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Rapid Method Development With the BioLogic DuoFlow Chromatography System for the Purification of His-Tagged Proteins

from crude E. coli lysate on a Profinity IMAC Ni2+-charged resin. In the present study, the concentration of a competitive eluting agent, imidazole, was optimized using the BioLogic DuoFlow system by executing multiple repetitive chromatography runs with a nine-buffer system. The plumbing setup of the system included an SV5-4 select valve (5-port, 4-position) and an AVR9-8 select valve (9-port, 8-position), which were both designed for flexible automated operation. This multiple inlet port configuration, coupled with the buffer scouting feature of the BioLogic DuoFlow software, allowed us to complete this experiment with a single linked scouting run and virtually no user intervention.


Methods
Setup
The column, UV detector, conductivity monitor, BioFrac fraction collector, SV5-4 select valve, AVR7-3 automated sample injection valve, and AVR9-8 select valve were connected to the workstation and controller as illustrated in Figure 1. All nine buffers, with the following compositions, were filtered and connected to the indicated SV5-4 and AVR9-8 valve ports:

A1: loading or equilibration buffer (50 mM potassium phosphate, 50 mM EDTA, 300 mM NaCl, pH 7.5)
A2: cleaning buffer (50 mM potassium acetate, 300 mM NaCl, pH 4.0)
A3: charging buffer (100 mM NiSO4, 300 mM NaCl, pH 4.3)
A4: loading buffer (50 mM potassium phosphate, 300 mM NaCl, pH 8.0)
B1: elution buffer 1 (loading buffer + 75 mM imidazole)
B2: elution buffer 2 (loading buffer + 100 mM imidazole)
B3: elution buffer 3 (loading buffer + 250 mM imidazole)
B6: elution buffer 4 (loading buffer + 50
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