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.