FabRICATOR® in Middle-Up Quantitative N-Glycan Profiling of Therapeutic Monoclonal Antibodies

Glycosylation is one of the most important post-translational modifications (PTM) which can occur during the production of therapeutic monoclonal antibodies (mAbs). Attached glycan motifs are highly heterogeneous and can significantly influence effector functions and circulating half-life. In addition, certain cell lines express non-human glycan types, which can lead to severe immunogenic effects in patients. These potential impacts on safety, clinical efficacy and stability, make glycosylation a key critical quality attribute (CQA) that needs to be addressed. Currently, the gold standard method for glycan characterisation and quantification is released glycan analysis, where glycans are enzymatically released from the protein, fluorescently labelled and analysed using HILIC with either fluorescence or mass spectrometric detection.
 
Recently, the use of HILIC-MS at protein subunit level has emerged as a potential technique for qualitative glycan analysis in the characterisation of mAbs, biosimilars, fusion proteins and ADCs. In this paper, the authors propose a middle-level approach to glycan analysis which may serve as a more cost- and time-effective alternative to widely used released glycan methodologies.
 
FabRICATOR is a highly effective tool for middle-level analysis of mAbs and similar types of molecules. Below hinge digestion using FabRICATOR, followed by chemical reduction, generates Fd’, LC and Fc/2 subunits that can be chromatographically separated using HILIC. With a molecular weight of around 25 kDa, the subunits are highly amenable to high resolution mass spectrometry (HRMS). For glycosylation analysis at the subunit level, HILIC is an excellent choice for chromatographic separation as, unlike reversed-phase (RP) chromatography, separation of the Fc-glycoforms is achieved allowing for more accurate glycoform detection and increased sensitivity.
 

 
The scientists in this study set out to confirm the suitability of using the FabRICATOR-generated subunits. On a range of products containing highly varied glycosylation profiles, they evaluated the performance of their middle-level HILIC-MS method for the identification and quantification of N-glycans compared to the released glycan method.
 
The results demonstrated that the middle-level approach showed strong correlation, with respect to both glycan identification and relative abundance, with the released glycan approaches and the data were consistent with previously published data on the tested molecules. Overall, HILIC-MS analysis performed at the subunit level not only provides a method for fast, qualitative evaluation of mAb glycosylation profiles, but also provides the basis for a robust relative quantification technique.
 
A potential further advantage of the middle-level approach, that has yet to be fully explored, is in its ability to be used as a multi-attribute monitoring (MAM) approach. Keeping the glycans attached to the protein allows for simultaneous analysis of other PTMs, particularly when performing mass spectrometry. An example of this was highlighted within the study, where a glycated variant of the LC domain was identified after it presented as an additional chromatographic peak. Both the identification of this glycation and its localisation to the LC would not have been possible at the intact level, and highlights both the advantage of using FabRICATOR for middle-level analysis of mAbs, and the MAM potential of this type of method.

Reference

Duivelshof et al., 2021. Quantitative N-Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis. Pharmaceutics. DOI: 10.3390/pharmaceutics13111744.

FabRICATOR® – Below hinge digestion of IgG