Chemometrics approach for optimization of capillary electrophoretic conditions for the separation of insulin analogues.

Six insulin analogues with high degree of chemical similarity were separated using Capillary Electrophoresis (CE). In order to improve the method performance, optimization and development the Design of Experiment (DoE) approach has been used and this strategy provided related statistical models. This methodology delivered the information regarding the influence of main factors in the method development and explained the interaction of relevant factors in terms of shortening the analysis time. The response surface methodology (RSM) design employing Central Composite Face Centered (CCF) Design analyzed the effect of the most influencing factors, including background electrolyte pH and concentration, applied voltage and temperature. This study demonstrated that the approach of analyzing the influence of each parameter in the migration behavior of analyzed peptides was capable to assess the best electrophoretic conditions for the separation of insulin analogues.

Determination of deamidated isoforms of human insulin using capillary electrophoresis.

The applicability of capillary zone electrophoresis (CZE) for the separation of the deamidated forms of insulin has been studied. 50 mM NH4Ac (pH=9) with 20 % v/v isopropylalcohol was found optimal for efficient separation of insulin from its even 10 deamidated forms. The developed method was efficiently applied for monitoring the degradation rate of insulin and the formation of different deamidation isoforms. Two months after the acidification more than thirty peaks can be observed in the electropherogram, because degradation products other than deamidated components were formed as well. The recorded mass spectra enabled us to assign the exact mass of the components, and thus the identification of insulin isoforms could be accomplished. We think that this study provides useful information on how the determination of several deamidation forms can be carried out with CE-MS, but the identification of the exact position of deamidation sites in the insulin molecule remains a challenge.

Application of the capillary zone electrophoresis (CZE) and capillary gel electrophoresis (CGE) for the separation of human insulin, insulin lispro and their degradation products.

Two capillary electrophoresis (CE) methods have been developed for the separation of charge and mass variants of human insulin and its recombinant analogue lispro. Since the capillary zone (CZE) and Capillary gel electrophoresis (CGE) are based on different principles of separation, they can be used to detect different impurities of insulin and its analogues. Application of CZE enabled a separation of compounds with different m/z ratio, therefore CZE is a suitable method for the separation of deamidation products of insulin. After the optimization, this method is validated according ICH requirements. CGE method was used for the separation of higher molecular weight transformation products. Experimental data have shown that CZE and CGE are simple, fast and robust methods which could be used as a routine analysis for quality control of insulin formulations.