Glycans play a key role in determining the structure and function of protein therapeutics. Even small modifications in the composition or location of these polysaccharide chains can have a large impact on the efficacy and safety of biopharmaceutical products. Accurate methods for characterizing glycoprotein-based therapeutics are essential to ensure product quality and safeguard patient health.

Glycoprotein monosaccharide content determination using HPAE-PAD

High-performance anion-exchange (HPAE) chromatography coupled with pulsed amperometric detection (PAD) is a powerful carbohydrate analysis technique that combines the highly selective separation performance offered by HPAE with the ability to directly quantify analytes using PAD, eliminating the need for time-consuming sample derivatization.

Determination of the monosaccharide composition of a glycoprotein pharmaceutical is a key quality control assay for glycoprotein-based therapeutics. HPAE-PAD is widely used for the determination of glycoprotein monosaccharide composition in glycoproteins. However, accurate analyses require complete and reproducible hydrolysis of glycoprotein samples, as small variations in hydrolysis conditions can affect monosaccharide yields and lead to inconsistent results. Convenient methods for determining and optimizing the efficiency of glycoprotein hydrolysis reactions are therefore vital for obtaining reliable results.

Optimizing glycoprotein hydrolysis efficiency using a glycopeptide standard

One of the most effective ways to determine the efficiency of glycoprotein hydrolysis reactions involves the use of glycopeptides as positive controls. By monitoring the release of monosaccharides from a glycopeptide standard with a known composition, the reaction efficiency can be calculated and improvements can be realized.

We recently demonstrated the potential of this approach using a commercially available glycopeptide standard, as reported in this application brief. A glycopeptide standard and two glycoproteins, fetuin and alpha-1 acid glycoprotein (AGP), were hydrolyzed for 4 hours using either hydrochloric acid (HCl) or trifluoroacetic acid (TFA). Following a short work-up procedure, the hydrolyzed samples were analyzed using a Thermo Scientific Dionex ICS-5000+ Reagent Free Ion Chromatography System running a Dionex CarboPac PA20-Fast-4μm column. Representative chromatograms for the HCl and TFA hydrolyzed glycopeptide are shown in Figure 1. Peaks corresponding to all expected monosaccharides, including fucose, galactosamine, glucosamine, galactose, glucose and mannose, can be seen.

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Monosaccharide yields for the glycopeptide standard using each hydrolysis method were compared with those of fetuin and AGP. Amino sugar yields from the HCl hydrolysis reactions were close to the theoretical maximum, indicating complete release of amino sugars, whereas yields from the TFA reactions were lower for the glycopeptide control and both glycoproteins. This comparison revealed efficient hydrolysis using the tested conditions, demonstrating the effectiveness of this approach for method optimization.

Advances in HPAE-PAD column chemistries are delivering more accurate analyses

Achieving high-resolution chromatographic separation is critical for achieving accurate and reliable monosaccharide determinations using HPAE-PAD. The Dionex CarboPac PA20-Fast-4μm column used in this analysis is specifically designed for the determination of mono- and disaccharides in pharmaceutical, food/beverage and biofuel samples. Packed with smaller resin particles that produce more efficient peaks, the advanced column chemistry in the Dionex CarboPac PA20-Fast-4μm column supports more accurate signal integration and ultimately more accurate results. The smaller particle size and shorter column lengths also result in significantly shorter run times compared with standard Dionex CarboPac PA20 columns, without sacrificing performance.

Driving improvements in glycoprotein carbohydrate analysis

Determining the glycoprotein monosaccharide composition of biopharmaceutical proteins demands the highest standards of analytical accuracy. Thanks to improvements in column chemistries and the latest techniques for method optimization, HPAE-PAD analyses are helping researchers achieve even greater confidence in their results.

You can read more about this approach for improving accuracy in HPAE-PAD glycoprotein carbohydrate analysis in this application brief.

Figure 1. HPAE-PAD analysis of the glycopeptide standard hydrolyzed using trifluoroacetic acid (red trace) and hydrochloric acid (blue trace).

Figure 1. HPAE-PAD analysis of the glycopeptide standard hydrolyzed using trifluoroacetic acid (red trace) and hydrochloric acid (blue trace).