Pharmaceutics (Oct 2021)

Quantitative <i>N-</i>Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis

  • Bastiaan L. Duivelshof,
  • Steffy Denorme,
  • Koen Sandra,
  • Xiaoxiao Liu,
  • Alain Beck,
  • Matthew A. Lauber,
  • Davy Guillarme,
  • Valentina D’Atri

DOI
https://doi.org/10.3390/pharmaceutics13111744
Journal volume & issue
Vol. 13, no. 11
p. 1744

Abstract

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The identification and accurate quantitation of the various glycoforms contained in therapeutic monoclonal antibodies (mAbs) is one of the main analytical needs in the biopharmaceutical industry, and glycosylation represents a crucial critical quality attribute (CQA) that needs to be addressed. Currently, the reference method for performing such identification/quantitation consists of the release of the N-glycan moieties from the mAb, their labelling with a specific dye (e.g., 2-AB or RFMS) and their analysis by HILIC-FLD or HILIC-MS. In this contribution, the potential of a new cost- and time-effective analytical approach performed at the protein subunit level (middle-up) was investigated for quantitative purposes and compared with the reference methods. The robustness of the approach was first demonstrated by performing the relative quantification of the glycoforms related to a well characterized mAb, namely adalimumab. Then, the workflow was applied to various glyco-engineered mAb products (i.e., obinutuzumab, benralizumab and atezolizumab). Finally, the glycosylation pattern of infliximab (Remicade®) was assessed and compared to two of its commercially available biosimilars (Remsima® and Inflectra®). The middle-up analysis proved to provide accurate quantitation results and has the added potential to be used as multi-attribute monitoring method.

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