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 Dionex UltiMate 3000 Bio-LC system is applied for the Reverse phase analysis of Infliximab. The separation was performed on a Thermo Scientific MabPac RP column with UV detection at 214 nm. All oxidized and deamidated impurities are well separated.

This work describes an HPAE-PAD method for monosaccharide composition analysis using manually prepared eluent. Monosaccharide analysis using electrolytically generated eluent has been described before. Here, three commercially available proteins, IgG, fetuin, and alpha-1-acid glycoprotein, were individually subjected to two hydrolysis conditions using 1)HCl, for the amino sugars galactosamine and glucosamine, and 2)TFA, for the neutral sugars mannose, glucose, and galactose. Results for method linearity, robustness, and accuracy are presented here. An ICS-6000 can be used for this application.

The Thermo Scientific Vanquish Flex UHPLC system with UV detection is applied for the charge variants analysis of NISTmAb Humanized IgG1K monoclonal antibody using strong cation exchange-based chromatographic separation on a MAbPac SCX-10 column. The use of Thermo Scientific CX-1 pH gradient buffers enabled high resolution separation of reference material monoclonal antibody charge variants using a 30 minute gradient.

This study shows a fully automated UHPLC setup applying a simple and efficient online solid phase extraction method. Showcase assay performance by using a column cartridge for sample trapping and a Thermo Scientific™ MAbPac™ RP column for separation with water/acetonitrile-based gradients. Leverage the new technologies such as the Thermo Scientific™ Vanquish™ UHPLC platform in combination with the Thermo Scientific™ Q Exactive™ HF Hybrid Quadrupole Orbitrap mass spectrometer.