Optimization of a mass spectrometric analytical method for the quality assessment of insulin and its analogs.

The principal aim of this study was to optimize analytical methodology based on mass spectrometry for the evaluation of the quality of recombinant human insulin and its analogs. In this study ESI-MS was used to assess the quality of human insulin, short acting insulin analogs, insulin lispro, insulin aspart and insulin glulisine and long acting analogs including insulin glargine, insulin degludec, and insulin detemir, in respective pharmaceutical formulations. In this study, with the aimed to optimize analytical conditions, different factors influencing the analytical performance such as pH, ionic strength, sample dilution, organic solvent addition were addressed. The study results demonstrated that MS is a suitable technique for the analysis of biotechnological compounds like insulin and its analogs. Although the obtained results provide an important information regarding this methodology, further studies are needed to validate this analytical approach and check for its suitability to be used in the regulatory environment.

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.