The Molecular Chaperone α-Crystallin as an Excipient in an Insulin Formulation

PURPOSE: To investigate insulin fibrillation under accelerated stress conditions in the presence of a novel excipient, the molecular chaperone α-crystallin, in comparison with common excipients. METHODS: To induce fibrillation, recombinant human insulin (0.58 mg ml(−1)) formulations without excipient or with bovine α-crystallin (0.01–0.2 mg ml(−1)), human serum albumin (1–5 mg ml(−1)), sucrose (10–100 mg ml(−1)) or polysorbate 80 (0.075–0.3 mg ml(−1)) were subjected to stirring stress in a fluorescence well plate reader and formulation vials. Protein fibrillation was monitored by thioflavin T. The formulations were further characterized by size-exclusion chromatography, light obscuration, UV/Vis and circular dichroism spectroscopy. RESULTS: In both methods, insulin formed thioflavin T-binding species, most likely fibrils. Addition of α-crystallin in the well plate assay greatly improved insulin’s resistance to fibrillation, measured as a 6-fold increase in fibrillation lag time for the lowest and 26-fold for the highest concentration used, whereas all other excipients showed only a marginal increase in lag time. The stabilizing effect of α-crystallin was shown by all characterization techniques used. CONCLUSIONS: The effect of α-crystallin on insulin’s physical stability outperforms that of commonly used excipients. α-Crystallin is proposed to bind specifically to pre-fibrillation species, thereby inhibiting fibrillation. This makes α-crystallin an interesting excipient for proteins with propensity to fibrillate.