THE HYPOTHETICAL N-GLYCAN CHARGE - A NUMBER THAT CHARACTERIZES PROTEIN GLYCOSYLATION

The production of recombinant glycoprotein therapeutics requires chara cterization of glycosylation with respect to the lot-to-lot consistenc y. Here we introduce the 'hypothetical N-glycan charge Z' as a paramet er 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 vi a HPAE-PAD. In HPAEC, N-glycans are clearly separated according to the ir charge, i.e., their number of sialic acid residues, providing disti nct regions for neutral structures as well as for the mono- di-, tri, and tetrasialylated N-glycans (Hermentin et al., 1992a). Z is defined ae 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: [GRAPHICS] Thus, a glycoprotei n with mostly C4-4 structures will provide Z congruent to 400 (e.g., rhu EPO (CHO), Z = 361), a glycoprotein carrying largely C3-3: struct ures will amount to Z congruent to 300 (e.g;., bovine fetuin, Z = 290) , a glycoprotein with mostly C2-2 structures will have Z congruent 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 trunka ted structures will provide Z congruent to 0 (e.g., bovine pancreas ri bonuclease B, Z = 15, and hen ovomucoid, Z 15, respectively). The dete rmination of Z was validated in multiple repetitive experiments and pr oved to be highly accurate and reliable. Z may therefore be regarded a s a new and characteristic parameter forprotein N-glycosylation.