(7d) Suppressing Protein Aggregation by Reducing Local Concentration Fluctuations

Aggregation of therapeutic proteins is a ubiquitous concern during product purification, formulation, storage and administration.  Recent reports in the literature highlight the importance of conformational stability, so as to minimize the concentration of unfolded, aggregation-prone proteins.  In contrast, optimizing so-called colloidal stability –- e.g., the osmotic second virial coefficient (B₂₂) -- has been found to be less effective in reducing aggregation rates.   In addition, a direct theoretical basis for quantitatively relating B₂₂ (or more accurately, the corresponding Kirkwood Buff integral G₂₂) to observed aggregation rates has been lacking.  Here we propose a new approach to relate B₂₂ and G₂₂ to aggregation rates, based on an adaptation of statistical mechanical fluctuation theory.  Experimentally, we test the theory with a comparison of conformational stability, laser light scattering, and aggregation kinetics for two model therapeutic monoclonal antibodies as a function of pH and NaCl concentration.   The results show that one can achieve dramatic reductions in aggregation rates by maximizing colloidal repulsions, and thereby minimizing local fluctuations in protein concentration that are necessary to nucleate new aggregates.  The model also provides a novel means to predict the effects of changes in formulation conditions on aggregation rates quantitatively or semi-quantitatively, and to relate these to molecular-scale fluctuations that can be probed experimentally via techniques such as laser light scattering.