Protein Purification with SMBC

This versatile bench top multicolumn system is capable of simulated moving bed protocols at fluid pressures up to 300 psi. The system carries eight column positions arranged in series and connected through a proprietary pneumatic valve system. Fluid flow is controlled by four independent pumps, each of which is capable of flow rates from 0.05 to 10.00 ml/min with a precision of 0.5%. 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, from running individual columns to schemes employing multiple columns, including simulated moving bed chromatography (SMBC). The system easily accommodates continuous operation for multiple cycles of purification. All flow paths are made of metal-free biocompatible materials that are also compatible with organic solvents for chemical applications. The instrument brings the productivity of SMBC to the bench top.

SMBC emulates counter-current separation where the mobile phase flows in the opposite direction of the solid phase (Perrin and Nicoud, 2001). The solid phase is represented by individual columns connected in series, and the mobile phase by inlet streams of Feed and Desorbent and outlet streams of Raffinate and Extract. Valves between the columns are systematically switched open or closed at timed intervals (switch time) to introduce the inlet streams and withdraw the outlet streams between the separation zones, simulating counter-current movement of the columns. Separation occurs due to the differential migration of the Feed mixture components through the column material. Components that interact more strongly with the column material are carried into the Extract, whereas weaker-interacting components move into the Raffinate. By adjusting the stream flow rates, the switch time, and the Desorbent composition, a cycle is established in which Feed and Desorbent are continuously added and highly purified products are continuously recovered. Figure 1 shows a schematic diagram of an 8-column SMBC system in a “3-2-3” configuration at equilibrium, at two arbitrary positions 1 and 2. The 3-2-3 designation refers to the number of columns in each SMBC zone.

The zones are defined as follows (Perrin and Nicoud, 2001):

• Zone 1: Between Desorbent inlet and Extract outlet; where the more retained component is desorbed
• Zone 2: Between Extract outlet and Feed inlet; where the less retained component is desorbed and the more retained component is enriched
• Zone 3: Between the Feed inlet and Raffinate outlet; where the more retained component is adsorbed and the less retained component is enriched and desorbed

Figure 1. Two positions of a 3-2-3 SMBC system configuration at equilibrium. 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 Position 1 (Panel A) the Desorbent enters Column 1, the Feed mixture (purple) enters Column 6, and the separated components of the Feed mixture (red and blue) are withdrawn from Columns 3 and 8. After a defined interval (switch time) all streams are switched to the next column in the direction of fluid flow. Panel B shows the positions of the streams in the next step in the cycle (Position 2). The switch time is adjusted so that streams are added and withdrawn to match the movement of the separated components through the system. In this example the blue component has greater affinity for the column material and is carried into the Extract, whereas the red component moves in the direction of the fluid flow into the Raffinate. One cycle consists of 8 sequential positions in this system.

A fourth zone consisting of columns between the Raffinate outlet and Desorbent inlet is commonly included in large scale SMBC systems (see Perrin and Nicoud, 2001 for review) and can be configured into the Semba Octave System. This zone serves as a buffer between Zones 1 and 3 to ensure that no Raffinate enters Zone 1. It is also used in some configurations as a point to recycle the Desorbent. Because the Semba Octave System can effectively prevent flow of Raffinate into Zone 1 simply by closing off the valve between Zone 3 and Zone 1, and because volumes of Desorbent are generally low enough to eliminate the need for recycling, Zone 4 is not necessary for most applications.

The column resin can be anything that is used in conventional chromatography, including ion exchange and affinity resins. Semba Biosciences has used the Octave instrument to demonstrate the superior performance of SMBC for purification of recombinant proteins by immobilized metal affinity chromatography (IMAC).

In the IMAC application the feed is a bacterial cell lysate containing a histidine-tagged recombinant protein that has been overexpressed using standard molecular biology methods. In this case the sample (Feed) is treated as a binary mixture in which the tagged protein is the more retained component (Extract) and all other untagged proteins and unwanted cellular components are the less retained component (Raffinate). The recombinant protein is expressed from a plasmid that encodes a small peptide consisting of 6-10 consecutive histidine residues, which becomes incorporated into the protein. This “histidine tag” confers the resulting fusion protein with an affinity for metals such as nickel and cobalt. Solid phase resins containing chelated metal ions (usually nickel) can be used to purify the histidine-tagged proteins from crude samples such as bacterial lysates by IMAC. In a typical process a crude sample is passed through a nickel-chelate resin under conditions where the histidine-tagged target protein binds to the immobilized nickel. Untagged bacterial proteins are washed away, and then the purified target protein is released from the solid phase by increasing the concentration of imidazole, a small molecule which competes with the histidine tag for the metal binding sites on the resin. Semba Biosciences has applied IMAC to SMBC for protein purification using two different methods; Isocratic Mode and Step Mode. In Isocratic Mode (patent-pending), a single imidazole concentration is used throughout the system. The chosen concentration is high enough to cause the histidine-tagged target protein to move through the solid phase but still low enough for it to be preferentially retained relative to the untagged or weakly binding proteins. This method, which may also be applied to other protein affinity systems, is analogous to “classical” SMBC, and takes advantage of the resolving power of SMBC to produce extremely high purity with the convenience of a single buffer system. 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 2, below, shows a schematic diagram of an 8-column Step SMBC configuration at equilibrium, at two arbitrary positions in the cycle. 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 2. Two positions of a Step SMBC configuration at equilibrium. 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 (Panel A, Columns 5 and 6), while untagged proteins flow through and exit the system as Raffinate (red stream). In the Wash zone (Panel A, Columns 3 and 4), 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 (Panel A, Columns 1 and 2), the Desorbent stream provides a high imidazole concentration to elute the target protein as Extract (blue stream). In the Regeneration zone (Panel A, Columns 7 and 8), a low imidazole concentration provided by the Aux 2 stream re-equilibrates the columns to prepare them for the next binding cycle. Panel B shows the next position, in which all streams are switched forward one column. One cycle consists of 8 sequential positions in this system.

Both Step and Isocratic modes of SMBC operation have produced high target protein recovery and purity. Figure 3 shows the purification of histidine-tagged human enolase and annexin-1 from bacterial lysates using both methods. In this example the Isocratic Mode product (annexin 1) was > 95% pure, and the Step Mode product (enolase) 90% pure as determined by SDS-PAGE analysis.

	A. Annexin-1, Isocratic SMBC           B. Enolase, Step SMBC
	Figure 3. Isocratic and Step SMBC purification of histidine-tagged proteins from bacterial lysates. Histidine-tagged human annexin-1(Panel A) and enolase (Panel B) were expressed in E. coli. Crude bacterial cell lysates were prepared by standard methods and applied to Ni-chelate columns on the Semba Octave System in a 3-2-3 Isocratic (Panel A) and Step (Panel B) SMBC configurations. Panel A, Isocratic Mode fractions. M, Markers 10-225 kDa; Feed, crude E. coli lysate containing recombinant His-annexin 1; P2-P5, Extract fractions containing purified annexin-1; R2-R4, Raffinate. Panel B, Step Mode fractions. M, Markers 10-225 kDa; Feed, crude E. coli lysate containing recombinant His-enolase; P7-P10, Elute fractions containing purified enolase.

Figure 4 shows side-by-side comparisons of three proteins purified using Isocratic Mode SMBC vs. the conventional single column method. In all three cases the Isocratic Mode produced higher purity, with an increase of 7%, 17%, and 25% for human enolase, annexin-1, and PKI-alpha, respectively. Importantly, SMBC Isocratic and Step modes can be effectively combined to obtain concentrated highly purified preparations from feedstocks containing low levels of dilute target protein. Advantages of these SMBC approaches over standard linear or batch procedures include increased throughput, efficiency, and purity.

Figure 4.Comparison of Isocratic SMBC and conventional single column purification of histidine-tagged proteins from bacterial lysates. The indicated human histidine-tagged fusion proteins were expressed in E. coli. Crude bacterial cell lysates were prepared by standard methods and applied to Ni-chelate columns on the Semba Octave System in a 3-2-3 Isocratic SMBC configuration and to an identical single column. The single columns were processed manually per manufacturers’ instructions. Sample of Feed and purified products were analyzed by SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. Equivalent amounts of protein were loaded for each pair of purified samples. Lanes are indicated in the legend. Purities were determined by scanning densitometry of separate gels in which 3 different sample loads were run side by side in triplicate. Yield data were obtained from extended Isocratic SMBC runs of enolase and annexin-1.

The SMBC methods developed for the immobilized metal affinity system can also be applied to other protein purification strategies, including other specific affinity approaches as well as group-specific methods such as ion exchange, hydrophobic interaction, and size exclusion chromatography. Purification of monoclonal antibodies has also been achieved in our laboratory using cation exchange chromatography adapted for SMBC. In this application the sample (Feed) can be derived from ascites fluid, serum or tissue culture supernatant. The principle is similar to Isocratic Mode, where a Desorbent condition is found that differentiates the target protein (antibody) from the contaminants (serum proteins) with respect to its affinity for the solid phase. In the case of ion exchange this can be accomplished by adjustment of pH and salt concentrations (Stein and Kiesewetter, 2007). Isocratic and Step Mode procedures for continuous antibody purification using Protein A or G affinity chromatography can also be utilized with the Semba Octave Chromatography System