RECOMBINANT HUMAN INSULIN .5. OPTIMIZATION OF THE REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC SEPARATION

The applicability of reversed-phase high-performance liquid chromatogr aphy (HPLC) to the analysis of the products of recombinant insulin was studied. The influence of several mobile phases in reversed-phase and ion-pair HPLC on selectivity, resolution and sensitivity was investig ated. Optimum conditions for the separation of insulin-related protein s on commercial and laboratory-made supports were established by means of three-dimensional optimizations of selectivity and resolution as a function of pH and ionic strength (mu). A mechanism for the separatio n of proteins with a mobile phase containing a high salt concentration and a pH near the isoelectric point of proteins is proposed. The ques tions of scaling up are considered. The proposed techniques allow the analysis of the main impurities and ensures a high quality of active i nsulin production.