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Correlated Analytical and Functional Evaluation of Higher Order Structure Perturbations from Oxidation of NISTmAb



Tsega Solomon, Frank Delaglio, John Giddens, John Marino, Yihua Bruce Yu, Marc Taraban, Robert Brinson


The 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.


low field water NMR, 2D NMR, biologics, NISTmAb, higher order structure, surface plasmon resonance, antigen binding


Solomon, T. , Delaglio, F. , Giddens, J. , Marino, J. , Yu, Y. , Taraban, M. and Brinson, R. (2023), Correlated Analytical and Functional Evaluation of Higher Order Structure Perturbations from Oxidation of NISTmAb, mAbs, [online],, (Accessed April 18, 2024)
Created January 22, 2023, Updated February 28, 2023