Tuning selectivity in cation-exchange chromatography applied for monoclonal antibody separations, part 2: Evaluation of recent stationary phases

Revealing charge heterogeneity of stressed trastuzumab at the subunit level

“…Other chromatographic approaches may also be employed for this purpose, such as hydrophobic interaction chromatography [11][12][13][14] and displacement chromatography [15,16]. Cation-exchange chromatography (CEX) is the method of choice due to the basic pI of most mAbs [8,[17][18][19][20][21][22][23][24][25][26][27][28][29]. Charge variants of mAbs can be eluted from the cation-exchange column either by salt [17,19,27] or pH gradients [18,[20][21][22][23][24][25][26][27].…”

Characterization of Complex Proteoform Mixtures by Online Nanoflow Ion-Exchange Chromatography-Native Mass Spectrometry

“…Hyphenating native separations to native MS allows the measurement of complex samples, resolving proteoforms according to specific mechanisms (therefore aiding identification) and increasing the dynamic range of the measurement. Cation-exchange chromatography (CEX), in particular, is a nondenaturing separation mode that is considered the benchmark method for separating charge variants of proteins. , Minor modifications of proteins often result in changes of iso-electric points (pI), resulting in alterations of their surface-charge distributions and, therefore, influencing the retention in CEX. , To obtain a detailed characterization of native proteins, CEX can be coupled to high-resolution MS. , The use of volatile salts facilitates direct coupling of CEX to MS. , This approach has been successfully applied to the characterization of biotechnological protein products available in relatively large amounts (e.g., several tens of μg of purified biopharmaceuticals) but is not sensitive enough for broad application to biological systems. , …”

“…21,22 Minor modifications of proteins often result in changes of iso-electric points (pI), resulting in alterations of their surface-charge distributions and, therefore, influencing the retention in CEX. 23,24 To obtain a detailed characterization of native proteins, CEX can be coupled to high-resolution MS. 25,26 The use of volatile salts facilitates direct coupling of CEX to MS. 27,28 This approach has been successfully applied to the characterization of biotechnological protein products available in relatively large amounts (e.g., several tens of μg of purified biopharmaceuticals) but is not sensitive enough for broad application to biological systems. 29,30 In this work, we have developed a method to characterize intact proteins and complexes in their native states, covering a broad MW range with a small sample consumption (down to 33 ng injection for reference monoclonal antibodies).…”