The characterization and monitoring of charge variants of mAb biosimilars are critical quality requirements to ensure the stability and process consistency in drug development. Conventionally, ion exchange chromatography (IEX) with salt-gradient is the gold standard method for charge-sensitive antibody analysis. A key challenge of salt-gradient is that the method parameters often need to be optimized for each individual protein. In 2009, Genentech first reported the use of IEX with pH-gradient for mAb charge variant separations. It was found that mAbs with isoelectric points in the range of pH 7-9 could be well separated. Moreover, the approach was more generic and could easily be used to separate different charge variants in a wide range of antibodies.
In this study, we describe both salt-gradient and pHgradiet methods for separating the charge variants of bevacizumab biosimilar using Shimadzu Nexera UHPLC and a Shim-pack Bio IEX column (4.6 mm x100 mm, 3 μm).