Minimization of Shaking-induced Formation of Insoluble Aggregates of Insulin by Cyclodextrins

Abstract

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 gyrotory shaker. Visible aggregation was quantitated by measuring optical density in the visible range on a spectrophotometer. Solutions were then filtered through a 0.22 μ 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, α-, β-, γ- and hydroxypropyl-β-cyclodextrin, each at 1.5% level, were used to prevent aggregation. The efficacy of cyclodextrins in preventing aggregation (% insulin aggregated in parentheses), was: hydroxypropyl-β- (15) ～ β- (18) > α- (54). No protection was observed with γ-cyclodextrin.