The hypothetical N-glycan charge: a number that characterizes protein glycosylation.
The production of recombinant glycoprotein therapeutics requires characterization of glycosylation with respect to the lot-to-lot consistency. Here we introduce the ¿hypothetical N-glycan charge Z' as a parameter that allows to characterize the protein glycosylation in a simple, however, efficient manner. The hypothetical N-glycan charge of a given glycoprotein is deduced from the N-glycan mapping profile obtained via HPAE-PAD. In HPAEC, N-glycans are clearly separated according to their charge, i.e., their number of sialic acid residues, providing distinct regions for neutral structures as well as for the mono- di-, tri, and tetrasialylated N-glycans (Hermentin et al., 1992a). Z is defined as the sum of the products of the respective areas (A) in the asialo, monosialo, disialo, trisialo, tetrasialo, and pentasialo region, each multiplied by the corresponding charge: [formula: see text] Thus, a glycoprotein with mostly C4-4* structures will provide Z approximately equal to 400 (e.g., rhu EPO (CHO), Z = 361), a glycoprotein carrying largely C3-3* structures will amount to Z approximately equal to 300 (e.g., bovine fetuin, Z = 290), a glycoprotein with mostly C2-2* structures will have Z approximately equal to 200 (e.g., human serum transferrin, Z = 207, or human plasma AT III, Z = 180), and a glycoprotein carrying only high-mannose type or trunkated structures will provide Z approximately equal to 0 (e.g., bovine pancreas ribonuclease B, Z = 15, and hen ovomucoid, Z = 15, respectively). The determination of Z was validated in multiple repetitive experiments and proved to be highly accurate and reliable. Z may therefore be regarded as a new and characteristic parameter for protein N-glycosylation.
Hermentin
P
P
Research Laboratories of Behringwerke AG, Marburg, Germany.
Witzel
R
R
Kanzy
E J
EJ
Diderrich
G
G
Hoffmann
D
D
Metzner
H
H
Vorlop
J
J
Haupt
H
H
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D002417:Cattle; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006834:Hydrazines / Q000378:metabolism; D008956:Models, Chemical; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results
D006023:Glycoproteins; D006834:Hydrazines; D011134:Polysaccharides; D011994:Recombinant Proteins; C029424:hydrazine; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase