High-throughput technologies have become an integral part in the development of chromatographic purification steps. With miniaturized columns, a resin screening tool exists which applies the packed bed principle of conventional chromatography but on a microliter scale. Applications of micro columns mostly refer to sample preparation, dynamic binding capacity determination, screening for cleaning regimens, and optimization of step elution. Academic groups have proven early on that also complex separations are possible with this format employing pseudo-linear gradients. Especially for the wider use of the latter, a practical approach for the successful combination of robot, scale-down, and chromatography is required. This case study describes the development of pseudo-linear gradient elution for resin selectivity screening using RoboColumns®. It gives a guideline for the implementation of this method on a Freedom EVO® robotic platform, addressing fundamental aspects of scale down and pitfalls of liquid handling. The creation of a flexible script for buffer preparation and column operation plus efficient data processing provided the basis for this work. Based on the concept of discretization, linear gradient elution was transformed into multi-step gradients. The impact of column scale, flow rate, multi-step gradient, and fractionation scheme on separation efficiency was investigated, using a ternary model protein mixture. We identified key parameters and defined optimal settings for effective column performance. For proof of concept, we examined the selectivity of several cation exchange resins for varying buffer conditions. The final protocol qualified for a clear differentiation of resin selectivity on micro column scale (see figure) and achieved similar resolution compared to linear gradient elution experiments with lab scale columns.

Method development for resin selectivity screening in RoboColumn® format

251st National Meeting (2016)

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