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Tutorials : Oct 15, 2009 ( )
Simulated Moving Bed Chromatography
Semba's SMBC System Attacks the Bottleneck Created by Upstream Protein Increases!--h2>
The number of protein-based pharmaceuticals reaching the marketplace has dramatically increased over the past few years. New developments in protein expression and bioprocessing technologies have led to significant advancements in protein manufacturing and production. The development of highly efficient downstream protein purification processes, however, has not kept pace with the significant increases in recombinant protein expression from bacterial and yeast systems and high titers of monoclonal antibody from recombinant mammalian cell culture.
Inducible expression systems enable routine overexpression of fusion-tagged proteins at up to 50% of the total cell protein, and monoclonal antibody titers can exceed 10g/L using specialized cell lines and fed batch cultures. This dramatic rise in protein productivity has been a mixed blessing for biomanufacturers. Although desirable from a cost-of-goods perspective, these huge productivity increases stress the capabilities of all downstream steps originally designed for much lower titers. The biomanufacturing bottleneck has shifted further away from upstream protein expression productivity and toward downstream protein capture and purification.
Protein purification using affinity media in a single-column bind-wash-elute chromatography mode is a well-established technique that has been pushed to its productivity limit by expression increases upstream. Throughput at high target protein concentration requires larger chromatography columns, more process buffer, additional purification steps, or multiple purification campaigns translating into higher cost, longer lead times, and decreased efficiency.
Some of these process problems have been overcome by employing high-capacity, high-flow affinity supports such as monoliths, membranes, and perfusion particles. Ion exchange, mixed-mode, and precipitation methods are less expensive alternatives to protein A capture, but antibodies purified by these methods require additional polishing steps to achieve the same level of purity possible with highly specific protein A resins.
Recombinant proteins purified through histidine (His) or glutathione-S-transferase fusion tags often are insufficiently pure for functional characterization, x-ray crystallography, or drug target validation. Improvements in purity and efficiency at any stage of these purifications would increase throughput and lower costs.
Simulated moving bed chromatography (SMBC) combined with high capacity, high linear velocity chromatography supports can help relieve the bottleneck created by upstream protein increases. The countercurrent flow, central to SMBC, enables the highest yields of purified proteins with the smallest investment in chromatography resins and mobile phases, bringing dramatic increases in operational efficiency and productivity. In SC systems, separation only occurs in a small fraction of the column at any one time, with the rest of the column performing no function other than occupying solvent and broadening bands. Repeated stacked injections necessary when crude product mass exceeds SC capacity amplify process delays.
With SMBC, a series of small columns is used instead of one large column. Typically 50–70% of the affinity media’s capacity is actively engaged in the separation, while the rest of the column media is being prepared for the next cycle of purification. Moreover, SMBC essentially provides an “infinite” column bed length for increased resolution in theoretical plate-dependent separations without the costs associated with obtaining, operating, and maintaining much larger single columns.
Finally, SMBC purification functions continuously until the desired volume of feedstream has been loaded. These advantages of SMBC over SC purification translate into 10- to 20-fold increases in throughput and productivity, respectively, and corresponding reductions in labor and QC costs.
SMBC was developed by D. B. Broughton and C. G. Gerhold almost 50 years ago and commercialized as the Sorbex process by UOP for industrial petrochemical separations. Since then, SMBC has been successfully applied to separations of sugars, chiral compounds, organic molecules, nucleic acids, and proteins. To date, the SMBC process has been successfully utilized in the large-scale production of several single enantiomer active pharmaceutical ingredients (APIs).
Although applications of SMBC for bioseparation have been increasing, it has not been used frequently for protein purifications. SMBC has primarily been used to separate binary component mixtures, and the complex mixture of molecules in biological feed streams is certainly not binary. However, affinity fusion tags and highly selective affinity chromatography media can be used to simplify SMBC purification of proteins. Highly specific affinity interactions simplify the fractionation behavior of complex biochemical feed stocks to binary mixtures for purposes of SMBC protein purification.
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.
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.
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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