Minimization of shaking-induced formation of insoluble aggregates of insulin by cyclodextrins.
Aggregation is known to complicate insulin delivery and the processing and formulation of biotechnology-derived peptide/protein drugs. Shaking-induced formation of insoluble aggregates in bovine insulin and the potential role of cyclodextrins in preventing such aggregation were investigated. Insulin, dissolved in phosphate buffer, pH 7.2, and preserved with 2 mg/ml of phenol was aggregated, in triplicate, by shaking at 450 rpm for 2.5 days on a gyratory shaker. Visible aggregation was quantitated by measuring optical density in the visible range on a spectrophotometer. Solutions were then filtered through a 0.22 mu filter and the amount of insulin remaining in filtrate was determined by HPLC. Aggregation increased at lower concentrations, with solutions turning milky at 0.5 mg/ml; HPLC assay of filtrate indicated a complete loss of insulin. Under the same conditions, except for shaking, control solutions exhibited no insulin loss, excluding absorption as a cause of the insulin loss. The use of cyclodextrins (0.5 mg/ml) to stabilize insulin was investigated. alpha-, beta-, gamma- and hydroxypropyl-beta-cyclodextrin, each at 1.5% level, were used to prevent aggregation. The efficacy of cyclodextrins in preventing aggregation (% insulin aggregated in parentheses), was: hydroxypropyl-beta- (15) approximately beta- (18) > alpha- (54). No protection was observed with gamma-cyclodextrin.
Banga
A K
AK
Department of Pharmacal Sciences, Auburn University, AL 36849.
Mitra
R
R
D000327:Adsorption; D000818:Animals; D002417:Cattle; D002626:Chemistry, Pharmaceutical; D002851:Chromatography, High Pressure Liquid; D003505:Cyclodextrins / Q000737:chemistry*; D004339:Drug Compounding; D007328:Insulin / Q000008:administration & dosage* / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet
D003505:Cyclodextrins; D007328:Insulin