Troels Kornfelt

Analysis of the glycoforms of human recombinant factor VIIa by capillary electrophoresis and high-performance liquid chromatography

The carbohydrate-dependent microheterogeneity of recombinant coagulation factor VIIa (rFVIIa) has been characterized by capillary electrophoresis (CE) of the native protein and by high-performance liquid chromatography (HPLC) of tryptic peptides and of oligosaccharides released by hydrazinolysis. The development of the CE analysis is reported here. We have found that application of 1,4-diaminobutane (putrescine) as additive to the CE separation buffer is essential for the separation of the various glycoforms. Under optimum conditions rFVIIa migrates as a cluster of six peaks or more. By CE of neuraminidase-treated rFVIIa a faster-moving double peak is observed. This indicates that the separation obtained is primarily based upon the different content of N-acetyl-neuraminic acid of the oligosaccharide structures in rFVIIa. By reversed-phase HPLC of tryptic digested neuraminidase treated rFVIIa the glycopeptides containing the heavy chain N-glycosylated site elute as two peaks compared to the four peaks corresponding to glycopeptides with 0 to 3 N-acetyl-neuraminic acids seen for untreated rFVIIa. In high-pH anion-exchange HPLC of the oligosaccharides released from native rFVIIa by hydrazinolysis the major peaks elute as oligosaccharides with 1 or 2 N-acetyl-neuraminic acids. Oligosaccharides released from neuraminidase treated rFVIIa elute earlier compared to oligosaccharides from native rFVIIa, but separated into several peaks, indicating heterogeneity for the oligosaccharide structures without N-acetyl-neuraminic acid.

Improved peptide mapping using phytic acid as ion-pairing buffer additive in capillary electrophoresis

By digestion of the highly basic polypeptide aprotinin or bovine pancreatic trypsin inhibitor (BPTI) with endoproteinase Lys-C after unfolding, reduction and pyridylethylation, five fragments are obtained. These fragments are separated by free solution capillary electrophoresis using a phosphate buffer at neutral pH. The effect of the ion-pairing buffer additive phytic acid on the separation was investigated. It is shown that phytic acid through ion-pair formation influences the mobility of only those peptide fragments having a net positive charge at the pH of the separation buffer. The affinity of phytic acid to the peptides correlates with their isoelectric point and the charge to mass ratios. Hence, by changing the concentration of phytic acid, it is possible to manipulate the migration order and the separation of the peptides.