As monoclonal antibody-based drugs are becoming more prevalent in the treatment of many illnesses, the need to characterize the quality attributes of mAb therapeutics through high resolution techniques is also becoming evident. Among the high-resolution biophysical methods, NMR spectroscopy is a powerful technique that provides atomic-level structural information that can be used to evaluate clinically relevant structural changes in mAb therapeutics including misfolding, aggregation and chemical modifications. While low to moderate resolution analytical techniques are commonly used in the pharmaceutical industry to assess the activity and quality attributes of mAbs, integration of high-resolution NMR with these methods will enable reliable and precise characterization of mAb drug candidates and biosimilars. Employing a pharmaceutically relevant forced degradation method, the present study demonstrates how 2D NMR fingerprinting and a number of analytical techniques, including low-field benchtop NMR, Tycho, SPR and SEC, can be implemented in parallel to evaluate a model mAb therapeutic, NISTmAb, when subjected to an oxidative stress condition. This case study involves monitoring the time dependent oxidation of methionine residues of NISTmAb by hydrogen peroxide in a benchtop NMR instrument and analyzing time course aliquots through 2D <sup>1</sup>H-<sup>13</sup>C HSQC Methyl fingerprint spectra in addition to the different analytical methods. The resulting 2D methyl spectra trace the oxidation of individual methionine residues as well as changes to different regions of the spectra reflecting additional structural perturbation while ligand binding and thermal stability analyses show functional and quality changes that correlate with the structural assessment. The complementary measurement outputs from the different techniques demonstrate the utility of combining high-resolution NMR with other analytical tools to extract structural changes of therapeutic mAbs that are functionally relevant and reliably decide the efficacy and safety of mAb drugs.

Integration of high-resolution NMR with other analytical and functional methods for the characterization of therapeutic mAbs: A case study of NISTmAb oxidation

ACS Spring 2022

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