Kinetics of insulin aggregation in aqueous solutions upon agitation in the presence of hydrophobic surfaces.

Abstract

The stability of protein-based pharmaceuticals (e.g., insulin) is important for their production, storage, and delivery. To gain an understanding ofinsulin's aggregation mechanism in aqueous solutions, the effects of agitation rate, interfacial interactions, and insulin concentration on the overall aggregation rate were examined. Ultraviolet absorption spectroscopy, high-performance liquid chromatography, and quasielastic light scattering analyses were used to monitor the aggregation reaction and identify intermediate species. The reaction proceeded in two stages; insulin stability was enhanced at higher concentration. Mathematical modeling of proposed kinetic schemes was employed to identify possible reaction pathways and to explain greater stability at higher insulin concentration.The stability of protein-based pharmaceuticals is essential for the efficacy of conventional therapeutic preparations (1), continuous infusion pumps, and controlled release polymeric devices. Insulin aggregation, accompanied by drastic reduction of biological potency and obstruction of delivery routes, creates serious problems for drug delivery systems (2-4). Although insulin aggregation has been investigated (5-16), its molecular mechanism remains speculative.This study aims at elucidating the fundamental nature of this phenomenon using a rigorous kinetic analysis. Based on experimental observations, a reaction mechanism was formulated and possible destabilizing pathways were identified. Mathematical modeling was used to verify the predictive powers of the proposed scheme.MATERIALS AND METHODS Solution Preparation. Bovine Zn-insulin (specific activity, 24.4 international units/mg; Zn2+ content, <0.5%) was used.Phosphate-buffered saline (PBS) (0.14 M NaCI/0.1% NaN3 preservative, pH 7.4) was sterilized by filtration through a 0.45-,um Millipore HV filter and degassed by sonication. Stock solutions were prepared by adding Zn-insulin to PBS; the resulting cloudy mixture was sealed with Parafflm, placed in a shaker, and gently agitated for 3 hr at 37°C. Zn-insulin dissolved completely at concentrations up to 0.6 mg/ml. The stock solutions were filtered through sterile 0.22-,m Millex GV low-protein binding filters; lower concentrations were obtained by dilution with PBS prior to final filtration. All glassware was rinsed with 0.01 M HCl, followed by drying at 100°C. The initial concentrations of Zn-insulin solutions were determined by UV absorbance at 280 nm (e = 5.53 mM-'cm-1).Concentration-Dependence Studies. Air-water interface. Glass 1.1-ml HPLC vials were filled with 0.75 ml of insulin solution, capped, sealed with Parafilm, taped horizontally to the shaker platform, and agitated at 250 rpm and 370C. Every 20 min, one vial was removed and the extent of aggregation was determined by size-exclusion isochratic HPLC analysis [Bio-Rad's SEC-125 column; mobile phase consisting of 10% acetonitrile and 90% aqueous solution containing 0.02 M NaH2PO4 and 0.05 M Na2SO4 (pH 6.8), flow rate of 1.2 ml/min, detection at 280 and 217 nm]. Ins...

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