Articles tagged ”HILIC”

FabRICATOR, SialEXO and OglyZOR in Middle-up HILIC/HRMS Approach

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In an article by Valentina D’Atri et al. recently published in Analytical Chemistry (2019), the scientists developed a middle-up HILIC/HRMS workflow for detailed characterisation of the Fc fusion protein etanercept.  The etanercept molecule consists of an IgG1 Fc domain fused to a tumour necrosis factor receptor (TNFR) and is used in the treatment of autoimmune diseases such as rheumatoid arthritis. The protein is highly glycosylated and contains numerous O- and N-glycosylation sites that require extensive characterization.

 

To develop a strategy that would work with a mass spec instrument of limited resolution, the authors used FabRICATOR enzyme to specifically digest the etanercept molecule and generate TNFR and Fc/2 subunits. Combinations of the O- and N- glycosidases SialEXO, OglyZOR and PNGaseF were applied to allow evaluation of the O- and N-glycosylation patterns of TNFR and Fc/2 respectively. In addition, complete deglycosylation allowed for primary structure analysis. By using a wide-pore HILIC stationary phase, appropriate separation of the subunits with different degrees of remaining glycans was achieved, and this significantly facilitated spectra deconvolution.

 

Applying this workflow, D’Atri and colleagues were able to assess the main PTMs, the subunit distribution of glycans, the overall N/O glycan composition and the sialylation profiles of each subunit.

 

Read more about the SmartEnzymes in this publication

 

Reference:

D’Atri, V. et al., 2018. Orthogonal Middle-up Approaches for Characterization of the Glycan Heterogeneity of Etanercept by Hydrophilic Interaction Chromatography Coupled to High-Resolution Mass Spectrometry. Analytical Chemistry, 91(1), pp.873–880.

ADC Subunit Characterization of Drug Load and Glycosylation using HILIC-MS

FabRICATOR-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

D’Atri, V. et al., 2018. Characterization of an antibody-drug conjugate by hydrophilic interaction chromatography coupled to mass spectrometry. Journal of Chromatography B, 1080, pp.37–41.

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.