Biophysical and bioinformatics analysis of antibody colloidal and conformational stability

Abstract

There has been an explosion in the development of therapeutic monoclonal antibodies (mAbs) in recent years due to their high affinities, specificities, stabilities and other drug-like properties. The development of antibody therapeutics, however, is a long, arduous and complicated process. The overall goal of this research is to address several key fundamental challenges related to generating and formulating antibodies for therapeutic applications. First, antibodies are commonly affinity maturated in vitro using monovalent antibody fragments (Fabs) prior to reformatting them as bivalent mAbs for therapeutic applications. A common problem is that engineered Fabs display poor biophysical properties (e.g., low solubility or high viscosity) when reformatted as bivalent antibodies. Therefore, we have sought to develop methods for profiling the biophysical properties of monovalent antibody fragments in a manner that is predictive of their properties as bivalent antibodies prior to reformatting. We have developed a nanoparticle-based assay (self-interaction nanoparticle spectroscopy, SINS) to measure Fab self-association in a multivalent format with the goal of sampling colloidal interactions that are similar to those for bivalent mAbs. Moreover, we have extended these biophysical studies to evaluate the effects of chemical modifications (e.g., deamidation and oxidation) on the aggregation behavior of mAbs. In particular, we have investigated how chemical modifications of antibodies lead to aggregation during challenging downstream processing conditions (e.g., low pH). Interestingly, we find little correlation between the impact of these chemical modifications on antibody conformational stability and aggregation. Methionine oxidation leads to significant reductions in antibody conformational stability while having little impact on antibody aggregation except at extreme conditions (low pH and elevated temperature). Conversely, tryptophan oxidation and asparagine deamidation have little impact on antibody conformational stability while promoting aggregation at a wide range of solution conditions, and the aggregation mechanisms appear linked to unique types of reducible and non-reducible covalent crosslinks and, in some cases, to increased levels of attractive colloidal interactions. We expect that our findings will improve the generation of safe and potent antibody therapeutics.