Middle level analysis of site-specific ADC

May 29, 2017 | References |

In a collaboration between Universite de Strasbourg, Pierre-Fabre, and Catalent Biologics a multilevel mass spectrometry analysis of a site-specific conjugated ADC was recently published in the journal mAbs. As a first step in the analysis, FabRICATOR (IdeS) was used to perform middle-up analysis and confirm the drug to antibody ratio, DAR. The scientists write on p.7: “Middle level analysis, which is now the first line strategy used in most laboratories for mAb and ADC analytical characterization, revealed a highly homogeneous sample in terms of drug load, with mainly DAR4 detected and low amounts of DAR3” (Botzanowski et al. 2017).

For intact mass analysis using top-down mass spectrometry, the ADC was deglycosylated using IgGZERO for rapid removal of the glycan heterogeniety on the intact ADC. For this approach the newer and improved GlycINATOR enzyme could have been used. Studies have shown that GlycINATOR (EndoS2) removes all glycans from IgG whereas IgGZERO (EndoS) leaves the high-mannose glycans (Sjögren et al. 2015).



Botzanowski, T. et al., 2017. Insights from native mass spectrometry approaches for top- and middle- level characterization of site-specific antibody-drug conjugates. mAbs, ePublished ahead of print.

Sjögren, J. et al., 2015. EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans. Glycobiology, 25(10), pp.1053–1063.