Simulated moving bed (SMB) chromatography is a continuous process, which for preparative purposes can replace the discontinuous elution chromatography. Furthermore, the countercurrent contact between fluid and solid phase used in SMB chromatography maximizes the mass transfer driving force, leading to a significant reduction of mobile phase consumption and solid phase requirements when compared with elution chromatography. With the developed theory and technique,1–5 the resolution of similar products on preparative scale (ktons/year) can be performed efficiently using SMB chromatography. Successful examples are the separation of p-xylene from a mixture of C8 isomers, the separation of glucose and fructose, and the resolution of enantiomers on chiral stationary phases.1, 2, 6–8 The new challenge for the SMB technology is its application to the separation and purification of biomolecules; examples of products that are considered for SMB separation and purification are therapeutical proteins, antibodies, nucleosides, and plasmid DNA.9–17
Research on the separation and purification of proteins by SMB technology started with size-exclusion SMB (SE-SMB) chromatography, for its design simplicity in terms of the liquid and solid flow rate ratios (linear distribution coefficients for all proteins on porous stationary phases). Experimental examples are: insulin purification by SE-SMB packed with Sephadex G50 gel,12 purification of plasmid DNA by SE-SMB packed with Sepharose FF particles,13, 16 and separation of bovine serum albumin (BSA) and myoglobin by SE-SMB packed with Sepharose Big Beads.17 Because of the absence of ligand in these particles, the distribution coefficients of proteins depend only on the accessible porosity in the particles. A large protein has a smaller distribution coefficient, as weakly retained component, and will elute in the raffinate stream; in contrast, a small protein has a bigger distribution coefficient, as strongly retained component, will elute from the extract stream in the SE-SMB. It is easy to obtain a large protein molecule with high purity from the raffinate stream, but it is difficult to recover the smaller protein molecule with high purity protein in the extract stream because of the limitation of the mass transfer resistance of the larger protein molecule.17
As a result of the limitation of SE-SMB technology for the separation and purification of proteins, research was extended to the application of ion-exchange SMB (IE-SMB), reversed-phase SMB (RP-SMB), and affinity SMB (A-SMB).11, 18, 19 By carefully selecting the buffer, pH value, solvent strength, and ligand of adsorbent to ensure that the larger protein molecule elutes from the extract stream, while the smaller protein is recovered from the raffinate stream, the two proteins can be separated efficiently by the SMB technology. When the binding capacities of proteins on the adsorbent are close to each other, an isocratic SMB mode may be used to separate the proteins, where the adsorbents have the same binding capacity to protein in all sections of the SMB unit, as shown in Figure 1a. However, the binding capacities of proteins on adsorbents are usually so different in ion-exchange, hydrophobic-interaction, reversed-phase, and affinity chromatography (IEC, HIC, RPC, and AC) that we cannot separate them in the isocratic mode with a reasonable retention time. In the conventional elution chromatography, a gradient mode should be used for the separation of proteins; for example, organic solvent gradient in RPC, and salt gradient in IEC, either in step-wise gradient or linear gradient. In the SMB unit, a step-wise gradient can be formed easily by introducing a solvent mixture with a lower strength at the feed inlet port compared to the solvent mixture introduced at the desorbent port; then the adsorbent has a lower binding capacity for protein in section I and II to improve the desorption, and a stronger binding capacity in section III and IV to increase the adsorption, as shown in Figure 1b. Some authors11, 18, 20–24 stated that the solvent consumption by the gradient mode can be decreased significantly when compared with the isocratic SMB chromatography. Moreover, when a given feed is applied to the gradient SMB chromatography, the protein obtained from the extract stream can be enriched if the protein has a medium or high solubility in the solution with the stronger solvent strength, while the raffinate protein is not diluted at all.18
Experimental research for the separation of proteins by salt gradient IE-SMB11 and for the separation of antibodies by solvent gradient RP-SMB25 allowed a qualitative analysis of process feasibility. Furthermore, theoretical analysis for gradient SMB reported by some authors11,18,20–24 confirmed the potential application of the gradient SMB technology in bioseparation. Up to now, this research is just underway partly because experiments are expensive (expensive proteins, special adsorbent, and SMB unit) for the practical separation and purification of proteins by the gradient SMB chromatography. Therefore, a detailed mathematical simulation, using a gradient SMB model instead of an equivalent gradient TMB model, is more significant to understand the performance of the gradient SMB chromatography. One objective of this paper is to simulate the salt gradient IE-SMB processes by using the gradient SMB model.
The open loop configuration was used by many authors to avoid the accumulation of contaminants in the columns of SMB for the separation and purification of proteins, as shown in Figure 2a, where the liquid stream from section IV is discarded, instead of being recycled to the desorbent stream allowing the reduction of desorbent consumption. It is well known that one of the biggest advantages of SMB chromatography compared with fixed bed chromatography is the lower desorbent consumption. This can be achieved in the closed loop configuration by recycling the liquid stream from section IV to the desorbent inlet of section I, as shown in Figure 2b, which is also important for RP-SMB because of the large amount of organic solvent being consumed. However, the recycle of liquid stream is more complicated for the gradient SMB chromatography. The solvent strength in the eluent is different between sections I and IV, and the composition of the stream leaving section IV varies in a dynamic manner during the switching interval, which complicates the direct recycling of the eluent.25 In Figure 2c, a holding vessel with a given volume is added to the system to mix online the desorbent with the recycled liquid stream from section IV during the switching interval, to reduce the fluctuation of the solvent strength in the columns. Another objective of this paper is to evaluate and compare three different configurations of gradient SMB: open loop (Figure 2a), closed loop (Figure 2b), and closed loop with a holding vessel (Figure 2c).