Cation exchange chromatography (CEX) is a non-denaturing technique used for separation and isolation of protein charge variants before characterisation via MS. This is usually a two step process, where the specific charge variant peak of interest has to be isolated prior to characterisation via mass spectrometry because in CEX mostly non-volatile buffers are used, which are not compatible with MS. The coupling of MS to CEX would save time and exclude possible artefacts of the long isolation process. In addition, it is possible to overlook minor species of e.g. monoclonal antibodies (MAb) that do not exhibit distinctive UV peaks in this two-step approach.
In this application note based on a study by Yan et al., high resolution SCX (strong cation exchange chromatography) was coupled directly to an ultrasensitive online native MS [1].
A combined pH and salt gradient based on a MS compatible buffer system was utilised. YMC’s BioPro IEX SF, a non-porous strong cation exchange column, was used for the characterisation of the NIST mAb.
The mobile phase composition and gradient were optimised to achieve an efficient separation of charge variants by changing the pH and the salt concentrations in a range where MAbs are maintained in their native states.
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