Antibody Drug Conjugate Characterization
FabRICATOR digestion of antibody drug conjugates (ADCs) into subunits allows detailed characterization of this complex class of drugs.
Form an analytical perspective; a 150 kDa antibody is a challenging molecule to characterize and coupling drugs to the antibody further increases the complexity and thus requires careful and detailed characterization. Nevertheless, antibody drug conjugates is a rapidly growing field and appropriate analytical workflows are required.
For increased resolution and reduced complexity of ADCs, the FabRICATOR protease has been applied to specifically digest the lower hinge region. After digestion and reduction the ADC appears in three 25 kDa fragments, the Fc/2, Fd and LC. FabRICATOR digestion and reduction coupled to liquid chromatography and mass spectrometry allows high-resolution analysis and has been applied to study a range of ADC quality attributes.
Wagner-Rousset et al. studied ADCs using FabRICATOR digestion to generate antibody subunits and separated these on liquid chromatography prior to mass spectrometry analysis (LC/MS). The scientists analyzed a model of an ADC using a reporter dye, and successfully characterized dye loading and distribution, as well as dye to antibody ratio. In the same run, a full glycoprofile of the Fc/2 fragment was obtained (Wagner-Rousset et al. 2014).
Janin-Bussat et al. used FabRICATOR digestion followed by reduction and LC/MS analysis on an approved ADC, brentuximab vedotin (Adcetris). The analytical workflow allowed characterization of subunit drug load and could for the first time determine the payloads positional isomers (Janin-Bussat et al. 2015). For thiol conjugated ADCs, Firth et al. developed a workflow based on deglycosylation with IgGZERO followed by FabRICATOR digestion, reduction, and LC/MS analysis. The researchers suggest this method as a rapid tool for lot-to-lot comparison (Firth et al. 2015).
By applying endoglycosidases such as IgGZERO or GlycINATOR the ADC can rapidly be stripped from the major glycan complexity and the intact ADC can be studied using native-MS, IM-MS or top-down MS to determine the ratio of drugs per antibody (Marcoux et al. 2015; Beck et al. 2016). Several research groups have applied this approach and the reduced complexity leads to increased resolution when studying the intact antibody drug conjugate.
FabRICATOR is available as a lyophilized enzyme, immobilized on spin columns or in 96-well plate format for convenient sample preparation.
IgGZERO is available as lyophilized enzyme and in spin column formats, called deGlycIT, for easy removal of Fc glycans from IgG.
GlycINATOR is available as a lyophilized enzyme and cleaves all Fc glycans from IgG, including high-mannose and hybrid type glycans.
- Beck, A. et al., 2016. Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates. Expert review of proteomics, 13(2), pp.157–183.
- Firth, D. et al., 2015. A rapid approach for characterization of thiol-conjugated antibody–drug conjugates and calculation of drug–antibody ratio by liquid chromatography mass spectrometry. Analytical Biochemistry, 485(C), pp.34–42.
- Janin-Bussat, M.-C. et al., 2015. Characterization of antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC-MS analysis. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 981-982, pp.9–13.
- Marcoux, J. et al., 2015. Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine-linked antibody drug conjugate. Protein science : a publication of the Protein Society.
- Wagner-Rousset, E. et al., 2014. Antibody-drug conjugate model fast characterization by LC-MS following IdeS proteolytic digestion. mAbs, 6(1), pp.173–184.