The molecular chaperone alpha-crystallin as an excipient in an insulin formulation

To investigate insulin fibrillation under accelerated stress conditions in the presence of a novel excipient, the molecular chaperone alpha-crystallin, in comparison with common excipients.

To induce fibrillation, recombinant human insulin (0.58 mg ml(-1)) formulations without excipient or with bovine alpha-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.

In both methods, insulin formed thioflavin T-binding species, most likely fibrils. Addition of alpha-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 alpha-crystallin was shown by all characterization techniques used.

The effect of alpha-crystallin on insulin's physical stability outperforms that of commonly used excipients. alpha-Crystallin is proposed to bind specifically to pre-fibrillation species, thereby inhibiting fibrillation. This makes alpha-crystallin an interesting excipient for proteins with propensity to fibrillate.