Background

The biotechnology industry produces proteins for therapeutic and diagnostic use by using biological systems such as bacteria, yeast, and mammalian cells. A common bottleneck in the production of these proteins is the initial purification, during which the proteins are recovered from large volumes of cell- and debris-containing feedstocks, such as fermentation broths and homogenates. The purification processes are usually based on packed-bed chro-matography techniques in which the protein is bound to an adsorbent packed in a column. A packed bed (fixed bed) cannot handle particulates very well and would eventually become totally blocked if an unclarified feedstock was passed through it. Another concern with these types of feedstocks is that because of the sometimes low production level of the protein of interest, the feedstock volume is large. A large volume takes a long time to pass through a chromatography column, and a long processing time increases the risk of proteolytic breakdown of the protein. Unit operations to clarify and reduce the volume of these feedstocks are therefore vital for a successful purification process. It is not uncommon for a purification process to consist of a sequence of unit operations just to achieve initial recovery. The techniques traditionally used for feedstock clarification are centrifugation and filtration (1).

Centrifugation is well suited to industrial processes because the throughput is high and it can be performed in a contained environment (a requirement when handling recombinant host organisms). The efficiency of the centrifugation process will depend on the properties of the feedstock, but it is usually difficult to remove all particulates by centrifugation, and the resulting fluid may therefore not be sufficiently free of particulates for direct application to a packed chromatography bed (2). Whole cells are relatively easy to remove by centrifugation but disrupted cells are not, and cells (especially mammalian) may even be damaged by the centrifugation process (3) because of shear stress and centrifugal forces. New and improved centrifuges that are more gentle can be used for mammalian cells (4), but often the clarification problem remains because at harvest, culture fluids usually contain relatively large amounts of subcellular particulates, such as debris. When the feedstock is secreting bacteria or a bacterial ho-

mogenate, for example, clarification is even more difficult and centrifugation is not very efficient.

When filtration is used for clarification of feedstocks, it is usually performed in cross-flow mode (5,6). The fluxes that can be used will depend on the properties of the feedstock, but it is often difficult to reach an acceptable economic limit (100 L/m2/h) (7) for a cross-flow filtration process because fluxes are typically less than 50 L/m2/h. Cross-flow filtration can easily be operated as a completely contained system, and it yields a particulate-free solution. The fouling of the filtration membranes is a major concern, and the resulting decrease in flux per filter area often leads to long processing times. In addition, the cost of membranes must be taken into account in a process where membrane fouling is severe because the membranes have to be replaced frequently Because of the drawbacks associated with both the centrifugation and the filtration techniques, they are often used in combination in a purification process to achieve the best clarification of the feedstock.

The volume of the feedstock is not significantly reduced by either centrifugation or filtration, which is why product concentration does not usually increase until the protein has been adsorbed to a chromatography adsorbent in a packed-bed column. Because the feedstock volume is not reduced before application to the packed bed, processing time will be long. Thus, for a number of reasons, the best option for successful initial recovery of a protein from a large-volume cell- or debris-containing feedstock is direct adsorption of the protein.

A variety of methods for direct recovery have been tried. One such method is to use a batch procedure in which an adsorbent is added directly to the unclarified feedstock in a stirred tank (8,9). The advantage of using a batch procedure is that the feedstock does not have to be clarified. The disadvantage is that adsorption of protein to the chro-matography adsorbent is inefficient, because the stirred tank acts as one single theoretical plate in a separation process (10). Several attempts, however, have been made to improve batch adsorption, for example, continuous-batch adsorption processes (11).

Another alternative for direct recovery is fluidized bed adsorption. This technique uses high-density adsorbent particles in an upflow fluidized bed column (12,13). As with a batch procedure, a fluidized bed behaves like a column with a low number of theoretical plates. The adsorbent particles and the feedstock are constantly "back-mixed," which usually results in poor adsorption of the protein by the adsorbent particles, making it necessary to recirculate the feedstock through the column, leading to long processing times. A number of solutions to improve the efficiency of fluidized beds have been suggested, such as columns fitted with perforated horizontal plates (14). This creates a column with a number of smaller, each completely back-mixed, fluidized beds. The number of fluidized beds depends on the number of plates inserted. The end result is a column with more than one theoretical plate and consequently a more efficient process. Another way to improve the efficiency of a fluidized bed is to use magnetically stabilized fluidized beds (15). The adsorbent particles are magnetically susceptible and are fluidized while being stabilized by a magnetic field. Stabilizing the particles in this way prevents back-mixing and gives the column several theoretical plates. There are, however, drawbacks that have prevented this technique from being used at industrial scale, such as the heat generated by the magnetic field.

The concept of expanded-bed adsorption (or expanded-bed chromatography) was introduced after another attempt to stabilize and prevent back-mixing in a fluidized bed. An expanded bed is a chromatography bed in which the bed voidage is larger than in a settled or packed bed. Furthermore, it displays no or very low back-mixing of adsorbent and liquid. An expanded bed can be also be described as a stable fluidized bed because the liquid flow shows a constant velocity profile over the whole cross-section of the bed (i.e., plug flow) (Worth mentioning is that the terms fluidized bed and expanded bed are used differently in the literature, and sometimes authors do not differ between the expressions.) The early expanded bed experiments were performed using Sepharose® Fast Flow (Amersham Pharmacia Biotech, Uppsala, Sweden) as the adsorbent and a sintered glass filter as the liquid flow distributor at the bottom of the column (16). The results showed that if the bed expansion was limited to approximately twice the settled bed height, the liquid flow through the column was close to plug flow and the protein adsorption efficiency was similar to that of a packed bed. These promising results showed that in an expanded bed it is possible to combine the fluidized bed's ability to handle unclarified feedstocks with a packed bed's characteristic of efficient protein adsorption. The adsorbent used in these early experiments is intended for use in packed beds. The density of the particles is relatively low, which meant that the flow through the expanded bed column also had to be low, otherwise the bed would expand to an extent that adsorbent would start to pack at the top of the column. For this technique to be attractive at industrial scale, the flow velocities must be significantly increased. The sintered glass filter that was used as a flow distributor is not appropriate for large-scale operations for a number of reasons: particulates may get trapped in the filter, the filter is difficult to clean, and it is difficult to produce large homogenous sinters. Hence, it soon became apparent that for expanded-bed adsorption to be successful at industrial scale, new types of adsorbent particles and new column designs were needed.

Many different types of adsorbents have been tested: agarose (17,18), zirconia, silica (19), glass (20,21), hydro-philized perfluorocarbon (22), and composites such as kie-selguhr/agarose (23), cellulose/titanium dioxide (24), and dextran/silica (25). These adsorbents are suitable for certain applications, but they lack the combination of properties needed for an efficient expanded-bed adsorption process. Agarose adsorbents are well suited for protein recovery but they are not dense enough to allow high flow velocities. Silica cannot withstand the harsh cleaning and sanitization conditions that are used in early recovery processes (26). Zirconia particles are the most dense, are stable at high pH, and are sterilizable. Porous glass and the described perfluorocarbons have promising sedimentation properties, but porous glass sometimes shows quite low protein-binding capacities (21), and the perfluorocarbons have low protein capacities because of the low particle porosity (22).

With the recent development of STREAMLINE® (Amersham Pharmacia Biotech) adsorbents (agarose/ crystalline quartz composite, agarose-dextran/crystalline quartz composite, and agarose/metal alloy composite) and STREAMLINE® columns, it was possible to secure a recovery system that has the properties needed for an efficient expanded-bed process. The columns have a flow distributor in the bottom that generates an even flow distribution over the whole cross-section of the bed. The density of the adsorbent permits process flow velocities (300-500 cm/h), and the size distribution creates a classified bed that enhances the stability of the expanded bed. The result is a bed with protein adsorption characteristics similar to those of a packed chromatography bed. Ex-panded-bed adsorption is a scalable, one-passage technique that is intended for industrial-scale operations (27).

There have been a number of applications reported that use expanded bed adsorption with various feedstocks: secreting bacteria (28), periplasmic preparations of bacteria (29), bacterial homogenates (30,31), secreting yeast (3234), yeast homogenates (35,36), mammalian cells (37-45), milk/whey (46-48), and inclusion body preparations (49).

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