Chiral Separation 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.

Figure 2 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.

Enantiomers of a chiral active pharmaceutical ingredient (API) could have dramatically different biological activities. Drug development regulations demand single enantiomer testing and purification if bioactive differences are detected. In 2004 nine of the top 10 drugs had chiral APIs and six of these were single enantiomers (Rouhi, 2004). SMBC is ideally suited for chiral API development since the fractionation process was developed for binary mixtures. Significant advantages of SMBC over single column batch chromatography are more efficient use of expensive chiral stationary phases (CSP), continuous sample processing, reduced solvent requirement, and more reliable scalability (Juza et al., 2000). The counter-current flow of mobile and solid phases in SMBC gives increased resolution of enantiomers from the racemate. Semba Biosciences’ Octave instrument has been used to demonstrate the superior performance of SMBC for chiral separation (Fig. 3).

	Figure 3. Separation of (+)/(–) 5-methyl 5-phenylhydantoin enantiomers by SMBC on the Semba Octave™ System

Enantiomers were separated using the indicated Astec CHIROBIOTIC™M V2 columns on the Octave System in a 3-2-3 SMBC configuration. Samples of Feed (Racemate), Raffinate, and Extract were analyzed by HPLC on an Astec CHIROBIOTIC V2 25 cm x 4.6 mm, 5 μ column using 100% MeOH as eluent. CHIROBIOTIC is a trademark of Sigma-Aldrich/Supelco. Data courtesy of Sigma-Aldrich/Supelco