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