Step SMBC

In contrast to Isocratic Mode, the Step Mode uses multiple solvents and establishes four independent zones for binding, washing, elution, and regeneration steps analogous to conventional batch chromatography protocols, but operated in a continuous cycle. Figure 1 shows a schematic animation of an 8-column Step SMBC configuration during initial loading and running at equilibrium. The main advantage of this method is the ability to achieve high recovery and concentration of target proteins, and it is especially useful when working with dilute samples. As in Isocratic Mode, “column switching” is actually performed by simultaneously switching all fluid streams one column forward at defined intervals, which has the effect of “moving” the solid phase in the opposite direction of the fluid flow.

Figure 1. Animation of Step SMBC

Fluid streams, represented by coloured lines and arrows, indicate the direction of fluid flow. The columns are fixed in place and connected in series to form a continuous loop. In Step mode, four independent zones having different buffer conditions are established by closing connections between them.

For IMAC protein purification each of the four zones has a different inlet stream composition. The Feed (purple stream) enters the system at a low imidazole concentration, which allows adsorption of the target protein to the resin, while untagged proteins flow through and exit the system as Raffinate (red stream). In the Wash zone, the Aux 1 stream provides a slightly higher imidazole concentration to remove untagged proteins and low affinity contaminants, which exit the system at Aux 1 Out. In the Elute zone, the Desorbent stream provides a high imidazole concentration to elute the target protein as Extract (blue stream). In the Regeneration zone, a low imidazole concentration provided by the Aux 2 stream re-equilibrates the columns to prepare them for the next binding cycle. The animation shows initial loading of the system and then operation at equilibrium.