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@PHDTHESIS{Vo:1004548,
author = {Voß, Leonie Fidelis Johanna Margarete},
othercontributors = {Büchs, Jochen and Schillberg, Stefan Johannes},
title = {{H}ochskalierung der {H}erstellung eines zellfreien
{P}roteinsynthesesystems aus {T}abakzellen und {E}ntwicklung
eines {V}erfahrens zur {D}urchführung der {R}eaktion im
{L}iter-{M}aßstab},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
publisher = {RWTH Aachen University},
reportid = {RWTH-2025-01489},
pages = {1 Online-Ressource : Illustrationen},
year = {2025},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University; Dissertation, RWTH Aachen University, 2025},
abstract = {In this work, an upscaling of the lysate preparation and
the CFPS reaction of Nicotiana tabacum cv. BY-2 cell lysate
was investigated to enable the production of
difficult-to-produce proteins in larger protein quantities.
For the preparation of lysate with a final volume of 1 L,
important preparation steps such as the cultivation and
protoplast formation of the BY-2 cells were transferred from
the shake flask process to a stirred tank reactor (STR)
process. The transfer of the cultivation process for the
purpose of lysate production was successful, but with up to
30 $\%$ lower biomass formation compared to the shake flask
and a negative influence of media sterilization using heat
on the quality of the lysates. The lower biomass formation
was compensated for by e. g. increasing the culture volume
by 25 $\%.$ For media sterilization, a combination of
filtration and heat sterilization was developed so that
influences on the lysate quality were minimized. An
influence of the stirring frequency and the aeration rate
was identified for the protoplast formation, and process
parameters of 180 rpm and 0.1 vvm (laboratory fermentation
system) were defined. Five processes were carried out for
the preparation of 1 L of lysate per cycle, in each of which
50 L of suspension culture could be processed into one liter
of lysate. The lysates showed variations in quality with
yields for eYFP $11\%$ better and $80\%$ worse and for GOx
$44\%$ better and $69\%$ worse compared to the reference. In
the early development phase of this upscaled preparation
process, optimizations of the individual steps were
continuously implemented and data for possible quality
parameters were recorded during the process. These data,
such as PCV values of the cultivation, protoplast and
evacuated protoplast number, could be evaluated
retrospectively and possible quality parameters identified.
In order to carry out the CFPS reaction in a volume of 1 L,
three different reactor vessels were tested: a roller
bottle, a wave-mixed bag bioreactor (CELL-tainer) and an
STR. It was found that the CFPS reaction was possible in
both the CELL-tainer and the STR with the same efficiency
compared to the reference in a microtiter plate. The CFPS
reactions in the CELL-tainer showed a yield comparable to
the reference when expressing eYFP, while the expression of
GOx showed a 62 $\%$ loss in efficiency. Initially, the
process in the STR was associated with foam formation, which
could be minimized by adapting the stirrer design. The
reaction in the roller bottle showed a significant
difference in performance with $50\%$ lower yields compared
to the reference in a microtiter plate. Finally, the method
was evaluated on two product candidates, bovine enterokinase
(EKL) and the fusion protein of MSP4-specific scFv antibody
and granzyme B (GrB-scFv). The yields of GrB-scFv were
comparable to cell-based yields with 1.4 mg of purified
protein per liter of lysate, while those of EKL were below
cell-based yields with 0.4 mg of purified protein per liter
of lysate. In summary, the scaling up of the system from
BY-2 cells and its application on a larger scale could be
demonstrated. The cell lysate has great potential as a
platform technology to enable important aspects of the
bioeconomy in the long term, such as the establishment of
resource-saving biological processes for industry.},
cin = {416510 / 162910 / 160000},
ddc = {570},
cid = {$I:(DE-82)416510_20140620$ / $I:(DE-82)162910_20140620$ /
$I:(DE-82)160000_20140620$},
typ = {PUB:(DE-HGF)11},
doi = {10.18154/RWTH-2025-01489},
url = {https://publications.rwth-aachen.de/record/1004548},
}
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