Clarifying the role of cryo- and lyo-protectants in the biopreservation of proteins

摘要

Biopharmaceuticals are frequently stored in the frozen state to avoid rapid degradation. Moreover, therapeutic proteins are frequently made into a dried form to provide long-term storage. However, both freezing and drying stresses can result in protein unfolding and aggregation. Thus, a proper formulation, containing suitable excipients, must be used to avoid loss of activity. Here, the conformational stability of a model protein, human growth hormone, is studied during freezing, and in the dried state as well, using molecular dynamics. The impact of the ice-water interface and of water removal is deeply investigated, and the role of protectants in preventing denaturation phenomena is addressed. We found that good cryo-protectants not always are equally effective as lyo-protectants, and experimental data confirmed simulation results. From this analysis, we also discovered that the interaction of stabilizers with specific amino acid sequences of the protein, rather than with the molecule as a whole, seems to be a crucial issue in the preservation of protein structure. This finding was confirmed for another protein, i.e., lactate dehydrogenase, thus suggesting that it is a generally applicable result. Remarkably, those sequences which unfolded during freezing and drying, generally coincided with the aggregation prone regions of the protein.