Efficient recovery and purification of recombinant proteins from cell culture fluid is the key to downstream process. Capture step for mAb and Fc-fusion protein is well-developed due to the highly selective Protein A chromatography. However, capture step development for other recombinant proteins is still challenging due to the wide varieties of the protein physicochemical properties. Multimodal chromatography provides “pseudo-affinity” selectivity by utilizing multiple interactions for protein separation, and is a good candidate for capture step. However, application of multimodal chromatography is hurdled by the complexity of the binding behavior and the extensive experiment work required to identify the operation conditions. An automated high-throughput system overcomes the obstacle by testing in parallel up to 96 conditions in one experiment with very small amount of material and manual labor work. In this work, a methodology of developing capture steps for non-mAb recombinant proteins was investigated with several multimodal resins and model proteins. An initial well-plate based binding experiment was conducted for all resins with pure protein samples. Then, the resins with high bind capacity and well-defined binding condition ranges were chosen for selectivity mapping by loading clarified cell culture fluid on a well-plate resin plate. The identified loading and elution conditions were then verified by Robo-Column and further optimized in terms of operation parameters, including loading capacity, flowrate and elution. Final confirmation was performed on bench scale columns. A workflow of automated high throughput development was established through this work, which could greatly reduce the time and work load of chromatography process development. The application of multimodal chromatography potentially can be a platform approach for non-mAb recombinant protein capture.

Automated high throughput development of multimodal chromatography for capture step in non-mAb recombinant protein purification

2019 Spring National Meeting

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