Monitoring antibody aggregation in early drug development using Raman spectroscopy and perturbation-correlation moving windows.

In this study, we demonstrate the sensitivity of two-dimensional perturbation-correlation moving windows (PCMW) to characterize conformational transitions in antibodies. An understanding of how physiochemical properties affect protein stability and instigate aggregation is essential for the engineering of antibodies. In order to establish the potential of PCMW as a technique for early identification of aggregation mechanisms during antibody development, five antibodies with varying propensity to aggregate were compared. Raman spectra were acquired, using a 532 nm excitation wavelength as the protein samples were heated from 56 to 78 °C and analyzed with PCMW. Initial principal component analysis confirmed a trend between the observed spectral variations and increasing temperature for all five samples. Analysis using PCMW revealed that when spectral variations were directly related to temperature, distinct differences in conformational changes could be determined between samples related to protein stability, providing a greater understanding of the aggregation mechanisms of problematic antibody variants.