379733 Computational Studies on Protein Hydration of Trp-Cage Miniprotein

Tuesday, November 18, 2014: 10:29 AM
Crystal Ballroom A/F (Hilton Atlanta)
Sang Beom Kim and Pablo G. Debenedetti, Chemical and Biological Engineering, Princeton University, Princeton, NJ

Therapeutic proteins have emerged as one of the fastest growing segments of the pharmaceutical industry over the last several decades. Despite recent advances, however, the formulation of stable drugs for long-term storage still remains as a challenging problem. The standard approach for enhancing the stability is lyophilization, or freeze-drying, in which proteins in aqueous solution are dehydrated at cold temperatures to produce vitrified powders [1]. Vitrification greatly enhances stability, arresting aggregation and chemical degradation processes that often occur when proteins are stored in aqueous environments.  Lyophilized proteins, however, may exhibit significant loss in their therapeutic activity when reconstituted with water from the freeze-dried state.  Since the physical mechanisms responsible for the activity loss are not yet fully understood, developing rational formulation strategies to mitigate the damaging effects of lyophilization remains a significant challenge.

We introduce a new computational method for the study of water sorption on protein systems, which allows us to systematically explore protein behavior during dehydration and rehydration process. This method is applied to investigate the hydration-dependent behavior of model lyophilized protein matrices comprised of Trp-cage miniproteins. To understand the impact of the underlying structure of the protein matrix on the hydration process, water sorption isotherms are calculated for several Trp-cage matrices with varying degrees of monomeric denaturation and translational and rotational ordering at the monomer level [2]. We demonstrate that the water sorption isotherms are relatively insensitive to nature of the protein matrix, which is consistent with the experimental observation that the qualitative features of water sorption isotherms are nearly universal for different lyophilized proteins [3].  We show that each model protein matrix undergoes similar macroscopic structural transformations during the dehydration and rehydration process, and we describe how such transformation influence microscopic properties including solvent accessibility and hydrogen bonding. Finally, we discuss the broader implications of our findings with regards to protein lyophilization, and we present preliminary results from our investigation of the protein hydration/dehydration in the presence carbohydrates, which have been experimentally found to stabilize proteins during the freeze-drying process. 

[1] C. J. Roberts and P. G. Debenedetti, AIChE J. 48 (6), 1140 (2002)

[2] M. Tarek and D. J. Tobias, Biophys. J., 79 (6), 3244 (2000).

[3] J. A. Rupley and G. Careri, Adv. Protein Chem. 41, 37-172 (1991).

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