NMR mapping of the highly flexible regions of 13C/15N-labeled antibody TTAC-0001-Fab

Cha, Soyoung; Lee, Weon Sup; Choi, Joonhyeok; Jeong, Jong Geun; Nam, Ju Ryoung; Kim, Jihong; Kim, Hak-Nam; Lee, Joon‐Hwa; Yoo, Jin-San; Ryu, Kyoung‐Seok

doi:10.1007/s10858-020-00313-1

Cited by 2 publications

(1 citation statement)

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“…Nevertheless, several approaches were developed to improve refolding in bacteria, including the addition of fusion proteins, protein expression at lower temperature, the inclusion of a signal peptide for refolding in the periplasm (by the periplasmic oxidoreductase DsbA), and more. In most cases, yields remain low when they are reported [9][10][11][12][13], which may become abysmally low when attempting to produce fully deuterated proteins. In addition, the yeast K. phaffii has the ability to produce N-glycosylated Fc fragments [14], which are different from N-glycans in humans.…”

Section: Introductionmentioning

confidence: 99%

Strategies for the production of isotopically labelled Fab fragments of therapeutic antibodies in Komagataella phaffii (Pichia pastoris) and Escherichia coli for NMR studies

Gagné,

Sarker,

Gingras

et al. 2023

PLoS ONE

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The importance and fast growth of therapeutic monoclonal antibodies, both innovator and biosimilar products, have triggered the need for the development of characterization methods at high resolution such as nuclear magnetic resonance (NMR) spectroscopy. However, the full power of NMR spectroscopy cannot be unleashed without labelling the mAb of interest with NMR-active isotopes. Here, we present strategies using either Komagataella phaffii (Pichia pastoris) or Escherichia coli that can be widely applied for the production of the antigen-binding fragment (Fab) of therapeutic antibodies of immunoglobulin G1 kappa isotype. The E. coli approach consists of expressing Fab fragments as a single polypeptide chain with a cleavable linker between the heavy and light chain in inclusion bodies, while K. phaffii secretes a properly folded fragment in the culture media. After optimization, the protocol yielded 10–45 mg of single chain adalimumab-Fab, trastuzumab-Fab, rituximab-Fab, and NISTmAb-Fab per liter of culture. Comparison of the 2D-1H-15N-HSQC spectra of each Fab fragment, without their polyhistidine tag and linker, with the corresponding Fab from the innovator product showed that all four fragments have folded into the correct conformation. Production of 2H-13C-15N-adalimumab-scFab and 2H-13C-15N-trastuzumab-scFab (>98% enrichment for all three isotopes) yielded NMR samples where all amide deuterons have completely exchanged back to proton during the refolding procedure.

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