TY - JOUR
T1 - The molecular chaperone a-crystallin as an excipient in an insulin formulation
AU - Rasmussen, Tue
AU - Tantipolphan, Ruedeeporn
AU - van de Weert, Marco
AU - Jiskoot, Wim
PY - 2010/7
Y1 - 2010/7
N2 - 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.
AB - 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.
KW - Former Faculty of Pharmaceutical Sciences
U2 - 10.1007/s11095-010-0116-8
DO - 10.1007/s11095-010-0116-8
M3 - Journal article
C2 - 20333453
SN - 0724-8741
VL - 27
SP - 1337
EP - 1347
JO - Pharmaceutical Research
JF - Pharmaceutical Research
ER -