This technical note reports an HPAE-PAD method to profile IgG N-linked oligosaccharides and N-linked high-mannose type oligosaccharides that are atypical for human polyclonal IgG but sometimes found on mAbs. This HPAE-PAD method is orthogonal to other oligosaccharide methods applied to IgG. More information on the development and application of the separation described in the technical note can be found in Rohrer, et al. as referenced in the document.

The Thermo Scientific Vanquish H system with fluorescence detection is applied for the glycan profile comparison of a biosimilar candidate produced ‘in house’ using Chinese hamster ovary (CHO) cell line using HILIC-based chromatographic separation on Accucore 150-Amide-HILIC column. Developed methodology offers an excellent analytical platform for glycan profile comparison of multiples lots of the biosimilar product.

O-glycans released by reductive ß-elimination to preserve structural integrity can be quantitatively measured by a simple direct method that minimizes sample manipulation and cleanup. Released glycans are separated with high resolution by charge, size and polarity on a mixed mode column employing both ion exchange and HILIC separation modes, using a simple, mass spectrometry compatible mobile phase.<enter final 90 characters of description>

The work shown here describes glycoprotein deglycosylation with PNGase F, followed by an efficient sample pretreatment using desalting and detergent removal spin columns to remove high concentrations of salt and detergents from small-scale PNGase F digestions. The HPAE-PAD technique was used to separate and detect the oligosaccharides. Oligosaccharides were separated using the Dionex CarboPac PA200 column. The PA200 is a nonporous, high-efficiency, pellicular anion-exchange column that provides high-resolution separations required for oligosaccharide mapping and analysis.

Here, oligosaccharides released from two glycoproteins are labeled with a fluorophore followed by their purification and HPAE-FLD analysis. Two different methods for purification of the labeled oligosaccharides from unbound labeling reagent were tested along with two elution conditions. The data show that the variations tested here do not significantly affect the retention time profiles and that the method described here is suitable for routine profiling of oligosaccharides released from glycoproteins. An ICS-6000 can be used for this application.