Abstract
An anion-exchange high-performance liquid chromatographic method using a TSKgel DEAE-2SW column and zone electrophoresis using a fused-silica capillary are described for the preparative and analytical separations of the products of the reaction between insulin and exo-3,6- epoxy-1,2,3,6-tetrahydrophthalic anhydride (ETPA), an amino protective reagent. Four stable products were identified having exo-3,6-epoxy-1,2,3,6-tetrahydrophthalic group(s) on insulin at the i) Gly(A1), ii) Gly(A1) and Phe(B1), iii) Gly(A1) and Lys(B29), and iv) Gly(A1), Phe(B1) and Lys(B29) sites, together with four labile products produced by the reaction of ETPA with the non-amino groups of the stable products. The usefulness of capillary electrophoresis was demonstrated in the evaluation of the effect of the molar ratios of anhydride to insulin on the production of the insulin derivatives.
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