LAUSR

Monoclonal antibodies (mAbs) have been used as therapeutics for the last few decades. It is necessary to investigate the stability of these mAbs under stress conditions, and to elucidate aggregation mechanisms as a means of developing approaches which minimise the problem. Attenuated Total Reflection (ATR)-FTIR spectroscopic imaging allows probing of a sample at a depth of penetration of around 0.5-5 µm, which makes it suitable for the study of aggregated proteins when accumulated as a layer close to the surface of the ZnSe internal reflection element (IRE). Here, macro ATR-FTIR spectroscopic imaging, along with a variable angle of incidence accessory, have been used to differentiate between the secondary structure of proteins in bulk solution, and those that have precipitated onto or near the ZnSe IRE surface. IgG spectra obtained from protein samples in individual wells have been averaged, extracted and pre-processed, and the Amide I bands of protein samples compared and further analysed to reveal protein distribution at the ZnSe IRE surface. These findings show depth profiling of IgG aggregates at the ZnSe IRE surface (0.5-5 µm), do not follow a trend of decreasing protein presence with increasing angle of incidence or increasing depth of penetration, suggesting an irregular distribution of aggregates in the z-direction.