Characterizing ADCs using FabRICATOR and middle-down MS

September 24, 2019 | Applications, Products |

The process of characterizing an antibody drug conjugate (ADC) requires the evaluation of critical quality attributes including primary sequence analysis and drug conjugation assessment. Addressing glycoprofile determination as well as drug load distribution and drug-to-antibody ratio (DAR) is however challenging using peptide mapping. In addition, further challenges arise from the increased hydrophobicity of the ADC and the risk of drug-linker dissociation in an MS/MS experiment.


In an article by Hernandez-Alba et al. (2019) the authors characterized a site-specific ADC using middle-down MS. They combined three different fragmentation strategies for improved sequence coverage and drug conjugation assessment. The site-specific ADC (DAR=4) was digested using FabRICATOR and reduced to generate homogenous Fc/2, Fd’ and LC fragments for analysis by multiple ion activation techniques. By combining MS/MS data obtained with HDC (hydrodynamic chromatography), ETD (electron-transfer dissociation) and UVPD (ultraviolet photodissociation) fragmentation modes, the scientists obtained valuable information with the advantages of minimal sample preparation and analysis time using middle-down MS. 


The UVPD mode showed better performance compared to ETD and HDC. This indicated that the performance of this activation technique was unaffected by the hydrophobicity of the ADC. The complementarity between UVPD and ETD was further highlighted for drug conjugation assessment by allowing primary sequence validation and accurate identification of drug conjugation and glycosylation sites. These results highlight the potential of middle-down MS as a complement in next-generation strategies for the characterization of  mAb-based compounds including ADCs. 


Read more about FabRICATOR and Applications of FabRICATOR.


Hernandez-Alba, O. et al., 2019. A Case Study to Identify the Drug Conjugation Site of a Site-Specific Antibody-Drug-Conjugate using Middle-Down Mass Spectrometry. American Society for Mass Spectrometry, 30(8). pp. 1-11.