Recombinant human insulin V Optimization of the reversed-phase high-performance liquid chromatographic separation |
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Authors: | V. E. Klyushnichenko S. A. Yakimov A. M. Arutyunyan A. E. Ivanov K. V. Maltsev A. N. Wulfson |
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Abstract: | The applicability of reversed-phase high-performance liquid chromatography (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 investigated. Optimum conditions for the separation of insulin-related proteins 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 (μ). A mechanism for the separation of proteins with a mobile phase containing a high salt concentration and a pH near the isoelectric point of proteins is proposed. The questions of scaling up are considered. The proposed techniques allow the analysis of the main impurities and ensures a high quality of active insulin production. |
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