Analysing the structure of antibodies using circular dichroism with an antibody augmented reference set and the algorithm SELCON

Proper folding of therapeutic monoclonal antibody (mAb) drugs is key to their efficacy and safety. Circular Dichroism (CD) spectroscopy is well suited for investigating the secondary structure of proteins, but the adequacy of the technique for antibody molecules has been severely impacted by the lack of suitable CD reference sets that include spectra of proteins of similar structure. Here we report on the performance of an expanded reference set that includes CD spectra of 14 mAbs, and uses structures derived from homology models. This enhanced reference set is part of a freely available Python-based software package, SSCalcPy-mAb, that includes the new SP-mAb178 reference set for secondary structure analysis using the SELCON3 method. We propose to employ both the secondary structure analysis results and a generated list of the proteins in the reference set that SELCON3 applies to derive the results that ascertain whether an antibody of interest is well-folded or not.