Liposomes as formulation excipients for protein pharmaceuticals: a model protein study.

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The advent of recombinant DNA technology has made possible the pharmaceutical use of a wide range of proteins and peptides. However, the complex structure of proteins renders them susceptible to physical instabilities such as denaturation, aggregation and precipitation. We tested the hypothesis that partial unfolding and exposure of hydrophobic domains leads to physical instability, and investigated approaches to stabilize protein formulations. KP6 beta, an 81 amino acid killer toxin from Ustilago maydis, was used as a model protean. Circular dichroism and fluorescence spectroscopy were used to study the temperature dependent folding/ unfolding characteristics of KP6 beta. ANS (1,8 anilinonaphthalene sulfonate), a fluorescent probe that partitions into hydrophobic domains, was used to detect exposure of hydrophobic domains. As the temperature was elevated, near-UV CD indicated progressive loss of KP6 beta tertiary structure, while far-UV CD indicated retention of secondary structure. Increasing exposure of hydrophobic domains was observed, as indicated by the penetration of ANS. At elevated temperatures (60 degrees C), KP6 beta3 conserved most secondary structural features. However, tertiary structure was disordered, suggesting the existence of a partially folded, structured intermediate state. Liposomes bound to partially unfolded structures and prevented the formation of aggregates. Partial unfolding resulted in increased exposure of hydrophobic domains and aggregation of KP6 beta, but with preservation of secondary structure. Liposomes interacted with the structured intermediate state, stabilizing the protein against aggregation. These results suggest a general formulation strategy for proteins, in which partially unfolded structures are stabilized by formulation excipients that act as molecular chaperones to avoid physical instability.