Anisotropy Resolved Multidimensional Emission Spectroscopy (ARMES) for the analysis of Immunoglobulin G (IgG) type proteins

Abstract

Immunoglobulin G (IgG) is the main antibody used in the biopharmaceutical industry for therapeutic purposes. Monoclonal antibodies (mAb) are growing in the marked due to its high specificity and safety. Physical and chemical stresses can lead to unfolding of the tertiary structure, which could then refold into a different structure, and/or aggregate. Changes in protein structure are dangerous and can cause adverse immunogenicity issues. One key factor to ensure efficacy and safety is to understand and measure the stability of mAbs in solution. A combination of different analytical techniques, some of them are expensive and time consuming, is necessary to monitor mAbs stability, and to assess protein aggregation. The aim of this project was to apply a newly developed fluorescence-based method, Anisotropy Resolved Multidimensional Emission Spectroscopy (ARMES), for the rapid characterisation of IgG type proteins in solution, which could be used to monitor IgG structure. This method combined polarized multidimensional fluorescence spectroscopy (pMDF), anisotropy, and chemometric analysis, such as Parallel factor analysis (PARAFAC), to try to resolve emission from different fluorophores, which were used to follow protein structure. ARMES measurements of IgG solutions in its native state were carried out with polarized Excitation Emission Matrix (pEEM) and polarized Total Synchronous Fluorescence Spectroscopy (pTSFS) measurements. It was the first time that PARAFAC analysis was attempted on IgG, as there was no consensus about the optimal pre-processing method for this type of data, we evaluated several methods. Although there was insufficient fluorescence fluctuation in the native state for PARAFAC analysis to resolve different fluorophore populations, we were able to photophysically characterise the IgG, which served as a baseline for monitoring protein stability using ARMES. Thus, for the IgG protein, even if ARMES could not recover individual fluorophore emission, it was showed to be a suitable technique for monitoring protein stability.