Changes in protein-protein interactions, protein unfolding, and nonnative aggregation were assessed for a series of human IgG1 antibodies as a function of pH and solution ionic strength (I). Unfolding transitions were characterized with differential scanning calorimetry. Protein-protein interactions were characterized with the apparent second virial coefficient (A(2)) from light scattering. Aggregation pathways were assessed using size-exclusion chromatography and multi-angle laser light scattering, aggregation kinetics, and structural changes monitored by circular dichroism spectroscopy and thioflavine T (ThT) binding. Ionic strength had relatively minor qualitative effects on unfolding, while pH had large effects for all four antibodies. A(2) was sensitive to both pH and I, and indicated that electrostatic interactions and nonuniform surface-charge distributions were important near neutral pH. Depending on solution pH and I, distinct aggregation pathways were found for each antibody, and these shared similar patterns versus pH, I, and A(2). Main differences observed across different antibodies included thermal unfolding transitions in DSC and the effects of pH and I on aggregation kinetics and pathways. These correlated strongly with whether aggregates of a given antibody bound ThT, suggesting possible differences with respect to conformational changes and/or regions of the proteins that are structurally involved in stabilizing the aggregates.
© 2010 Wiley-Liss, Inc. and the American Pharmacists Association