The glycan moieties attached to the monoclonal antibodies (mAbs) can directly affect their stability, bioactivity, and immunogenicity. Glycan analysis can profile the glycan structure and monitor the relative quantities of a particular set of glycans to ensure product quality. As a forward-looking company as well as a market leader in the field of antibody glycan analysis, Creative Biolabs has successfully developed a versatile technology platform to illustrate the structure and relative quantities of glycan in mAbs and screen the changes in glycosylation. Intact glycoprotein profiling is a good way to ascertain general glycan patterns and glycan heterogeneity.

Background of Intact Glycoprotein Profiling

Glycosylation is considered a critical quality attribute of therapeutic mAbs, which is vital in the determination of physicochemical properties (solubility, viscosity, protein folding, antigen binding, secretion, antigenicity, etc.) and biological functions [antibody-dependent-cell mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)]. Analysis of released glycans and glycopeptides provides information about the composition and/or position of the glycan, while intact glycoprotein analysis offers complementary structural information and allows assignment of individual proteoforms. The analysis of intact glycoproteins is the most straightforward approach to providing a general overview on the glycome, the total glycosylation pattern of glycoproteins, glycolipids, or other types of biomolecules, and then to revealing the complexity of a glycoprotein sample.

With the development in mass spectrometry technology, intact glycoprotein analysis is more advanced, which is fast, robust, does not require enzymatic digestion (preventing undesired modifications induced by enzymatic treatments), and enables high-throughput analysis.

Fig.1 MS-based glycoproteomic analyses. (Tabang, Megan & Lingjun, 2021)Fig.1 A representative workflow for mass spectrometry-based glycoproteomic analyses.1

Technologies for Intact Glycoprotein Profiling

Intact glycoproteins can be directly analyzed for general glycan patterns and glycan heterogeneity, which is widely used in the analysis of the entire repertoire of glycans attached to proteins. Moreover, it has the diagnostic potential for different stages of cancers and various other disease states, helping to discover new biomarkers. At Creative Biolabs, we can provide diverse technologies to perform intact glycoprotein analysis, including but not limited to:

  • ESI-MS
    Electrospray ionization mass spectrometry (ESI-MS) is the most common method for intact glycoprotein analysis, which enables the precise mass determination and characterization of intact glycoproteins. The ability of ESI-MS to place multiple charges on particular species enables large intact glycoprotein analysis via conventional mass ranges. Particularly, it has been commonly used coupled with reversed-phase (RP) HPLC or size-exclusion chromatography (SEC). RPLC-ESI-MS provides better chromatographic separation of protein variants and has shown ability in the detection of intact mAb with 10 ppm accuracy, while SEC-MS using non-denaturing or denaturing mobile phase is operated at room temperature and produces good quality of mass spectra.
  • MALDI-TOF MS
    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a high-throughput method to analyze the structural characterization of glycosylated compounds with high sensitivity and robustness. It can identify the entire set of glycans in small intact proteins.

Glycomics workflow by MALDI-TOF MS. Fig.2 Glycomics workflow by MALDI-TOF MS. (Jansen, 2016)

  • HILIC-MS
    Hydrophilic interaction chromatography-mass spectrometry (HILIC-MS) provides detailed glycoform profiles of intact pharmaceutical proteins, including metabolites and peptides. In the context of glycoprotein characterization, HILIC-MS has been widely used for the determination of released glycans and glycopeptides.

Why Choose Us?

  • High throughput with sensitivity and specificity
  • Stability and consistency; simple, fast, accurate
  • One-stop service with fully customized design
  • Competitive prices and best after-sale service

If you are interested in our services, please contact us or directly sent us an inquiry.

References

  1. Tabang, Dylan Nicholas, Megan Ford, and Lingjun Li. "Recent advances in mass spectrometry-based glycomic and glycoproteomic studies of pancreatic diseases." Frontiers in chemistry 9 (2021): 707387.
    Distributed under Open Access License CC BY 4.0, without modification.
  2. Jansen, Bas C., et al. "Pregnancy-associated serum N-glycome changes studied by high-throughput MALDI-TOF-MS." Scientific Reports 6.1 (2016): 23296.
    Distributed under Open Access License CC BY 4.0, without modification.

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