The analysis of glycosylation patterns on glycoprotein therapeutics such as antibodies is important as glycans are critical to glycoprotein function and stability. Among the many techniques that have been used for glycan analysis, high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) is significant as it provides high-resolution separation and high-sensitivity detection for oligosaccharides without requiring sample labeling.
HPAE-PAD separations can be performed on Dionex CarboPac® columns, a family of anion-exchange columns developed for carbohydrate analysis. The newest member of the CarboPac column family—the CarboPac PA200 column—is designed for high-resolution oligosaccharide separations.
The CarboPac PA200 typically uses a NaOAc gradient of up to 250 mM in 100 mM NaOH. Some glycobiologists have observed that this gradient is not always effective for separating neutral N-linked oligosaccharides.
We have found that the eluting NaOH concentration has significant impact on neutral oligosaccharide separations. Decreasing the NaOH concentration from 100 to 50 mM greatly improved resolution.
Six neutral and two sialylated oligosaccharide standards were used in this study (Figure 1). These oligosaccharide structures are commonly found on monoclonal and polyclonal antibodies. All separations were performed on a Dionex ICS-3000 system, which includes an ICS-3000 DP gradient pump, an AS autosampler, and an ICS-3000 DC column compartment with an electrochemical detector cell.
The carbohydrates were detected by pulsed amperometry with a gold working electrode and an Ag/AgCl reference electrode using the standard 4-potential waveform developed at Dionex (t1=0.0s, E1= +0.1V; t2=0.2s, E2= +0.1V; t3=0.4s, E3= +0.1V; t4=0.41s, E4= -2.0V; t5=0.42s, E5= -2.0V; t6=0.43s, E6= +0.6V; t7=0.44s, E7= -0.1V; t8=0.5s, E8= -0.1V). Chromatography was controlled by Chromeleon® Chromatography Data System software.
The effect of NaOH concentration on oligosaccharide separation using the CarboPac PA200 column is shown in Figure 2A. With the typically used 100 mM NaOH, two pairs of oligosaccharides co-eluted and another pair was only partially resolved.
When 50 mM NaOH was used, the resolution was greatly improved and all the six neutral oligosaccharides were well resolved. The sodium acetate gradient was 0–5 mM NaOAc in 40 min for both. Adjusting the sodium acetate gradient did not significantly improve the resolution. For this set of neutral oligosaccharides, NaOH concentrations higher than 100 mM resulted in faster elution and more severe co-elution. Decreasing the NaOH concentration further to 30 mM again resulted in co-elution of some peaks.