Abstract
Controlling and preventing aggregation is critical to the development of safe and effective antibody drug products. The studies presented here test the hypothesis that protein A and protein G inhibit the agitation-induced aggregation of IgG. The hypothesis is motivated by the enhanced conformational stability of proteins upon ligand binding and the specific binding affinity of protein A and protein G to the Fc region of IgG. The aggregation of mixed human IgG from pooled human plasma was induced by agitation alone or in the presence of (i) protein A, (ii) protein G or (iii) a library of 24 peptides derived from the IgG-binding domain of protein A. Aggregation was assessed by UV spectroscopy, SDS-PAGE, high performance size-exclusion chromatography (HP-SEC), dynamic light scattering (DLS) and fluorescence spectroscopy. Additional information on IgG-ligand interactions was obtained using differential scanning fluorimetry (DSF) and competitive binding studies. The results demonstrate that protein A provides near-complete inhibition of agitation-induced aggregation, while protein G and two peptides from the peptide library show partial inhibition. The findings indicate that the IgG protein A-binding site is involved in the agitation-induced aggregation of IgG, and suggest a dominant role of colloidal interactions.