Quantitative twoplex glycan analysis using 12C6 and 13C6 stable isotope 2-aminobenzoic acid labelling and capillary electrophoresis mass spectrometry

Abstract

Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS). To ensure maximum resolution and excellent precision without the requirement for excessive analysis times, CE separation conditions including the concentration and pH of the background electrolyte, the effect of applied pressure on the capillary inlet and the capillary length were evaluated. Using readily available C stable isotopologues of 2-AA, the developed method can be applied for quantitative glycan profiling in a twoplex manner based on the generation of extracted ion electropherograms (EIE) for C 'light' and C 'heavy' 2-AA labelled glycan isotope clusters. The twoplex quantitative CE-MS glycan analysis platform is ideally suited for comparability assessment of biopharmaceuticals, such as monoclonal antibodies, for differential glycomic analysis of clinical material for potential biomarker discovery or for quantitative microheterogeneity analysis of different glycosylation sites within a glycoprotein. Additionally, due to the low injection volume requirements of CE, subsequent LC-MS analysis of the same sample can be performed facilitating the use of orthogonal separation techniques for structural elucidation or verification of quantitative performance.

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation, methodology and applications

“…In this regard, a CZE‐MS method was developed by Varadi et al. using 12/13 C 6 stable isotopologues of 2‐aminobenzoic acid as labeling agents . In this way, precise relative quantitative data on mAb glycosylation profiles could be achieved in a twoplex manner, including the batch‐to‐batch variation of Cetuximab.…”

“…Besides migration time index and the application of glycan standards and/or exoglycosidases, CE-MS methods have been established to facilitate structural identification, especially for unknown glycan structures and in complex samples. In this regard, a CZE-MS method was developed by Varadi et al using 12/13 C 6 stable isotopologues of 2-aminobenzoic acid as labeling agents [74]. In this way, precise relative quantitative data on mAb glycosylation profiles could be achieved in a twoplex manner, including the batch-to-batch variation of Cetuximab.…”

Insights from capillary electrophoresis approaches for characterization of monoclonal antibodies and antibody drug conjugates in the period 2016–2018

“…The last development of CE-MS methods highlighted the attractiveness to characterize and quantify the glycosylation heterogeneity with high accuracy and precision [107]. In 2016, a quantitative twoplex glycan analysis using heavy and light carbon stable isotopes of 2-aminobenzoic acid (2-AA) labelling have been developed to check the quality of different mAbs batches [118]. Among the possible explanations, the type of sialic acid attached to glycans has a significant impact on the IMS heat map.…”

Capillary Electrophoresis Separations of Glycans

“…In addition to APTS, other labeling reagents can be used with CE separations of carbohydrates. Recently, 2-aminobenzoic acid was used with a helium cadmium laser rather than an argon ion laser, 76 as well as for UV–visible absorbance detection 77 or CE coupled with MS. 78 The fluorescent label 7-amino-4-methylcoumarin 79 was also reported with an UV light emitting diode excitation source. This dye is neutral following conjugation and labeling, so the use of boric acid, which complexes with diols to form a negative complex in order to separate glycans, was required.…”