PEPTIDE-MAPPING AND EVALUATION OF GLYCOPEPTIDE MICROHETEROGENEITY DERIVED FROM ENDOPROTEINASE DIGESTION OF ERYTHROPOIETIN BY AFFINITY HIGH-PERFORMANCE CAPILLARY ELECTROPHORESIS

High-performance capillary electrophoresis (HPCE) has been employed to characterize the peptide map of recombinant human erythropoietin (rHu EPO) expressed from Chinese hamster ovary (CHO) cells. The methodology employs an ion pairing agent, 100 mM heptanesulfonic acid in 40 mM so dium phosphate buffer, pH 2.5, to increase peptide resolution, to decr ease analyte wall interactions, and to evaluate glycopeptide microhete rogeneity. The total tryptic map is segregated into two regions, nongl ycosylated and glycosylated peptides. Reproducibility of the peptide m ap is excellent; the map results in baseline separation of 16 tryptic peptides and one doublet peak composed of two peptides (resolution 0.2 2). The map furthermore allows for the evaluation of the microheteroge neity associated with the three rHuEPO glycopeptides. At least 12 glyc opeptide forms were separated in the initial peptide map. Peptides wer e identified by Edman sequencing, and the glycopeptides were further s ubjected to Dionex anion-exchange chromatography. To simplify the leve l of complexity associated with the glycopeptides, much of the charact erization employed asialoglycopeptides and employed several endoproteo lytic digestions. The relative percent distribution for each purified asialoglycopeptide was calculated to define the level of complexity an d to tentatively assign a known structure to the HPCE peak. The level of structural complexity of the asialoglycopeptides appears to increas e from the simplest O-linked form to the more complex N83, N38, and N2 4 glycosylation positions, respectively. HPCE evaluation of glycopepti de microheterogeneity appears to be simpler, faster, and just as sensi tive as other more frequently employed methods for glycopeptide charac terizations.