Glycoproteins play a key role in many important biological and physiological processes and are increasingly studied as promising therapeutics. However, manufacturing these complex molecules at scale can be challenging, as even small changes in their composition or the position of glycan linkages can impact safety and efficacy. To maintain batch-to-batch consistency and to protect patient health, the use of accurate, reliable, and convenient methods for glycoprotein characterization is essential.
Glycoprotein characterization using high-performance anion-exchange chromatography
High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is a well-established technique for glycan analysis. HPAE chromatography takes advantage of the weakly acidic nature of carbohydrates to achieve highly selective glycan separations and is particularly effective for the profiling of sialylated glycans, which regularly feature in therapeutic glycoproteins. Coupled with PAD, the technique enables sensitive, derivatization-free analyte quantification, avoiding the need for time-consuming sample derivatization steps.
Ongoing advances in sensitivity, resolution, and accuracy afforded by Orbitrap mass spectrometry (MS) raise the bar when it comes to glycoprotein characterization. The latest Orbitrap technologies offer unparalleled analytical selectivity for confident structural elucidation, providing valuable insight on branching patterns and potential substitutions. By combining HPAE chromatography with Orbitrap MS, analytical scientists can harvest synergies between these two powerful techniques to achieve rapid and reliable glycan profiling.
High-resolution glycan profiling using HPAE coupled with Orbitrap MS
We put the performance of HPAE-Orbitrap MS for glycan profiling to the test in a recent application note.
Following efficient separation by HPAE chromatography, the glycans passed through a desalting device to remove sodium from the eluent, which can compromise analytical performance. Using the Q Exactive Hybrid Quadrupole Orbitrap mass spectrometer to fragment the glycans by higher-energy collisional dissociation, information-rich MS2 spectra were obtained. These spectra were dominated by glycosidic and cross-ring fragments that revealed valuable glycan linkage information.
Figure 1 presents extracted ion chromatograms showing the resolution of major fetuin sialylated glycan structures under four different elution conditions and highlights how higher hydroxide concentrations and lower temperatures resulted in improved peak resolution. By annotating all major and detectable minor peaks for the four glycoproteins, we found that the improved resolution achieved with higher hydroxide concentrations could be attributed to the more efficient separation of glycan isomers. The high-resolution performance offered by the Dionex ICS-5000+ HPIC and Q Exactive Hybrid Quadrupole Orbitrap systems was essential for achieving these results. Ultimately, this approach may be an effective way to quickly screen the impact of changes in cell culture conditions on sialylation.
Figure 1. Extracted ion chromatogram showing the resolution of major fetuin sialylated glycan structures under four different elution conditions; (A) 100 mM NaOH, 30 °C; (B) 100 mM NaOH, 35 °C; (C) 150 mM NaOH, 35 °C; (D) 150 mM NaOH, 25 °C.
Confident glycan structural analysis by HPAE-Orbitrap MS
Improvements in HPAE and Orbitrap MS technologies are providing greater confidence in glycoprotein characterization. By facilitating rapid and reliable structural determination, these powerful techniques offer manufacturers greater certainty in the safety of glycoprotein therapeutics.
You can read more about this HPAE-Orbitrap MS method for glycan profiling in this application note.