Preparative and Analytical Separation of Insulin-Dextran Conjugates From Native Insulin: Application to Preparation and Characterization of Insulin-Dextran Conjugates

Abstract

A high-performance size exclusion chromatographic (HPSEC) method was developed for analysis and separation of insulin-dextran conjugates and native insulin. The separation is achieved on a size-exclusion analytical column with a mobile phase of 0.05 M phosphate buffer (pH 7.0): acetonitrile (80:20, v/v) delivered at a flow rate of 0.5 ml/min. The conjugates are detected using a UV detector at a wavelength of 280 nm, based on the UV absorbance of the attached insulin. Under these conditions, the conjugates of insulin with dextrans with MWs of 500 kD, 70 kD, and 40 kD eluted at 4.3, 4.6, and 4.9 min, respectively. The conjugates were resolved from the native insulin which eluted at 6.3 min. Additionally, a method for preparative separation of the conjugates from the native insulin was developed. The method is based on a glass column packed with silica gel powder and elution of the conjugates with distilled water in less than 80 min. The application of these methods to preparation and characterization of conjugates of insulin with various MWs of dextrans was also demonstrated. The conjugates were prepared using the periodate method and the reaction was monitored using the HPSEC method. After 72 h of reaction, the insulin content of the conjugates (w/w) were independent of the MW of dextrans and ranged from 20.2 to 22%. Reaction samples containing 44–50% of native insulin were purified using the preparative columns, resulting in less than 5% free insulin impurity in the final samples.