TY - JOUR
T1 - Scaling eukaryotic cell-free protein synthesis achieved with the versatile and high-yielding tobacco BY-2 cell lysate
AU - Gupta, Mainak Das
AU - Flaskamp, Yannick
AU - Roentgen, Robin
AU - Juergens, Hannes
AU - Armero-Gimenez, Jorge
AU - Albrecht, Frank
AU - Hemmerich, Johannes
AU - Arfi, Zulfaquar Ahmad
AU - Neuser, Jakob
AU - Spiegel, Holger
AU - Schillberg, Stefan
AU - Yeliseev, Alexei
AU - Song, Lusheng
AU - Qiu, Ji
AU - Williams, Charles
AU - Finnern, Ricarda
N1 - Publisher Copyright:
© 2023 LenioBio GmbH. Biotechnology and Bioengineering published by Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.
PY - 2023/10
Y1 - 2023/10
N2 - Eukaryotic cell-free protein synthesis (CFPS) can accelerate expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and difficulties scaling such systems have prevented their widespread adoption in protein research and manufacturing. Here, we provide detailed demonstrations for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in 48 h, complete with native disulfide bonds and N-glycosylation. An optimized version of the technology is commercialized as ALiCE® and advances in scaling of BYL production methodologies now allow scaling of eukaryotic CFPS reactions. We show linear, lossless scale-up of batch mode protein expression from 100 µL microtiter plates to 10 and 100 mL volumes in Erlenmeyer flasks, culminating in preliminary data from a litre-scale reaction in a rocking-type bioreactor. Together, scaling across a 20,000x range is achieved without impacting product yields. Production of multimeric virus-like particles from the BYL cytosolic fraction were then shown, followed by functional expression of multiple classes of complex, difficult-to-express proteins using the native microsomes of the BYL CFPS. Specifically: a dimeric enzyme; a monoclonal antibody; the SARS-CoV-2 receptor-binding domain; a human growth factor; and a G protein-coupled receptor membrane protein. Functional binding and activity are demonstrated, together with in-depth PTM characterization of purified proteins through disulfide bond and N-glycan analysis. Taken together, BYL is a promising end-to-end R&D to manufacturing platform with the potential to significantly reduce the time-to-market for high value proteins and biologics.
AB - Eukaryotic cell-free protein synthesis (CFPS) can accelerate expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and difficulties scaling such systems have prevented their widespread adoption in protein research and manufacturing. Here, we provide detailed demonstrations for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in 48 h, complete with native disulfide bonds and N-glycosylation. An optimized version of the technology is commercialized as ALiCE® and advances in scaling of BYL production methodologies now allow scaling of eukaryotic CFPS reactions. We show linear, lossless scale-up of batch mode protein expression from 100 µL microtiter plates to 10 and 100 mL volumes in Erlenmeyer flasks, culminating in preliminary data from a litre-scale reaction in a rocking-type bioreactor. Together, scaling across a 20,000x range is achieved without impacting product yields. Production of multimeric virus-like particles from the BYL cytosolic fraction were then shown, followed by functional expression of multiple classes of complex, difficult-to-express proteins using the native microsomes of the BYL CFPS. Specifically: a dimeric enzyme; a monoclonal antibody; the SARS-CoV-2 receptor-binding domain; a human growth factor; and a G protein-coupled receptor membrane protein. Functional binding and activity are demonstrated, together with in-depth PTM characterization of purified proteins through disulfide bond and N-glycan analysis. Taken together, BYL is a promising end-to-end R&D to manufacturing platform with the potential to significantly reduce the time-to-market for high value proteins and biologics.
KW - N-glycosylation
KW - biomanufacturing
KW - cell-free protein synthesis
KW - eukaryotic post-translational modifications
KW - protein biotechnology
KW - scaling
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U2 - 10.1002/bit.28461
DO - 10.1002/bit.28461
M3 - Article
C2 - 37376851
AN - SCOPUS:85163401457
SN - 0006-3592
VL - 120
SP - 2890
EP - 2906
JO - Biotechnology and bioengineering
JF - Biotechnology and bioengineering
IS - 10
ER -