Collision Induced Unfolding Enables the Quantitation of Isomass Biotherapeutics in Complex Biological Matrices

Quantitative mass spectrometry has been widely used to evaluate the concentrations of molecules within a variety of biological matrices. Typically, such quantitative mass spectrometry analyses are predicated upon the production of mass-resolved precursor or fragment ions, leading to challenges surrounding the quantification of isomeric or conformationally distinct analytes. As such, new approaches are required for the label-free quantification of isomass proteins. Native ion-mobility MS (nIM-MS) in combination with collision induced unfolding (CIU) is a potentially enabling approach for such quantitative mass spectrometry methods as the technique can rapidly separate and detect many biomacromolecule isoforms. CIU uses collisional activation to capture the unfolding trajectory of ions in the gas phase, producing different intermediate structures that can be leveraged to distinguish protein structures that exhibit identical sizes at lower energies. Here we describe the deployment of quantitative CIU methodology to measure the concentrations of isomass pairs of biotherapeutics and sequence homologues in both standard and biological matrices. Our results cover three antibody pairs and include examples of mixed therapies where multiple biologics are commonly provided to patients. In all cases, CIU enables the production of resolved features for each antibody mixture probed, producing calibration curves with correlation coefficients ranging from 0.92 to 0.99, limits of detection ranging from 300 to 5000 nM and sensitivities ranging from 8.7 × 10^-5 nM^-1 to 6 × 10^-3 μM^-1. We conclude our report by projecting the future utility of CIU-enabled quantitative MS methods.