The Molecular Chaperone α-Crystallin as an Excipient in an Insulin Formulation
Open Access
- 24 March 2010
- journal article
- research article
- Published by Springer Science and Business Media LLC in Pharmaceutical Research
- Vol. 27 (7), 1337-1347
- https://doi.org/10.1007/s11095-010-0116-8
Abstract
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.Keywords
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