This study evaluates the applicability of the liquid handling workstation Tecan Evo Freedom 200 and 200 lL Media Scout RoboColumns to dynamic chromatographic operations such as frontal analysis (breakthrough) and elution experiments in a high throughput screening mode. Breakthrough experiments were conducted using BSA as a model protein and the stationary phases Poros 50 D, Q Ceramic HyperD and DEAE Sepharose FF. The obtained dynamic capacities at 10 and 50 % breakthrough matched well with reference data. Elution experiments were performed applying three protein mixtures: a) lipolase and BSA, b) human growth hormone and a process-related impurity, and c) an insulin analogue and a process-related impurity. The resins used resembled a variety of different ion exchange resins. In all cases, the resulting elution curves matched well with reference measurements performed on an ÄKTA explorer system at 1 mL or 2 mL scale.

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.

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