title: Analysis of monoclonal antibody by a novel CE-UV/MALDI-MS interface.
abstract: mAbs are highly complex proteins that present a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product- and time-consuming. CE-MS couplings, especially to MALDI, appear really attractive methods for the characterization of biological samples. In this work, we report the last instrumental development and performance of the first totally automated off-line CE-UV/MALDI-MS/MS. This interface is based on the removal of the original UV cell of the CE apparatus, modification of the spotting device geometry, and creation of an integrated delivery matrix system. The performance of the method was evaluated with separation of five intact proteins and a tryptic digest mixture of nine proteins. Intact protein application shows the acquisition of electropherograms with high resolution and high repeatability. In the peptide mapping approach, a total number of 154 unique identified peptides were characterized using MS/MS spectra corresponding to average sequence coverage of 64.1%. Comparison with NanoLC/MALDI-MS/MS showed complementarity at the peptide level with an increase of 42% when using CE/MALDI-MS coupling. Finally, this work represents the first analysis of intact mAb charge variants by CZE using an MS detection. Moreover, using a peptide mapping approach CE-UV/MALDI-MS/MS fragmentation allowed 100% sequence coverage of the light chain and 92% of the heavy chain, and the separation of four major glycosylated peptides and their structural characterization.
authors: Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), CNRS-UMR 7140, Université de Strasbourg, Strasbourg, France.
authors: Michael
authors: M
authors: Biacchi
authors: Ricky
authors: R
authors: Bhajun
authors: Nassur
authors: N
authors: Saïd
authors: Alain
authors: A
authors: Beck
authors: Yannis Nicolas
authors: YN
authors: François
authors: Emmanuelle
authors: E
authors: Leize-Wagner
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000032:analysis* / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D010449:Peptide Mapping / Q000295:instrumentation* / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation* / Q000379:methods; D014357:Trypsin
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D010446:Peptide Fragments; D014357:Trypsin
keywords: CE/MALDI-MS; Capillary electrophoresis; Intact protein; Monoclonal antibody; Peptide mapping