Comparative study on the deamidation of three recombinant human insulins using capillary electrophoresis

The applicability of capillary zone electrophoresis (CZE) for the separation of different recombinant human insulins and their deamidated isoforms was studied. The high resolving power of CZE is demonstrated by its ability to separate insulin isoforms differing only by 0.984 Da (different-fold deamidated forms) and even components having the exacts same mass but slightly different shapes (same-fold deamidated forms). From among the several insulins available, humulin, glargine and glulisine were selected for our study because their sequences and chemical parameters are quite similar, however, the small differences present in their amino acid sequences influence the deamidation processes. Using a background electrolyte with basic pH was favourable not only for the separation of the different types of insulin but also for the separation of deamidated protein forms even in a bare fused silica capillary. The LOD values ranged between 0.6 -0.93 mg/L and 2.17 -4.37 mg/L for UV and ESI-MS detection, respectively. At-20 --80 degrees C, the deamidation is minimal, but at temperatures above +5 degrees C deamidation is accelerated. At +5 degrees C only 1-fold deamidation forms could be observed for each insulin. Acidified samples incubated for 1-month at room temperature showed varying levels of deamidation: 1-fold, 1-2-fold and 1-2-3-fold forms for glargine, glulisine and humulin, respectively.

Automated summary

The applicability of capillary zone electrophoresis (CZE) was studied for the separation of different recombinant human insulins and their deamidated isoforms. CZE demonstrated high resolving power, separating insulin isoforms with small differences in mass and shape. Humulin, glargine, and glulisine were selected for study due to their similar sequences and chemical parameters, and the deamidation processes were influenced by minor differences in their amino acid sequences. The use of a basic pH background electrolyte enabled not only the separation of different types of insulin but also the separation of deamidated protein forms even in a bare fused silica capillary. The LOD values for UV and ESI-MS detection ranged from 0.6-0.93 mg/L and 2.17-4.37 mg/L, respectively. Deamidation was minimal at temperatures between -20 to -80 degrees C but accelerated above +5 degrees C. Acidified samples incubated for a month at room temperature showed varying levels of deamidation for different insulins.