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Recombinant monoclonal antibodies (MAbs) can be highly heterogeneous due to modifications such as sialylation, deamidation and C-terminal lysine truncation. As biotherapeutic drugs are becoming more popular, chemists in biopharmaceutical laboratories are coming under increasing pressure to adopt a fast, generic, and robust approach to clone screening, method development, and subsequent method transfer to QA/QC.
Traditionally cation-exchange chromatography using salt gradients has been successfully used to characterize MAb charge variants. However, additional effort is often required to tailor the salt gradient method for individual charge variants. Thermo Scientific™ pH buffer solutions and kits can be used to generate highly reproducible, linear pH gradients using cation-exchange chromatography. This generic, LC-based platform approach saves time in method development and facilitates method transfer to QA/QC for a wide range of MAb charge variants. Unlike traditional salt gradients, it is possible to predict the pI and the expected retention of the charge variants and use a narrow pH range to get a higher resolution separation.
The building blocks of the pH gradient platform are two multicomponent zwitterionic buffer concentrates, prepared using a patent-pending formulation. Buffer A is titrated to pH 5.6 and Buffer B is titrated to pH 10.2. In this pH range, each buffer species is either neutral or negatively charged. Therefore, they will not be retained by the cation-exchange column stationary phase and will serve as good buffers for the phase and the stationary phase. All that is required to generate a pH gradient is a 1:10 dilution of the pH buffer concentrates—then you’re ready to go! A linear pH gradient from pH 5.6 to 10.2 can simply be delivered by running a pump gradient from * eluent A to *eluent B.