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

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 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.

Building a Cell Culture Process with Stable Foundations: Searching for Certainty in an Uncertain World

“…Attempts have been made to develop summary statistics that reduce the dimensionality of these complex data sets. For example, the Z number (Hermentin et al 1996) has been proposed as a parameter that describes protein glycosylation in terms of numbers of charged glycans. Such parameters have been suggested as being suitable for product consistency monitoring, although some workers have reported poor correlation with bioactivity (Yuen et al 2011).…”

Systematic Approach to Optimization and Comparability of Biopharmaceutical Glycosylation Throughout the Drug Life Cycle

“…For that exercise the levels of the glycosylation CQAs were measured for product made using different manufacturing conditions and the statistical tools (e.g., 17.3 Scheme 2: Comparability of Drug Glycosylation Using QbD DS 553 principal component analysis) were used to determine the relationships between the CPPs and the CQAs.…”

“…• Comparability of glycosylation between drug batches is primarily determined using single -number glycometrics (such as the Z -number score for measuring the degree of sialylation) [17] .…”

Ibio-Number Assay: A Physicochemical Assay that Predicts the Bioactivity of Erythropoietin with High Precision and Accuracy and May Replace the Mouse Bioassay in the Quality Control of EPO Batch Release

“…In recent years, there have been efforts to replace the highly contested (consumption of animals) and highly inaccurate (CV ≈ 25% [1,2], ≈ 20% [3]; uncertainty 15-30% [4]; as stated by Zimmermann et al [3]) polycythaemic and normocythaemic mouse bioassays in the quality control of erythropoietin [5] by more precise and more accurate physicochemical methods such as Z-number determination based on ion-exchange chromatography of the isolated N-glycans (glycan mapping) [2,6,7] or I-number determination [8] based on the native protein and the peak areas of the various isoforms separated in CZE [9,10]. This approach has recently been supported by a study from Roche [3] that aimed at replacing the normocythaemic mouse bioassay for epoetin beta batch release on the basis of primarily CZE data.…”