Systematic generation of buffer systems for pH gradient ion exchange chromatography and their application

pH gradient protein separations are widely used techniques in the field of protein analytics, of which isoelectric focusing is the most well known application. The chromatographic variant, based on the formation of pH gradients in ion exchange columns is only rarely applied due to the difficulties to form controllable, linear pH gradients over a broad pH range. This work describes a method for the systematic generation of buffer compositions with linear titration curves, resulting in well controllable pH gradients. To generate buffer compositions with linear titration curves an in silico method was successfully developed. With this tool, buffer compositions for pH gradient ion exchange chromatography with pH ranges spanning up to 7.5 pH units were established and successfully validated. Subsequently, the buffer systems were used to characterize the elution behavior of 22 different model proteins in cation and anion exchange pH gradient chromatography. The results of both chromatographic modes as well as isoelectric focusing were compared to describe differences in between the methods.

► An in silico method for the development of pH gradient buffer systems is shown.
► Buffer systems for pH gradient IEC spanning 7.5 pH units are successfully developed.
► The applicability of the buffer systems for pH gradient IEC is validated.
► The buffer systems are used for characterization of proteins via pH gradient IEC.
► Comparison of pH gradient AEC, CEC and IEF clarifies mechanistic differences.