Stabilization of proteins against aggregation by solution additives

• Main Author: Baynes, Brian M., 1976-
• Other Authors: Bernhardt L. Trout and Daniel I.C. Wang.
• Format: Others
• Language: en_US
• Published: Massachusetts Institute of Technology 2005
• Subjects: Chemical Engineering.
• Online Access: http://hdl.handle.net/1721.1/28764

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2005. === Includes bibliographical references (p. 123-132). === (cont.) additives. === In order to develop protein formulations that limit aggregation, researchers heuristically screen potential solution additives (excipients). Such screening is necessary because current understanding of mechanisms of aggregation and molecular-level effects of additives on aggregation is limited. In this study, we developed a statistical-mechanical method in order to model the thermodynamic effects of additives in molecular-level detail. This method uses no adjustable parameters and was validated by quantitative comparison with experimental data on proteins in glycerol and urea solutions. We then applied our molecular simulation technique to study the mechanism by which arginine, a common refolding buffer additive, deters protein aggregation. We find that arginine acts as a weak surfactant at the protein-solvent interface, with its guanidino group tending to face the protein. We propose that arginine is a member of a class of anti-aggregation additives, which we term "neutral crowders," characterized by their (1) negligible effect on the free energy of isolated protein molecules and (2) large size relative to water. With a simplified statistical-mechanical model, we have shown that such additives selectively increase the free energy of protein-protein encounter complexes by being preferentially-excluded from the gap between the protein molecules in such complexes. This "gap effect" will therefore slow protein association reactions. We showed experimentally that, in accordance with the gap effect model predictions, arginine slows association of model globular proteins (antibody+antigen) and of folding intermediates and aggregates of carbonic anhydrase II. We predict that neutral crowders larger than arginine will be superior anti-aggregation === by Brian M. Baynes. === Ph.D.