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.
Klausen
N K
NK
Biologics Development, Novo Nordisk A/S, Gentofte, Denmark.
Kornfelt
T
T
D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary; D015942:Factor VIIa / Q000737:chemistry* / Q000235:genetics; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D009439:Neuraminidase / Q000378:metabolism; D009844:Oligosaccharides / Q000032:analysis; D010446:Peptide Fragments / Q000737:chemistry; D011700:Putrescine / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D012795:Sialoglycoproteins / Q000032:analysis; D014357:Trypsin / Q000378:metabolism
D006023:Glycoproteins; D009844:Oligosaccharides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; D009439:Neuraminidase; D015942:Factor VIIa; D014357:Trypsin; D011700:Putrescine