Rational design of additives for inhibition of protein aggregation
Massachusetts Institute of Technology. Dept. of Chemical Engineering.
Bernhardt L. Trout.
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Protein based therapeutics hold great promise in the treatment of human diseases and disorders and subsequently, they have become the fastest growing sector of new drugs being developed. Proteins are, however, inherently unstable and the degraded form can be quite harmful if administered to a patient. Of the various degradation pathways, aggregation is one of the most common and a cause for great concern. Aggregation suppressing additives have long been used to stabilize proteins, and they still remain the most viable option for combating this problem. However, the mechanisms by which the most commonly used additives inhibit aggregation still remain a mystery for the most part. It is clear that additive selection and the development of better performing additives will benefit from a more refined understanding of how commonly used additives inhibit or enhance aggregation. Aqueous arginine solutions are widely used to suppress protein aggregation and protein-protein interactions. Attempts have been made to develop cosolvents that are similar to arginine, but more effective at inhibiting aggregation. Therefore, a clear picture of the mechanism by which arginine inhibits protein aggregation is desirable. Baynes and Trout have proposed the design of a novel class of additives called "Neutral Crowder", which does not affect the free energy of isolated protein molecules but selectively increases the free energy of the protein-protein encounter complex. They proposed that arginine can be a "Neutral crowder" as the magnitude of the observed aggregation suppression effect of arginine is quantitatively equivalent to a neutral crowder of its size. On the basis of the results obtained in this thesis, we have been able to show that self-interaction of arginine plays a critical role in the mechanism by which it inhibits aggregation. The preferential interaction between protein and arginine is also influenced by the intrasolvent interactions in aqueous arginine solutions, something that is often overlooked and yet essential to understanding the effect of additives on aggregation. Furthermore, the linking together of arginine clusters into bigger clusters by hydrogen bond accepting counterions enhances its aggregation suppressing ability. According to the "Neutral Crowder" theory, large molecules that have the same concentration on the protein surface as the bulk solution should be effective at inhibiting protein association. However, large molecules naturally tend to be excluded from protein surfaces (e.g. polyethylene glycol) due to steric exclusion. We theorized, though, that if functional groups which tend to preferentially bind to proteins (e.g. guanidinium, urea, etc.) were added to the surface of a large, core structure that the resulting molecule could potentially behave as a neutral crowder. Therefore, creating a neutral crowder molecule requires a balance between attraction and repulsion with respect to the surface of a protein. Choosing a proper balance of interactions allowed us to produce compounds which have been shown to be potent aggregation suppressors, slowing aggregation by an order of magnitude more than the commonly used additives. Such potent aggregation suppressing additives might be useful during production and formulation, as they could improve yield and extend the shelf-life of protein therapeutics.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2011.Cataloged from PDF version of thesis.Includes bibliographical references (p. 189-197).
DepartmentMassachusetts Institute of Technology. Dept. of Chemical Engineering.; Massachusetts Institute of Technology. Department of Chemical Engineering
Massachusetts Institute of Technology