Traditional analytical methods including circular dichroism, Fourier transform infrared spectroscopy and differential scanning fluorometry provide low to moderate structural resolution. Recently, NMR spectroscopy has emerged as a powerful tool for HOS characterization. In this work, we assess the ability of two-dimensional (2D) 1H-13C methyl NMR to discern subtle differences in the HOS resulting from forced degradation of an IgG1 mAb and compare the method performance to some of the established biophysical methods for HOS assessments. The sample set was prepared by peroxide exposure, UV irradiation, and pH and thermal stress across a range of intensities and time periods. Data suggest that except for UV irradiation, most of the stressors do not affect bioactivity. The study further shows that unlike other biophysical methods, 2D-NMR has a unique ability to observe subtle modification between stressed samples and can therefore be used to compare and characterize protein therapeutics.
Learning Objectives:
Appreciate the use of 2D-NMR for characterizing subtle modifications between stressed samples.
Assess use of NMR as a high throughput method to compare and characterize protein therapeutics.
Assess structure- function relationships across a range of forced degradation conditions.
