The hypothetical N-glycan charge: a number that characterizes protein glycosylation

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Abstract
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 struc tures 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: Thus, a glycoprotein with mostly C4-4* structures will provide Z≅400 (e.g., rhu EPO (CHO), Z=361), a glycoprotein carrying largely C3-3* structures will amount to Z≅300 (e.g., bovine fetuin, Z=290), a glycoprotein with mostly C2-2* structures will have Z≅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≅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.

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