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SEC-MALS of Antibody Therapeutics – A Robust Method for In-Depth Sample Characterization


Monoclonal antibodies (mAbs) are effective therapeutics for cancers, auto-immune diseases, viral infections and other diseases. Recent developments in antibody therapeutics aim to add more specific binding regions (biand multispecificity) to increase their effectiveness and/or the molecule is downsized to the specific binding regions (e.g. scFv or Fab fragment) to achieve better penetration of the tissue. As the molecule gets more complex, the possible high and low molecular weight (H/LMW) impurities become more complex, too. In order to accurately analyze the various species, more advanced detection than UV is required to characterize a mAb sample.

Multi-angle light scattering (MALS) is suited to determine the molecular weight of sample components to better understand molecule aggregation as well as fragmentation. MALS detects the light a molecule scatters if hit by a laser beam. The intensity of scattered light depends on its concentration and on its molecular weight. In this application note, we coupled the TSKgel® UP-SW3000-LS column and the LenS3 MALS detector for the analysis of a mAb standard. Thereby we demonstrate which data can be derived from adding MALS detection, how robust the method is and which equipment is suited to easily integrate MALS detection into the mAb analysis workflow.

Experimental Conditions

To demonstrate the benefits of MALS detection combined with size exclusion analysis of a monoclonal antibody, we employed a reference mAb and a universal method for separation as described below. Adaptations were required to optimize MALS data quality as light scattering is more sensitive for particles such as salt crystals, microbes or column shedding than UV detection: the mobile phase was filtered twice and a light-scattering dedicated UHPLC column (TSKgel UP-SW3000-LS) was employed. The TSKgel UP-SW3000-LS column had previously proven the lowest noise levels in comparison to two other columns. The LenS3 MALS detector was selected for its high sensitivity to achieve characterization of low abundance impurities.

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SEC-MALS of Antibody Therapeutics – A Robust Method for In-Depth Sample Characterization
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