Capillary electrophoresis/Mass spectrometry: proteomic, intact proteins and monoclonal antibodies applications

Development of CE/MALDI-MS coupling: application to characterization of intact protein

Protein identification and characterization seem to be one of the most important challenges posed to analytical sciences these last years. Capillary electrophoresis (CE) coupled with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is a technique highly suitable for the separation and detection of intact proteins. Team LSMIS has developed a new automated CE/MALDI-MS interface, equipped with a deported UV/visible cell, and an integrated automatic distribution of matrix.

This new system has been evaluated on different mixtures of intact protein, digest protein and monoclonal antibodies (mAbs). The results obtained during this evaluation showed the complementarity of the new interface with conventional analytical systems. Furthermore, we have shown the first separation and analysis of mAbs by CE/MALDI-MS. This new interface appears to be very promising for the characterization by Top-Down approach of intact protein and mAbs. Finally the combination of the new CE/MALDI-MS interface with CESI-MS system has enabled the development in the laboratory of an original 2D strategy with separation followed by infusions using nanoelectrospray. This strategy opens the way for precise characterization of intact protein maintaining high resolution properties of electrospray

Development of high sensitivity CE/ESI-MS coupling: application to characterization of intact protein

Full protein characterization, as now requested by regulation agencies, is still an actual challenge for analytical sciences. Capillary Electrophoresis (CE) coupled with Electrospray Mass Spectrometry (ESI-MS) is a technique highly suitable for the separation and detection of intact proteins and peptide mixtures. Since 1988, several approaches for coupling CE to ESI-MS have been described. However, due to its miniaturized format; CE suffers from a lower loading capacity and a difficulty to maintain the electrical field.

A novel sheathless CE-ESI-MS platform, referred to as CESI-MS, allowing hyphenation of CE to ESI-MS, has been developed by Sciex Separation. In a first time, LSMIS Team have evaluated the suitability of CESI-MS platform as the nanoelectrospray emitter to analyze with high sensitivity intact proteins and non-covalent complexes. This opens new pathways in the study of biological mega-structure by nanoESI detection.In a second time, we have demonstrated the suitability of CESI-MS to analyse a complex mixture of digested proteins (Yeast Mitochondrial proteome) using a bottom-up proteomic approach. the CESI-MS/MS method performances were systematically compared to a conventionally used nanoLC-MS/MS using the same MS. The data collected by CESI-MS/MS were complementary to those obtained in nanoLC-MS/MS. Also the use of CE separation mechanism allowed to increase the number of identified peptides. This result has enlighten the capacity of the CESI-MS platform to increase the identification confidence and enlarge the amount of data attainable during the analysis of such complex samples.

Since October 2011, the LSMIS group was selected by International Beckman Coulter Inc. (Now Sciex Separation) to test the new CESI-MS interface. The expertise of Dr. Yannis François in CE and laboratory members in MS allowed the LSMIS group to become one of their reference center in Europe in the field of intact proteins (therapeutic proteins), and noncovalent complexes.

Characterization of monoclonal antibodies and related product using CE-MS

Monoclonal antibodies (mAbs) have become one of the most rapidly growing classes of biotherapeutics in the treatment of human disease. MAbs are highly heterogeneous proteins, thereby requiring a battery of sophisticated analytical technologies for their complete characterization.  Mass spectrometry (MS) has become an essential analytical tool for the structural characterization of mAbs. As regards capillary electrophoresis (CE), it has become a routine tool for the analysis of recombinant protein therapeutics in the biotechnology industry. 

LSMIS Team develops complementary and well-adapted CE-MS coupling-based approaches for the high resolution characterization of mAbs and related product (biosimilars, Antibody-Drug Conjugates (ADCs)). CE-MS approaches could appear as a viable and complementary alternative to investigate the characterization of these large intact proteins, and to ensure product quality with Top-down and bottom-up approaches and the research of heterogeneities in mAb products.

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