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Rapid Profiling of RapiFluor-MS Labeled NIST mAb N-glycans using UHPLC Coupled with an Orbitrap-based Mass Spectrometer

RapiFluor-MS Labeled NIST mAb N-glycans Profiling

September 08, 2020

In recent years, recombinant antibodies emerged as one of the front runners in the pharmaceutical industry. To perform quality control of these antibody drugs, a reference standard was needed. After rigorous analysis, the National Institute of Standards and Technology (NIST) has identified a protein for this purpose and labeled it as NIST monoclonal antibody (NIST mAb). Currently, this mAb is widely used as a gold standard in drug manufacturing. Using SimGlycan® software, we have developed a semi-automated workflow to identify the compositionally accurate NIST mAb N-glycans acquired using UHPLC coupled with an Orbitrap-based mass spectrometer.

Four raw files corresponding to the four technical replicates of RapiFluor-MS (RFMS) labeled NIST mAb sample were generated using the Thermo QExactive Plus instrument after the MS/MS analysis. The raw files were subjected to SimGlycan's robust LC-MS peak processing algorithm to generate the peak lists, and cluster MS/MS scans to the appropriate peaks. The peak lists were further aligned to find out and retain the valid peaks only. The aligned peaklist was subjected to MS/MS database search with a precursor and product ion tolerance of 10 ppm. Additional filters were applied to ensure that the software reported only those glycan structures that have 80% of their monosaccharide residues explained by structure specific characteristic ions in the MS/MS spectra. A total of 20 unique glycans were identified. These glycans were exported to Microsoft Excel file format and categorized in different groups based on Fucosylation, Galactosylation or Sialylation. Interactive charts and plots can be generated for different desired glycan groups using Microsoft Excel for further comparative studies.

Schematic Workflow for N-Glycan Identification of RFMS Labeled NIST mAbs

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