Strategies for the production of isotopically labelled Fab fragments of therapeutic antibodies in Komagataella phaffii (Pichia pastoris) and Escherichia coli for NMR studies.
Journal
PloS one
ISSN: 1932-6203
Titre abrégé: PLoS One
Pays: United States
ID NLM: 101285081
Informations de publication
Date de publication:
2023
2023
Historique:
received:
01
09
2023
accepted:
30
10
2023
medline:
1
12
2023
pubmed:
29
11
2023
entrez:
29
11
2023
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 38019850
doi: 10.1371/journal.pone.0294406
pii: PONE-D-23-28318
pmc: PMC10686436
doi:
Substances chimiques
Immunoglobulin Fab Fragments
0
Carbon-13
FDJ0A8596D
Nitrogen-15
0
Adalimumab
FYS6T7F842
Carbon Isotopes
0
Antibodies, Monoclonal
0
Trastuzumab
P188ANX8CK
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e0294406Informations de copyright
Copyright: © 2023 Gagné et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Déclaration de conflit d'intérêts
The authors have declared that no competing interests exist.
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