This report documents the development of a new monosaccharide separator column (CarboPac PA20, 3x150 mm) that allows fast, efficient monosaccharide separations with good spacing. It is based on a new chemistry with a reduced resin particle size (from 10 to 6.5 microm). Faster, more efficient separations of glycoprotein monosaccharides with better spacing were achieved across a range of isocratic NaOH concentrations at lower flow rates. Detection sensitivity was improved, enabling routine low to sub pmol monosaccharide determinations. Glycoprotein monosaccharides eluted in less than 10 min at a flow rate of 0.5 ml/min. Furthermore, when used with an AminoTrap guard column, the protein matrix consisting of amino acids and peptides (released by acid hydrolysis of glycoprotein) did not interfere with monosaccharide analysis. Compared to previous CarboPac columns (CarboPac PA1 and CarboPac PA10), the CarboPac PA20 has improved selectivity with respect to glycoprotein monosaccharides. The improved selectivity results in better separation of glucosamine and galactose, enabling the accurate determination of monosaccharide ratios for undergalactosylated glycoproteins. Finally, disposable gold working electrodes that eliminate the possibility of working electrode recession affecting peak area response were used.

Low picomole concentrations of glycoprotein monosaccharides are separated in less than 10 min by means of a new anion-exchange carbohydrate column. Pulsed electrochemical detection is simplified by means of disposable gold electrodes.

The high-resolution, high-efficiency ProPac(TM) ion-exchange columns are ideally suited to the characterization and quality control assessment of closely related protein variants. The rigid, nonporous pellicular resin provides exceptionally high resolving power, permitting the separation of proteins that differ by as little as one charged residue. In this brief application, we will examine the chromatography of a humanized monoclonal antibody.

We developed a set of cation exchange column packings (ProPac WCX-10 and ProPac SCX-10) that are based on a hydrophilic coated, pellicular polymeric support grafted with polymer chains bearing ion exchange functionalities. The supports are highly suited to resolving closely related protein variants. These column packings (1) afford minimal band spreading in conjunction with extremely high selectivity, (2) exhibit a very hydrophilic character, and (3) have moderate loading capacity. Cytochrome C variants (bovine, horse, rabbit) were baseline-separated, as was native ribonuclease A and its two deamidation products, the Asp67 and isoAsp67 forms. Humanized monoclonal antibody variants differing in the number of lysine residues at the C terminus of their heavy chains were baseline-resolved. Finally, the separation of hemoglobin variants found in a sample containing elevated levels of glycated hemoglobin was also demonstrated.

We developed a set of prototype cation-exchange column packings that are based on a hydrophilic coated, pellicular polymeric support with a grafted tentacular surface chemistry that is highly suited to resolving closely related protein variants. These column packings (1) afford minimal band spreading in conjunction with extremely high selectivity, (2) exhibit a very hydrophilic character and (3) have moderate loading capacity. Cytochrome c variants (bovine, horse, rabbit) were baseline-separated, as was native ribonuclease A and its two deamidation products, the Asp67 and isoAsp67 forms. Humanized monoclonal antibody variants differing in the presence of lysine at the C terminus of the heavy chains were baseline-resolved. Finally, the separation of hemoglobin variants found in a sample containing elevated levels of glycated hemoglobin was also demonstrated.

Presence or absence of N-acetylneuraminic acid (Neu5Ac) can change a sialylated glycoprotein's serum half-life and possibly its function. We evaluated the linearity, sensitivity, reproducibility, and accuracy of a HPAEC/PAD method to determine its suitability for routine simultaneous analysis of Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). An effective internal standard for this analysis is 3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (KDN). We investigated the effect of the Au working electrode recession and determined that linear range and sensitivity were dependent on electrode recession. Using an electrode that was 350 microm recessed from the electrode block, the minimum detection limits of Neu5Ac, KDN, and Neu5Gc were 2, 5, and 2 pmol, respectively, and were reduced to 1, 2, and 0.5 pmol using a new electrode. The response of standards was linear from 10 to 500 pmol (r2>0.99) regardless of electrode recession. When Neu5Ac, KDN, and Neu5Gc (200 pmol each) were analyzed repetitively for 48 h, area RSDs were <3%. Reproducibility was unaffected when injections of glycoprotein neuraminidase and acid digestions were interspersed with standard injections. Area RSDs of Neu5Ac and Neu5Gc improved when the internal standard was used. We determined the precision and accuracy of this method for both a recessed and a new working electrode by analyzing Neu5Ac and Neu5Gc contents of bovine fetuin and bovine and human transferrins. Results were consistent with published values and independent of the working electrode. The sensitivity, reproducibility, and accuracy of this method make it suitable for direct routine analysis of glycoprotein Neu5Ac and Neu5Gc contents.

A new quadruple-potential waveform is introduced for detection of carbohydrates using pulsed amperometry. The new waveform cleans the electrode by application of a potential more negative than the potential limit. In contrast to a commonly used triple-potential waveform, negative cleaning allows the time during which gold oxide is formed to be minimized, thus minimizing the dissolution and resulting recession of the gold working electrode as a result of gold oxide formation/reduction cycles. Preventing gold electrode recession is shown to improve long-term reproducibility. Waveform parameters were chosen to maximize signal-to-noise ratio and freedom from electrode fouling caused by matrix components in the sample. Compared to the triple-potential waveform, the quadruple-potential waveform shows similar minimum detection limits but greatly improved long-term reproducibility.

As discussed in Chapter 4 , exoglycosidases are useful reagents for the structural determination of glycoconjugates. Their anomeric, residue, and linkage specificity for terminal monosaccharides have been used to assess monosaccharide sequence and structure in a variety of glycoconjugates (1). Their usefulness depends on the absence of contaminating exoglycosidases and an understanding of their specificity. Digestions of oligosaccharides with exoglycosidases give two classes of products: monosaccharides and the shortened oligosaccharides. Most assays of such reactions have monitored the reaction by following oligosaccharides that are labeled at their reducing ends. In these assays, after exoglycosidase digestion the shortened oligosaccharide retains the label at its reducing end. The other digestion product, the released monosaccharide, does not carry a label and thus cannot be quantified. Additionally, identification of any other monosaccharide that could be the result of a contaminating exoglycosidase activity would not be possible. Quantitative measurement of all products (all released monosaccharide[s] as well as the shortened oligosaccharide product) would be useful because it would enable the determination of any contaminating exoglycosidase activities by determining the extent of release of other monosaccharides. High pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) detects the appearance of monosaccharide product(s), the shortened oligosaccharide product(s) as well as the disappearance of the oligosaccharide substrate(s) in a single chromatographic analysis without labeling. Thus, HPAEC-PAD has been used extensively to monitor the activities of several different exoglycosidases on glycocoqugates, usually using the CarboPac PA1 column to separate the digestion products (see refs. 1–8).

Protein glycosylation is a major post-translational modification that generates a potentially large group of glycoforms from a single polypeptide chain (1). Over 50% of catalogued protein sequences contain the requisite sequence for N-glycosylation (AsnXxxSer(Thr); Xxx ≠ Pro) (2), and any Ser or Thr can potentially be glycosylated (3). Since glycosylation can play an important role in the biological and pharmacologic activity of the glycoprotein, there is increasing interest in identifying and characterizing the oligosaccharide moieties of glycoproteins.

The monosaccharide content of a glycoprotein is often determined by acid hydrolysis at elevated temperature and subsequent high pH chromatography of the released, underivatized monosaccharides on pellicular anion-exchange resin (HPAE) using pulsed amperometric detection (PAD). We have found that for glycoproteins with low levels of glycosylation, monosaccharide quantitation can be compromised by amino acids fouling the working electrode surface. Specifically, lysine elutes on the CarboPac PA1 column just prior to galactosamine, whereas remaining amino acids and most peptides elute after the monosaccharides and do not interfere with monosaccharide quantification. A direct comparison of PAD vs Abs215detection of lysine using the CarboPac PA1 column as the separator reveals that lysine does not cleanly come off the working electrode. The monosaccharide response inhibition caused by lysine could be corrected by the posthydrolysis addition of a rhamnose internal standard and the determination of “correction factors.” We have developed a guard column with an altered selectivity for amino acids which, when used with a new separator, causes lysine to elute after the monosaccharides and also causes hydrophobic amino acids to elute further after the monosaccharides. Together the new separator and guard columns solve the lysine fouling problem, reduce sample-related baseline noise, and reduce the magnitude of correction factors.