Step SMBC Mode

Step Mode is suitable for “bind-elute” or flowthrough protocols associated with affinity chromatography or other high selectivity separations. The Step Mode uses multiple inlet solutions and establishes independent zones in different columns or groups of columns for the various process steps such as binding (loading), washing, elution, cleaning and regeneration analogous to conventional batch chromatography protocols, but operated in a continuous cycle.

With Semba’s unique valve design and ProComposer™ software, the number of columns and process steps can be easily configured and optimized to suit the protocol, target concentration in the Feed and mass transfer, capacity, and flow characteristics of the resin.

Benefits of Semba’s Step SMBC approach:

  • Productivity: up to 8-fold higher vs. batch
  • Flexibility: uses as few as 2 and as many as 8 columns
  • Simplicity: robust, compact valve block and easy-to-use software
  • Economy: saves up to 90% in resin and 40% in buffer volumes vs. batch
  • Scalability: from mg to kg of purified protein

Step SMB 7-column process animation

This animation shows a 7-column process for Protein A capture of monoclonal antibodies having three columns in the Load zone and one column each in the Wash, Elute, Clean, and Equilibration zones. The three columns in the Load zone are connected in series. High productivity is achieved by loading the first column to near capacity at a high flow rate (low residence time). Any breakthrough from the first column is captured by the downstream columns. Following the Load, a brief “Chase” with PBS buffer flushes any unbound antibody remaining in the first column on to the downstream columns and also serves as a first wash. Subsequent Wash, Elute, Clean, and Equilibration steps are performed in the other zones, which move through the columns by periodic switching of the inlet and outlet ports. The zone outlet streams are connected to UV, conductivity, and pH sensors to monitor the process.

More than one operation can be performed sequentially in a zone during a switch time. The animation below shows a 5-column Protein A capture process in which Wash and Elution occur in one column, Clean and Equilibration occur in the adjacent column, and Feed is loaded onto the other three columns during each switch time.

Step SMB 5-column process animation

The video below shows an 8-column Step Mode purification of a histidine-tagged recombinant protein using immobilized metal affinity chromatography (IMAC). The columns are divided into four 2-column zones. The Feed is an equal mixture of two bacterial lysates expressing fluorescent proteins; one contains histidine-tagged GFP fusion protein and the other contains untagged RFP fusion protein. The Feed enters the system at a low imidazole concentration, which allows adsorption of the green fluorescent target protein to the resin, while the bacterial and untagged red fluorescent 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.