N-glycan charge assay--an alternative for potency assays of therapeutic glycoproteins?

A new in vitro potency assay for erythropoietin has been developed and compared to the exhypoxic polycythemic mouse bioassay for rhuEPO batch release, required according to Ph. Eur. 2001. Evaluation of 14 batches of EPO has shown that the new assay is highly sensitive and able to reveal subtle changes in N-linked carbohydrates. In combination with regular EPO batch release assays (such as sialic acid determination, isoelectric focusing, RP-HPLC, and peptide mapping), this assay may enable to replace the highly variable exhypoxic mouse bioassay for EPO batch release and stability studies and thus reduce the use of laboratory mice.

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.

A strategy for the mapping of N-glycans by high-performance capillary electrophoresis.

We have evaluated high-performance capillary electrophoresis (HPCE) with respect to its suitability for use in establishing a carbohydrate-mapping database that would enable a carbohydrate structural analysis by mere comparison of migration times. The suitability of HPCE for carbohydrate structural assignments was ascertained by validation experiments. The migration times of distinct N-glycans, prepared and measured on different days, were shown to be highly reproducible, with a coefficient of variation of usually less than 0.20%, requiring only femtomoles of N-glycan per injection for reliable measurements. By including mesityl oxide and sialic acid as internal standards and a triple-correction method, HPCE fulfills the analytical requirements with respect to accuracy, precision, reproducibility, and sensitivity. The N-glycan-mapping database was established using a newly developed and optimized buffer system containing 1,5-diaminopentane as an organic modifier. Approximately 80 different sialylated N-glycans of known structure, which have thus far been measured and characterized, have been entered into our Lotus 1-2-3 mapping database. The database for structural determinations was tested using the N-linked carbohydrates released from recombinant human urinary erythropoietin (baby hamster kidney) by PNGase F treatment and from bovine serum fetuin and alpha 1-acid glycoprotein by automated and manual (large-scale) hydrazinolysis, respectively. The efficiency of the database and of the triple-correction method was further confirmed by HPCE measurements performed in a different laboratory and by a different analyst who used the HPCE system of a different manufacturer.

The mapping by high-pH anion-exchange chromatography with pulsed amperometric detection and capillary electrophoresis of the carbohydrate moieties of human plasma alpha 1-acid glycoprotein.

The reducing oligosaccharides released from alpha 1-acid glycoprotein (AGP) by conventional hydrazinolysis have been analyzed by two different mapping techniques, using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and capillary electrophoresis (CE) with uv detection at 190 nm. The CE measurements proved about 4000 times more sensitive than the measurements by HPAE-PAD. The N-glycan pool was fractionated by Mono Q anion-exchange chromatography, and individual fractions so obtained were desialylated using Vibrio cholerae neuraminidase. The resulting asialo-N-glycans were further analyzed by HPAE-PAD, revealing 2 major, 4 intermediate, and 4 small peaks and at least 3 spikes, which counted for at least 13 different asialo-N-glycans. The carbohydrate structures were tentatively assigned by comparison of the Mono Q-separated N-glycans with the known AGP carbohydrate structures and known structures contained in a mapping database that allows structural assignment of N-glycans by mere comparison of retention times. In addition to the hitherto known AGP carbohydrate structures, we have tentatively identified a number of sulfated N-glycans that are currently being analyzed in more detail. We have also compared the glycan pools recovered from AGP using hydrazinolysis and glycopeptidase F (PNGase F). Approximately 40 distinct peaks could be detected in the hydrazinolysis-derived N-glycan pool by either technique (HPAE-PAD and CE), while about 30 distinct peaks were detected in the N-glycan pool derived by PNGase F digestion of the tryptic AGP digest of the same batch of AGP. These differences were attributed to an increased desialylation (approximately 3 mol%) during hydrazinolysis, based on the detection by HPAE-PAD and CE of free sialic acid and monosialylated oligosaccharides in the glycan pool derived by conventional hydrazinolysis. The integrity of the N-glycans' chitobiose core was examined by 500-MHz 1H NMR spectoscopy. The hydrazinolysis procedure could be optimized such that the hydrazinolysis-derived N-glycan pool was chromatographically essentially identical to the PNGase F-derived N-glycan pool. Hydrazinolysis proved best, with practically no loss of N-acetlylneuraminic acid and the closest resemblance to the PNGase F-derived N-glycan pool, using an automated apparatus. Notably, it was recognized that, in our hands, PNGase F digestion in the presence of sodium dodecyl sulfate resulted in partial desialylation of the liberated N-glycans.

A strategy for the mapping of N-glycans by high-pH anion-exchange chromatography with pulsed amperometric detection.

We have evaluated the high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) with respect to its suitability to establish a carbohydrate mapping database that would enable carbohydrate structural analysis by mere comparison of retention times. The suitability of HPAE-PAD for carbohydrate structural analysis was ascertained by validation experiments. The retention times of distinct N-glycans, prepared and measured on different days, were shown to be highly reproducible, with a coefficient of variation (CV) of less than 0.5%, requiring less than 100 pmol of N-glycan per injection for reliable measurements. Including appropriate internal chromatographic standards, such as (Neu5Ac)1, (Neu5Ac)2, (Neu5Ac)3, and Neu5Gc, the HPAE-PAD method fulfills the analytical requirements with respect to accuracy, precision, reproducibility, and sensitivity. The N-glycan mapping database was established, using two optimized linear gradients "S" and "A" for sialylated and asialo N-glycans, respectively. Approximately 100 different N-glycans of known structure, which have thus far been measured and characterized, have entered our Lotus 1-2-3 mapping database. The efficiency of the database for structural determinations was tested, using the N-linked carbohydrates isolated from rhuEPO, expressed in BHK cells. Nine different sialylated N-glycans of rhuEPO (BHK) could be assigned with a deviation of less than +/- 0.5%, using gradient S, and six of the eight asialo N-glycans of rhuEPO (BHK) detected with gradient A could be assigned with an accuracy of less than +/- 1%, three of them even with an accuracy of less than 0.1%, providing the reliability of the established HPAE-PAD mapping database.

Spacing of Munsell Renotations in MacAdam's Geodesic Diagram.

The results of a brief experiment are described in which the perception of certain color intervals is compared with their corresponding intervals as computed from MacAdam's geodesic chromaticity diagram, the Munsell renotation system, using the Godlove formula and the 1964 CIE color-difference formula.