Step SMBC Mode

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.

4-Step SMB chromatography process animation

A 3-2-1-2 configuration is shown having 3 columns in the binding/loading zone, 2 columns in the wash zone, one column in the elute zone, and 2 columns in the equilibration zone. At defined intervals all ports are switched forward one column position to perform the next operation in the process.

The figure shows a schematic animation of an 8-column Step SMBC configuration 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.

Fluid streams, represented by colored 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 example, the video below shows a Step Mode purification of a histidine-tagged recombinant protein using immobilized metal affinity chromatography (IMAC). The eight columns are divided into four zones,  each of which has a different inlet stream composition. The Feed 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.

In the Wash zone, the inlet stream provides a slightly higher imidazole concentration to remove untagged proteins and low affinity contaminants. In the Elute zone, the Desorbent stream provides a high imidazole concentration to elute the target protein as Extract.

In the Regeneration zone, a low imidazole concentration re-equilibrates the columns to prepare them for the next binding cycle.

Continuous multicolumn affinity chromatography of proteins and mAbs with the Octave™ System

This video demonstrates affinity purification of green fluorescent protein (GFP) from a crude bacterial lysate using the Octave System and Ni NTA SF columns. The separation section was filmed under UV light to permit visualization of the purification process.