inst Biochem Engn, Allmandring 31, D-70569 Stuttgart, Germany.
Lindner, Robert; Moosmann, Anna; Boettinger, Heiner; Kontermann, Roland, Univ Stuttgart, Inst Cell Biol \& Immunol, D-70569 Stuttgart, Germany.
Dietrich, Alexander; Siemann-Herzberg, Martin, Univ Stuttgart, Inst Biochem Engn, D-70569 Stuttgart, Germany.}},
author = {Lindner, Robert and Moosmann, Anna and Dietrich, Alexander and Boettinger, Heiner and Kontermann, Roland and Siemann-Herzberg, Martin},
author-email = {{siemann@ibvt.uni-stuttgart.de}},
biburl = {https://puma.ub.uni-stuttgart.de/bibtex/29cbc1d3333e2aff678cdb9f78f5c1707/siemannherzberg},
da = {{2018-01-25}},
doc-delivery-number = {{AW0GW}},
doi = {{10.1016/j.jbiotec.2014.10.003}},
eissn = {{1873-4863}},
funding-acknowledgement = {{Bundesministerium fur Bildung und Forschung (BMBF), Germany}},
funding-text = {{We would like to thank Elke Gerlach<SUP>1</SUP> for providing us with
the A431 cells and for her help in the early morning experiments. We
also thank the fermentation engineers of the IBVT, Andreas
Freund<SUP>2</SUP> and Salaheddine Laghrami<SUP>2</SUP>, for their great
support during the HCDC. We would like to show our gratitude to M. Kinan
Hayani and Niki Francesco Macri for their proof reading. This work was
supported by the Bundesministerium fur Bildung und Forschung (BMBF),
Germany.}},
interhash = {efb834c12e07e05b356cc66a7819efc6},
intrahash = {9cbc1d3333e2aff678cdb9f78f5c1707},
issn = {{0168-1656}},
journal = {{JOURNAL OF BIOTECHNOLOGY}},
journal-iso = {{J. Biotechnol.}},
keywords = {Ion Mixed Periplasmic chain chromatography; disruption} exchange fragment mode myown variable; {Single},
keywords-plus = {{CELL-DENSITY FERMENTATION; THERAPEUTIC PROTEINS; E. COLI; EXCHANGE
CHROMATOGRAPHY; MONOCLONAL-ANTIBODIES; ENDOTOXIN REMOVAL; EXPRESSION
VECTOR; DRUG DEVELOPMENT; FV FRAGMENTS; PURIFICATION}},
language = {{English}},
month = {{DEC 20}},
number = {{A}},
number-of-cited-references = {{56}},
pages = {{136-145}},
publisher = {{ELSEVIER SCIENCE BV}},
research-areas = {{Biotechnology \& Applied Microbiology}},
times-cited = {{1}},
timestamp = {2018-01-25T12:38:18.000+0100},
title = {{Process development of periplasmatically produced single chain fragment
variable against epidermal growth factor receptor in Escherichia coli}},
type = {{Article}},
unique-id = {{ISI:000345970300023}},
url = {https://doi.org/10.1016/j.jbiotec.2014.10.003},
usage-count-last-180-days = {{1}},
usage-count-since-2013 = {{15}},
volume = {{192}},
web-of-science-categories = {{Biotechnology \& Applied Microbiology}},
year = {{2014}}
}