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

Purpose. The advent of recombinant DNA technology has made possible the pha rmaceutical use of a wide range of proteins and peptides. However, the comp lex structure of proteins renders them susceptible to physical instabilitie s such as denaturation, aggregation and precipitation. We tested the hypoth esis that partial unfolding and exposure of hydrophobic domains leads to ph ysical instability, and investigated approaches to stabilize protein formul ations. Methods. KP6 beta, an 81 amino acid killer toxin from Ustilago maydis, was used as a model protean. Circular dichroism and fluorescence spectroscopy w ere used to study the temperature dependent folding/ unfolding characterist ics of KP6 beta. ANS (1,8 anilinonaphthalene sulfonate), a fluorescent prob e that partitions into hydrophobic domains, was used to detect exposure of hydrophobic domains. Results. 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 observ ed, as indicated by the penetration of ANS. At elevated temperatures (60 de grees C), KP6 beta conserved most secondary structural features. However, t ertiary structure was disordered, suggesting the existence of a partially f olded, structured intermediate state. Liposomes bound to partially unfolded structures and prevented the formation of aggregates. Conclusions. Partial unfolding resulted in increased exposure of hydrophobi c domains and aggregation of KP6 beta, but with preservation of secondary s tructure. Liposomes interacted with the structured intermediate state, stab ilizing the protein against aggregation. These results suggest a general fo rmulation strategy for proteins, in which partially unfolded structures are stabilized by formulation excipients that act as molecular chaperones to a void physical instability.