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

2015, Analytical Biochemistry

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.

Sign up for access to the world's latest research.

checkGet notified about relevant papers

checkSave papers to use in your research

checkJoin the discussion with peers

checkTrack your impact