Development of an E. coli strain for cell-free ADC manufacturing
- Dan Groff,
- Nina Carlos,
- Rishard Chen,
- Jeff Hanson,
- Shengwen Liang,
- Stephanie Armstrong,
- Xiaofan Li,
- Sihong Zhou,
- Alexander Steiner,
- Trevor Hallam,
- Gang Yin
Dan Groff
Sutro Biopharma Inc South San Francisco
Corresponding Author:dgroff@sutrobio.com
Author ProfileStephanie Armstrong
Sutro Biopharma Inc South San Francisco
Author ProfileAbstract
Recent advances in cell-free protein synthesis have enabled the folding
and assembly of full-length antibodies at high titers with extracts from
prokaryotic cells. Coupled with the facile engineering of the E. coli
translation machinery, E. coli based in vitro protein synthesis
reactions have emerged as a leading source of IgG molecules with
non-natural amino acids incorporated at specific locations for producing
homogeneous antibody drug conjugates. While this has been demonstrated
with extract produced in batch fermentation mode, continuous extract
fermentation would facilitate supplying material for large-scale
manufacturing of protein therapeutics. To accomplish this, the
IgG-folding chaperones DsbC and FkpA, and orthogonal tRNA for
non-natural amino acid production were integrated onto the chromosome
with high strength constitutive promoters. This enabled co-expression of
all three factors at a consistently high level in the extract strain for
the duration of a five-day continuous fermentation. Cell-free protein
synthesis reactions with extract produced from cells grown continuously
yielded titers of IgG containing non-natural amino acids above those
from extract produced in batch fermentations. In addition, the quality
of the synthesized IgGs and the potency of ADC produced with
continuously fermented extract were indistinguishable from those
produced with batch extract. These experiments demonstrate that
continuous fermentation of E. coli to produce extract for cell-free
protein synthesis is feasible and helps unlock the potential for
cell-free protein synthesis as a platform for biopharmaceutical
production.26 May 2021Submitted to Biotechnology and Bioengineering 27 May 2021Submission Checks Completed
27 May 2021Assigned to Editor
30 May 2021Reviewer(s) Assigned
01 Jul 2021Review(s) Completed, Editorial Evaluation Pending
01 Jul 2021Editorial Decision: Revise Major
19 Aug 20211st Revision Received
19 Aug 2021Submission Checks Completed
19 Aug 2021Assigned to Editor
20 Aug 2021Reviewer(s) Assigned
30 Aug 2021Review(s) Completed, Editorial Evaluation Pending
30 Aug 2021Editorial Decision: Accept
Jan 2022Published in Biotechnology and Bioengineering volume 119 issue 1 on pages 162-175. 10.1002/bit.27961