PREPARATIVE AND ANALYTICAL SEPARATION OF INSULIN-DEXTRAN CONJUGATES FROM NATIVE INSULIN - APPLICATION TO PREPARATION AND CHARACTERIZATION OF INSULIN-DEXTRAN CONJUGATES

A high-performance size exclusion chromatographic (HPSEC) method was d eveloped for analysis and separation of insulin-dextran conjugates and native insulin. The separation is achieved on a size-exclusion analyt ical column with a mobile phase of 0.05 M phosphate buffer (pH 7.0): a cetonitrile (86:20, v/v) delivered ata now rate of 0.5 ml/min. The con jugates are detected using a UV detector at a wavelength of 280 irm, b ased on the UV absorbance of the attached insulin. Under these conditi ons, 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 conjuga tes were resolved from the native insulin which eluted at 6.3 min. Add itionally, a method for preparative separation of the conjugates from the native insulin was developed. The method is based on a glass colum n packed with silica gel powder and elution of the conjugates with dis tilled water in less than 80 min. The application of these methods to preparation and characterization of conjugates of insulin with various MWs of dextranswas also demonstrated. The conjugates were prepared us ing the periodate method and the reaction was monitored using the HPSE C method. After 72 h of reaction, the insulin content of the conjugate s (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 purifi ed using the preparative columns, resulting in less than 5% free insul in impurity in the final samples.