Towards a simple on-line coupling of ion exchange chromatography and native mass spectrometry for the detailed characterization of monoclonal antibodies

This work describes the direct hyphenation of cation exchange chromatography (CEX) with a compact, easy-to-use benchtop Time of Flight mass spectrometer (ToF/MS) for the analytical characterization of monoclonal antibodies (mAbs). For this purpose, a wide range of commercial mAb products (including expired samples and mAb biosimilars) were selected to draw reliable conclusions. From a chromatographic point of view, various buffers and column dimensions were tested. When considering pH response, buffer stability over time and MS compatibility, the best compromise is represented by the following recipe: 50 mM ammonium acetate, titrated to pH 5.0 (mobile phase A) and 160 mM ammonium acetate, titrated to pH 8.5 (mobile phase B). Despite the broader peaks observed with the 2.1 mm i.d. CEX column, this was preferentially selected for CEX-MS operation, since the efficiency loss (caused by extra-column dispersion) was still acceptable while MS compatibility was strongly enhanced (thanks to low flow rate). In terms of MS, it was important to avoid the use of glass-bottled mobile phases, laboratory glassware and glass vials to minimize loss of MS resolution, sensitivity, and mass accuracy due to metal contaminants. With this new CEX-MS setup, straightforward and rapid analysis (in less than 10 min) of charge variants was possible, allowing the separation and identification of several charge variants.