Jean-Luc Maloisel

Improving washing strategies of human mesenchymal stem cells using negative mode expanded bed chromatography

The use of human mesenchymal stem cells (hMSC) in clinical applications has been increasing over the last decade. However, to be applied in a clinical setting hMSC need to comply with specific requirements in terms of identity, potency and purity. This study reports the improvement of established tangential flow filtration (TFF)-based washing strategies, further increasing hMSC purity, using negative mode expanded bed adsorption (EBA) chromatography with a new multimodal prototype matrix based on core-shell bead technology. The matrix was characterized and a stable, expanded bed could be obtained using standard equipment adapted from what is used for conventional packed bed chromatography processes. The effect of different expansion rates on cell recovery yield and protein removal capacity was assessed. The best trade-off between cell recovery (89%) and protein clearance (67%) was achieved using an intermediate expansion bed rate (1.4). Furthermore, we also showed that EBA chromatography can be efficiently integrated on the already established process for the downstream processing (DSP) of hMSC, where it improved the washing efficiency more than 10-fold, recovering approximately 70% of cells after global processing. This strategy showed not to impact cell viability (>95%), neither hMSCs characteristics in terms of morphology, immunophenotype, proliferation, adhesion capacity and multipotent differentiation potential.

Amino Acid Sequencing of Sulfonic Acid-Labeled Tryptic Peptides Using Post-Source Decay and Quadratic Field MALDI-ToF Mass Spectrometry

Knowledge of protein and peptide sequences is fundamentally important for understanding many physiological and biochemical processes at the molecular level. Chemically assisted fragmentation by MALDI (matrix-assisted laser desorption/ionization mass spectrometry) is a new approach for amino acid sequencing of tryptic peptides. The technology is based on a new class of water stable sulfonation reagents, which strongly improves post-source decay (PSD) analysis and also simplifies the interpretation of the fragmentation spectra of the peptides. Convenient derivatization methods have been developed and optimized on a solid-phase support, enabling fast, simple and robust sample preparation.