Analysis of peptides and proteins by capillary electrophoresis-mass spectrometry : method optimization for the improved characterization of peptides and intact proteins in the pharmaceutical area

In this work capillary electrophoresis-quadrupole time-of-flight mass spectrometry (CE-QTOF MS) was applied for the characterization of pharmaceutical peptide and protein preparations. As a result, the impurities of the peptide hormone tetracosactide were fully identified and characterized in detail by exact mass and fragment ion spectra. These impurities included N- and C-terminally truncated peptide fragments and peptides showing residual protecting groups and additional amino acids. An unmodified fused silica capillary was also used for the separation and identification of rhGH and its deamidated isoforms, applying an alkaline BGE. Despite the good results on rhGH, the method bore difficulties arising from the complexity of other intact proteins. Hence, a different and widely applicable method for the analysis of intact proteins was developed by the optimization of several CE-MS parameters for the analysis of 8 model proteins and erythropoietin (EPO). Thereby, the influence of the capillary coating and the nebulizer were investigated in detail. The validation of the optimized method revealed highly suitable and reliable CE-MS conditions for the analysis of various intact proteins in the pharmaceutical area. Using the optimized method numerous different EPO glycoforms were separated. Furthermore the number and types of the present glycoforms in various commercial and pre-production EPO preparations were determined. In order to fully exploit the capabilities of CE-MS, principal component analysis and hierarchical agglomerative clustering were applied as supportive strategies for the detailed characterization of these EPO preparations. By this means, all evaluated preparations were differentiated clearly based on the relative peak areas of only few selected glycoforms out of a pool of more than 100 naturally occurring isoforms present in each sample.



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