(421f) Accelerated Physicochemical Characterization of Complex Protein Mixtures for Optimized Chromatographic Separation

Development of chromatographic separation for complex protein mixtures such as crude extract or lysate is typically very labor intensive and time consuming. Chromatography sorbent selection and the buffers to use with them are traditionally made based on knowledge from similar processes and a large number of small-scale chromatography runs. This could require a significant quantity of feed material and extensive analytical support, which at early development stage are likely in limited supply or not available.

This study outlines the use of Surface Enhanced Laser Desorption Ionisation-Time of Flight-Mass Spectrometry (SELDI-TOF-MS) technology to obtain physico-chemical properties of proteins in complex mixtures, which can then be utilized to highlight promising chromatographic conditions for purification of a target protein. The key benefits of this approach over the traditional methods are the speed and ease of ProteinChip preparation and data acquisition to generate a large amount of quality data within a few days using only microliters of the complex protein solution. Data from the SELDI are processed to provide detailed knowledge of the bind/release properties of each protein in the solution under various buffer conditions. This knowledge will eventually enable to the selection of sorbents and buffer conditions that exploit the greatest difference in adsorption/desorption characteristics between the protein of interest and impurities in order to find the most effective chromatographic separation step.