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

Abstract

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.

Backbone and methyl side-chain resonance assignments of the single chain Fab fragment of trastuzumab

“…However, more complex labelling schemes with yeast, such as methyl labelling, are not straightforward and thus require further developments. In parallel, we developed a method using Escherichia coli to produce isotopically labelled mAb Fab fragments for NMR resonance assignment (Gagné et al, 2023). The method, is based on the production of a single polypeptide chain in inclusion bodies constructed by the fusion of the heavy and the light chains with a removable linker to facilitate protein refolding.…”

“…The amino acid sequence of used for the production of samples of labelled trastuzumab-scFab has been described previously (Gagné et al, 2023). The heavy chain of the Fab domain, residues Glu1 to Pro230 (underlined) where Cys229 has been mutated to Ala229, was linked to residues Asp1 to Cys214 of the light chain via a linker made of ve (GGGGS) elements plus SSGLVPRGS.…”

“…Protein puri cations were conducted using a fast dilution approach at pH 9.0 and with 2 M L-arginine, as described in previously (Gagné et al, 2023). His tag was removed by incubating the protein with 100 units/mg of thrombin in phosphate buffer at pH 7.0 for 5h at 37℃ under light agitation.…”

Backbone and methyl side-chain resonance assignments of the Fab fragment of adalimumab

“…The amino acid sequence used for the production of samples of labelled adalimumab-scFab has been described in details previously (Gagné et al, 2023). The heavy chain of adalimumab Fab domain and a portion of the hinge region (see (Hodgson et al, 2019) for complete amino acid sequence), residues Glu1 to Pro234 where Cys233 has been mutated to Ala233, was linked to residues Asp1 to Cys214 of the light chain via a linker made of ve (GGGGS) elements plus SSGLVPRGS.…”