Characterization of monoclonal antibodies by a fast and easy liquid chromatography–mass spectrometry time-of-flight analysis on culture supernatant (original) (raw)

Rapid and efficient structural analysis is key to the development of new monoclonal antibodies. We have developed a fast and easy process to obtain MS profiles of antibodies from culture supernatant. Treatment of the supernatant with IdeS generates 3 fragments of 25 kDa that can be analyzed by LC-MS TOF in one run (LC, Fd and Fc/2). This process gives rapid access to isoform and glycoform profiles. To specifically measure the fucosylation yield, we have included a one-pot treatment with EndoS that removes the distal glycan heterogeneity. Our process has been successfully compared to HPCE-LIF, currently considered as the gold standard method. Monoclonal antibodies (MAbs) have been developed as therapeutic agents in several disease areas; such as cancer, rheumatoid arthritis, and Alzheimer's disease. Between 2010 and 2014, of the 54 biopharmaceuticals approved in Europe and United States, 17 were MAb-based products [1]. The growing popularity of recombinant proteins has sparked a demand for advanced and cost-effective protein analysis techniques. In particular, the initial stages of MAb development, that include clone selection during the cell-line generation and optimization of production parameters, require a systematic characterization of the produced antibodies in a screening mode. This step consists of primary amino-acid sequence confirmation, identification and quantification of isoforms and lastly post-translational glycosylation profiling.