Automated two-step chromatography using an ÄKTA equipped with in-line dilution capability

There has been a great emphasis on developing higher-throughput protein purification techniques to screen potential human therapeutics faster and more efficiently. Not only is it desirable to have high-throughput purification for initial screens but it is also desirable to efficiently purify selected protein therapeutics in the amounts and purity required for definitive assays. Current automated tandem technologies involve size exclusion as a second step that often fails to generate the required purity, is not robust and can only be operated at a limited scale. We have modified an ÄKTA to enable in-line dilution, assuring that the automated loading of a second column from a first column elution can be modified to a pH and ionic strength which is suitable for binding to the second column. For example, Protein A can be employed as a first step followed by direct loading on to a cation exchange column by conditioning the Protein A elution using the in-line diluter. Using this method as described, up to six samples of 1 L each can be purified through two columns without human intervention per day per machine, and the system produces good yields of purified protein over a wide range of loading levels (12-300 mg). In addition, the system employs guanidine HCl regeneration, followed by a sodium hydroxide wash between purification runs, minimizing the possibility of carryover contamination. The system is described at the 5 mL and the 10 mL column sizes; however, it could readily be programed for 100 mL columns to enable larger-scale purifications. Using this system to automate two-column purifications minimizes human intervention, increases efficiency and minimizes the risk of human error.