Mar 12, 2019 – MSDG Meeting

Mass spectrometry metabolomics, the direct measurement of the biochemical content of cells, tissues and biofluids by GC- or LC-MS, is a maturing discipline with the potential to quantitate and identify a wide range of biological small molecules. Metabolomic measurements are orthogonal to genomics and proteomic read-outs, and provide a unique and complementary view of biological state.  In contrast to the static maps of reactions that adorn textbooks and wall-charts, metabolism is a dynamic, regulated process that changes across space, time, and cellular environment.

Although metabolomic analysis can be used to search for new reactions and metabolic intermediates, actionable biological information can be gained from measuring both levels flux of previously observed compounds.  I will describe how we perform “targeted” analysis using a combination of triple-quadrupole and high-resolution accurate-mass platforms to enable assays that are widely deployable in the research lab and beyond.  I will center my discussion around how my laboratory is using a combination or mass spectrometric measurement of small polar metabolites levels and flux to generate predictive metabolic profiles of response to environmental stimuli and to determine mechanism of action of antimicrobials.

Liquid Chromatography (LC) and Mass Spectrometry (MS) play essential role and are the dominant techniques used for characterizing mAb-based bio-therapeutics. Hydrophobic Interaction chromatography (HIC) is a popular LC technique widely used in downstream process purification but recently gained interest in analytical scale analysis of mAbs and ADCs.  HIC is a useful characterization tool because it can often separate mAb variants, for example certain isomers or oxidized species, which are often different to isolate from other orthogonal chromatography methods. It is also a popular tool in determining drug-to-antibody ratio (DAR) value, which is considered a Critical Quality Attribute (CQA) of an ADC product. Such investigations often benefit from peak identification using MS analysis, but high salt buffer conditions required for HIC separations make it incompatible to couple with online MS.  With the Agilent 2D-LC solution, the second dimension enables automated desalting by the addition of reversed-phase (RP) chromatography for a straightforward online LC/MS workflow. In this presentation, we demonstrate the analysis of mAbs and ADCs using multiple heart-cutting 2D-LC approach coupled with online Q-TOF MS system.