Articles tagged ”HILIC-MS”
FabRICATOR® in Middle-Up Quantitative N-Glycan Profiling of Therapeutic Monoclonal Antibodies
A middle-level approach to glycan characterisation is described in this paper by scientists at the University of Geneva and collaborators. FabRICATOR (IdeS), a critical tool in this study, is used to generate Fd’, LC and the glycan-containing Fc subunits, which can be chromatographically separated using HILIC and characterised using mass spectrometry.
Read more »
FabRICATOR in an Evaluation of Mobile-phase Additives for LC-MS Characterization of mAbs
Biopharmaceuticals, including monoclonal antibodies (mAbs), have become an important class of therapeutics. The manufacturing procedure of mAbs is complex, and many possible variants of a particular mAb can be generated as a result of enzymatical and chemical modifications. Some of these modifications are critical for the efficacy and safety of the therapeutic mAb and are known as critical quality attributes (CQAs). CQAs need to be thoroughly monitored to ensure the quality and safety of the therapeutic agent.
ADC Subunit Characterization of Drug Load and Glycosylation using HILIC-MS
In a collaboration headed by Davy Guillarme at University of Geneva, scientists have explored the characterization of subunits derived from antibody drug conjugates (ADCs) using hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry (D’Atri et al. 2018).
The scientists used brentuximab vedotin (BV, Adcetris®), an approved ADC for treatment of Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (ALCL). The BV consists of an antibody directed towards CD30, coupled to the vedotin toxin using cysteine conjugation chemistry. The random cysteine conjugation method results in a heterogeneous attachment of the drug, with differences in efficacy depending on the drug load. For this reason, the amount of conjugated toxins requires careful characterization. A key quality attribute of both antibodies and ADCs is the glycosylation profile, that may affect the stability, efficacy and safety. In this paper, a method to study ADC drug load and glycan profiling in a single experiment was demonstrated.
The intact ADC is around 150 kDa, which makes it very complicated to study details with high resolution. For this reason, D’Atri and colleagues used FabRICATOR digestion and reduction to generate specific antibody subunits of around 25 kDa, with increased resolution in both separation and mass determination. New wide-pore HILIC phase has enabled separation of larger molecules such as antibody subunits, and the team has already published a glycoprofiling strategy using HILIC on naked antibodies (Periat et al. 2016).
The coupling of HILIC separation to MS of subunits resulted in more detailed characterization of the subunits as compared to reverse phase separation (RP-HPLC). The relative percentage of each subunit aligned well with both methods of separation. However, additional positional isomers of the Fd’ fragment were observed using HILIC separation. Also, the glycoforms of the Fc/2 fragments were chromatographically separated, making mass deconvolution and determination easier. The authors conclude the middle-up HILIC-MS method to be orthogonal to RP-MS with the benefit that the methodology allows simultaneous characterization of drug load and glycosylation of the antibody drug conjugate.
FabRICATOR is a protease with a single digestion site below the hinge of IgG. The enzyme is widely used in middle-level analytical workflows for characterization of antibody based biopharmaceuticals. Learn more about FabRICATOR.
References
Periat, A. et al., 2016. Potential of hydrophilic interaction chromatography for the analytical characterization of protein biopharmaceuticals. Journal of chromatography. A, 1448, pp.81–92.