1
Proteins Separation and Purification by Expanded Bed Adsorption and Simulated Moving Bed Technology

Ping Li, Pedro Ferreira Gomes, José M. Loureiro, and Alirio E. Rodrigues

1.1 Introduction

Proteins not only play an important role in biology, but also have large potential applications in pharmaceuticals and therapeutics, food processing, textiles and leather goods, detergents, and paper manufacturing. With the development of molecular biology technologies, various kinds of proteins can be prepared from upstream processes and from biological raw materials. However, there exist various proteins and contaminants in these source feedstocks, and the key issue is that proteins can be separated and purified efficiently from the source materials, in order to reduce the production cost of the high-purity protein. The development of techniques and methods for proteins separation and purification has been an essential prerequisite for many of the advancements made in biotechnology.

Most separation and purification protocols require more than one step to achieve the desired level of protein purity. Usually, a three-step separation and purification strategy is presented, which includes capture, intermediate separation and purification, and final polishing during a downstream protein separation and purification process. In the capture step the objectives are to isolate, concentrate, and stabilize the target proteins. During the intermediate separation and purification step the objectives are to remove most of the bulk impurities, such as other proteins and nucleic acids, endotoxins, and viruses. In the polishing step most impurities have already been removed except for trace amounts or closely related substances. The objective is to achieve final purity of protein.

In the capture step, as the primary recovery of proteins, the expanded bed adsorption (EBA) technology has been widely applied to capture proteins directly from crude unclarified source materials, such as, Escherichia coli homogenate, yeast, fermentation, mammalian cell culture, milk, and animal tissue extracts [1,2]. The expanded bed is designed in a way that the suspended adsorbent particles capture target protein molecules, while cells, cell debris, particulate matter, and contaminants pass through the column unhindered. After loading and washing, the bound proteins can be eluted by elution buffer and be concentrated in a small amount of elution solution, apart from the bulk impurities and contaminants in source materials. With specially designed adsorbents and columns, the adsorption behavior in expanded beds is comparable to that in fixed beds. Various applications of EBA technology have been reported from laboratory-scale to pilot-plant and large-scale production [1-9].

During the intermediate purification and final polishing steps, the techniques of the conventional elution chromatography have been applied successfully. A new challenge should be the application of simulated moving bed (SMB) to the separation and purification of proteins. SMB chromatography is a continuous process, which for preparative purposes can replace the discontinuous regime of elution chromatography. Furthermore, the countercurrent contact between fluid and solid phases used in SMB chromatography maximizes the mass transfer driving force, leading to a significant reduction in mobile and stationary phase consumption when compared with elution chromatography [10-14]. Examples of products that are considered for SMB separation and purification are therapeutic proteins, antibodies, nucleosides, and plasmid DNA [15-23].

When the binding capacities of proteins on adsorbent are close to each other, an isocratic SMB mode may be used to separate and purify the proteins, where the adsorbents have the same affinity capacity to proteins in all sections in SMB chromatography. However, usually the binding capacities of proteins are so different that we cannot separate them by the isocratic mode with a reasonable retention time. In conventional elution chromatography, a gradient mode should be used for the separation of proteins. It is most commonly applied in reversed-phase and ion exchange chromatography (IEC), by changing the concentration of the organic solvent and salt in a stepwise gradient or with a linear gradient, respectively. For SMB chromatography, only a stepwise gradient can be formed by introducing a solvent mixture with a lower strength at the feed inlet port compared with the solvent mixture introduced at the desorbent port; then the adsorbents have a lower binding capacity to proteins in sections I and II to improve the desorption, and have a stronger binding capacity in sections III and IV to increase adsorption in SMB chromatography. Some authors state that the solvent consumption by gradient mode can be decreased significantly when compared with isocratic SMB chromatography [17-19,24-29]. Moreover, when a given feed is applied to gradient SMB chromatography, the protein obtained from the extract stream can be enriched if protein has a medium or high solubility in the solution with the stronger solvent strength, while the raffinate protein is not diluted at all [24].

In this chapter, we shall describe the developments made at the Laboratory of Separation and Reaction Engineering (LSRE) for proteins separation and purification by expanded bed chromatography and salt gradient ion exchange simulated moving bed technology.

1.2 Protein Capture by Expanded Bed Technology

1.2.1 Adsorbent Materials

The design of a special adsorbent is a key factor to enhance the efficiency of expanded bed adsorption. The EBA process will be more effective for those adsorbents that have both high-density base matrix and salt-tolerant ligand. The high-density matrix means minimizing dilution arising from biomass or viscosity in feedstock and reducing dilution buffer consumption; the lack of sensitivity of the ligand to ionic strength and salt concentration means there is no need for dilution of feedstock [30-32].

"Homemade" adsorbents are commonly used for research purposes. Agarose and cellulose are the major components utilized on the tailoring of the adsorbents. Table 1.1 shows a list of such adsorbents.

Table 1.1 "Homemade" adsorbents.