Expression of

Labeling of proteins with deuterium is an essential tool in overcoming size limitations in the application of nuclear magnetic resonance (NMR) spectroscopy to proteins larger than 30 kilodaltons (kDa). A non-originator antigen-binding fragment (Fab) of NIST RM 8671 NISTmAb, so called yNIST-Fab, is a ~ 50 kDa protein, with 5 native disulfide linkages, that can be expressed in properly folded form in methylotrophic Komagataella phaffii (formerly Pichia pastoris). Further, the K. phaffii host can support the production of perdeuterated yNIST-Fab which is necessary to obtain well-resolved TROSY-based tripleresonance NMR spectra for chemical shift assignment of the peptide backbone resonances. Here, we examined growth conditions and effects of media composition to maximize biomass generation and expression yield of the 2H, 13C, 15N-enriched NIST-Fab fragment. Triple-labeled yNIST-Fab with ~93% deuteration reduced the 1HN, 15N and 13C-linewidths in the NMR spectra, allowing sequential NMR assignment of backbone resonance a key step toward sequence-specific structural and dynamic studies of Fab fragments and intact antibodies

Correlated analytical and functional evaluation of higher order structure perturbations from oxidation of NISTmAb

ABSTRACTThe clinical efficacy and safety of protein-based drugs such as monoclonal antibodies (mAbs) rely on the integrity of the protein higher order structure (HOS) during product development, manufacturing, storage, and patient administration. As mAb-based drugs are becoming more prevalent in the treatment of many illnesses, the need to establish metrics for quality attributes of mAb therapeutics through high-resolution techniques is also becoming evident. To this end, here we used a forced degradation method, time-dependent oxidation by hydrogen peroxide, on the model biotherapeutic NISTmAb and evaluated the effects on HOS with orthogonal analytical methods and a functional assay. To monitor the oxidation process, the experimental workflow involved incubation of NISTmAb with hydrogen peroxide in a benchtop nuclear magnetic resonance spectrometer (NMR) that followed the reaction kinetics, in real-time through the water proton transverse relaxation rate R2(1H2O). Aliquots taken at defined time points were further analyzed by high-field 2D 1H-13C methyl correlation fingerprint spectra in parallel with other analytical techniques, including thermal unfolding, size-exclusion chromatography, and surface plasmon resonance, to assess changes in stability, heterogeneity, and binding affinities. The complementary measurement outputs from the different techniques demonstrate the utility of combining NMR with other analytical tools to monitor oxidation kinetics and extract the resulting structural changes in mAbs that are functionally relevant, allowing rigorous assessment of HOS attributes relevant to the efficacy and safety of mAb-based drug products