Evaluation of complex antibody formats expression in E. coli
Year: 2024
Type: dissertation
Abstract: Antibody-based (AB) therapeutics have become fundamental in several medical fields in recent decades. Since the variable fragment (Fab) base formats do not need glycosylation to be functional, E. coli has been used as a production host for them. Currently, there are seven antibody-based therapeutics produced in E.coli on the market. This work aims to implement the knowledge of the complex AB format expression in E. coli in an industrial set-up, to find the bottlenecks in this process and address them. In particular in this thesis work, the production of two Fab based complex AB in E.coli periplasm was studied: a Fab with the C-terminus (C-T) of the heavy chain linked with a disulfide bond stabilized single-chain variable fragment ds scFv (BYbe™) and a Fab with both the C-T of the heavy and the light chain linked with a ds-scFv (TrYbe™). To address this aim, the expression of these AB formats and their building blocks was studied in diffent set up: shake flask IPTG induction, shake flask auto induction, Ambr®250mL. From the first test of these AB formats expression in W3110 the degradation of the heavy chain of both formats was detected, which remained constant with all the expression methods. A similar degradation product was also observed in the Fab expression in this strain. To investigate on the nature of this event, the Fab was expressed in the Keio selected mutants with non-essential periplasmic proteases knocked out. However, none of these strains showed an effected the HC degradation pattern. Fab with C-T of the HC linked with ds-scFv, the BYbe™ was expressed in three strains at fermentation scale (Ambr® 250mL): a wild-type XI strain, prc (periplasmic protease) KO strain and a prc (periplasmic protease) KO expression DsbC (periplasmic chaperon involved in DSB formation strain). While the prc absence resulted in no changes in the HC degradation of the BYbe ™ (Fab with C-T of the HC linked with ds-scFv) the co-expression of DsbC drastically increased the quality and the quantity of the expressed BYbe™.When the BYbe™ was produced in MXE016 DsbC, 10 % increment of intact BYbe™ HC and an ~300% increment the BYbe™ production was detected compared to W3110 (confirmed by WES and protein A and protein G quantification data). To complete the picture of the E. coli state during the formats expression, the proteome of the strains was studied, and the results showed that the co expression of DsbC also led to an upregulation of the expression of other periplasmic chaperones (FkpA and DsbA). Finally, the BYbe™ (Fab with C-T of the HC linked with ds-scFv) and all its related degradation species were purified with a 5-step purification and identified using mass fingerprint. This resulted in a better understanding of the degradation events happening during the BYbe™ expression. In conclusion, this study detected the bottlenecks in the BYbe™ expression in E.coli W3110, and how these can be mitigated co-expressing a DSB chaperone DsbC. In the future, the expression of this format can be futher optimised, in fact the the proteomic study suggests that FkpA and DsbA can also be beneficial. Moreover, proteomic data allowed us to select possible target proteins, which can be investigated in the future for the BYbe™ (Fab with C-T of the HC linked with ds-scFv) or for other Fab based formats expression optimization.
Authors: Pisent, Beatrice, thesis funded by Horizon 2020 EU-funded project 813979 conducted in UCB, S.A.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7084611955
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