Influence of the composition of the electrolyte solution on protein mobility and on capillary electrophoresis analysis of the synthetic therapeutic polypeptide calcitonin

This communication reports the results of a study performed to evaluate the interactions between proteins and ionic species in solution that may affect the electrophoretic mobility of these biomolecules. The investigation has been conducted with standard proteins and with synthetic human calcitonin, a 32-amino acid linear polypeptide hormone, which is currently employed for the treatment of chronic bone disorders, such as osteoporosis and hypercalcemia. The study covers a series of background electrolyte solutions (BGE) composed of phosphate, acetate, trifluoroacetate, chloride or formate salts of N,N,N'N'-tetramethyl-1,3-butanediamine (TMBD) that has been proven to be effective at preventing protein-capillary wall interactions in bare fused-silica capillaries. It is reported that several ionic species used as the components of the BGE may interact with proteins to different extents, depending on the chemical nature of both the protein and the ionic species in solution and on pH and concentration of the electrolyte solution. Such interactions are expected to influencing both charge and molecular size of the solubilized protein and, therefore, it charge-to-mass ration and, consequently its electrophoretic mobility. In addition, the components of the electrolyte solution affect the electric double layer at the interface between the capillary wall and the electrolyte solution with consequent variations of the electroosmotic flow. As a consequence, the migration behavior of proteins and their resolution are affected by both the variation of the electroosmotic flow and of the electrophoretic mobility of proteins, resulting by their interactions with the components of the electrolyte solution. It is shown that besides affecting the apparent mobility of the analytes, the appropriate selection of the concentration of TMBD in the running electrolyte can be useful to modulate the resolution of a given separation, such as the degradation products present in an aged sample of synthetic human calcitonin for therapeutic use, separated with BGEs of different compositions.