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Oct 15, 2009 (Vol. 29, No. 18)

Simulated Moving Bed Chromatography

Semba's SMBC System Attacks the Bottleneck Created by Upstream Protein Increases

  • Octave Chromatography System

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    Figure 1. Semba Octave Chromatography System

    The perceived complexity, cost, and scale of SMBC equipment and unpredictable behavior of proteins in the separations has reduced the number of scientists willing to adopt this technology for protein purification. Now the same benefits SMBC delivered to large-scale chromatography can be realized on a smaller scale with Semba Biosciences’ Octave™ System.

    The Octave Chromatography System (Figure 1) is a fully automated, benchtop-deployable, continuous-flow chromatography system for research and semipreparative scale purification of high-value chemical and biological compounds. This system is capable of a variety of SMBC protocols. Fluid flow is controlled by four independent piston pumps, each capable of flow rates up to 10 mL/min in the standard configuration or up to 100 mL/min in the high-flow version. 

    The Octave carries eight column positions arranged in series and connected through a pneumatic valve system. Each column is accessed by five inlet streams:  four arising from the external inlets and one from the upstream column. Outlet flow from each column can be directed to any of four outlet ports as well as to the next column in the series.

    The valve configuration provides ultimate flexibility in programming chromatographic protocols via the SembaPro™ software application and preprogrammed scripts to simplify purification under a wide variety of conditions. The pneumatic valve design contains no moving parts, occupies only 3 µL, and responds within 100 ms. All flow paths are made of metal-free biocompatible materials that are also compatible with most organic solvents and clean-in-place solutions.

    The Octave System is an open platform compatible with many commonly used separation chemistries and media; it is ideal for use with commercially available disposable 1- and 5-mL cartridges pre-packed with Protein A/G or other affinity resins.

  • Case Studies

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    Figure 2. Step-SMBC purification of monoclonal antibody with POROS® MabCapture™ A resin: Monoclonal antibody was produced in mammalian cell culture.

    Two continuous modes of SMBC useful for affinity purification are step and isocratic mode. The Step-SMBC mode establishes independent zones, each having a unique buffer condition for target capture, sorbent washing/contaminant removal, target elution, and column regeneration. The number of columns residing within a zone can be adjusted depending on the capacity and volumes required to achieve the desired separation.

    The zones accomplish “steps” analogous to those in SC affinity protocols, but operated in a continuous cycle. The Step-SMBC mode is ideal for protein A or protein G purification of monoclonal antibodies. Figure 2 shows protein A purification of a monoclonal antibody from concentrated tissue culture fluid using POROS® MabCapture™ A resin.

    Antibody recovery was 95%, purity was greater than 99%, and productivity of 200 mg/hr was achieved with 8 x 1 mL columns at 900 cm/h flow rate. With 8 x 5 mL columns and 2,000 cm/h flow rate, the productivity from a 1 g/L titer of monoclonal antibody culture fluid could exceed 2.8 g/h. These results represent significant reductions in column size, process time, and buffer consumption versus a single column protocol.  

    The Isocratic-SMBC mode utilizes a single eluent, and operating parameters (flow rates, switch times) are adjusted to preferentially slow the target protein migration through the matrix relative to nontarget proteins (raffinate). The countercurrent separation with the programmed advance of the product collection stream enables continuous peak shaving of the target molecule for optimum purity. Figure 3 shows IMAC purification of His-tagged proteins from bacterial cell lysates using Isocratic-SMBC. For three human kinase substrates, purity averaged 16% greater than that obtained by SC purification, and productivity averaged >60 mg/hr using 8 x 1.0 mL IMAC columns.

  • Conclusions

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    Figure 3. Isocratic-SMBC purification of histidine-tagged proteins by IMAC: The indicated human histidine-tagged fusion proteins were expressed in E. coli.

    With higher protein titers placing increasing demands on chromatography and process development scientists, SMBC has become an increasingly attractive alternative to single column methods. With the Octave System, researchers can now take advantage of SMBC to increase the efficiency and productivity of many protein affinity purification applications.



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