Figure 6. UV signal recording from the test procedure for determination of the number of theoretical plates (N). N = t2/r2, where t is the mean residence time and r is the standard deviation. Source: Courtesy of Amersham Pharmacia Biotech, Uppsala, Sweden.
After cleaning, the adsorbent can be stored in the column in an appropriate storage solution.
Particulate-containing feedstocks can be applied to an expanded bed without prior clarification steps such as cen-trifugation and filtration; however, there are certain physical properties a feedstock must have to make it suitable for an expanded-bed process. The size and shape of the particulates must be such that they can pass through the screen or net in the column, that is, the particulates must be smaller than approximately 60 im in diameter. Feedstocks containing small, round single cells or fine precipitates (e.g., renatured inclusion body preparations) will eas ily pass through the screens, but large aggregates of cells will not. If the particulates are too large, they will build up under the bottom screen and eventually block the column. When the feedstock contains particulates that are too large, they can be removed by centrifugation or by filtering through a simple mesh screen. It is of course desirable for these steps to be avoided completely, but they are relatively easy to perform and should not be compared to the complete clarification procedures needed for a packed-bed process. It is important to stir all feedstocks during application, otherwise the particulates may settle and disturb the expanded bed. Other parameters that will influence the performance of an expanded bed are the viscosity and biomass content of the feedstock. High values for these parameters will cause the bed to overexpand (relative to a buffer); if the bed expands up to the adaptor, the adsorbent particles will start to pack against the adaptor. This buildup will act as a filter and eventually cause the column to block. Furthermore, if the viscosity or the biomass content is too high, flow may become turbulent and channels may appear in the bed. If the turbulence is severe, the feedstock will pass through the channels in the bed, resulting in poor adsorption efficiency. The upper limits for feedstock viscosity and biomass content will vary from case to case, but for STREAMLINE® with homogenates of Escherichia coli, there are a few guidelines (30). A dry weight of 3 to 4% gives the best result, 5% gives an acceptable result, and 7 to 8% is the upper limit. Viscosities up to 10 mPa s (measured at a shear rate of 1 /s) give the best results; the upper limit is about 50 mPa s (at 1 /s). In cases where the viscosity or biomass content of the feedstock does not cause the bed to expand too much, build-up on the adaptor can be prevented if the column is intermittently back-flushed. The only way to lower the biomass content of the feedstock is to dilute it. The disadvantage is the resulting increase in feedstock volume. Dilution is only one of way of lowering viscosity. When the viscosity is caused by nucleic acids, the feedstock may be treated with a nuclease (30). When such a feedstock is a homogenate, further homogenization can be used to shear the nucleic acids and thereby lower the viscosity. The viscosity of a feedstock decreases with increased temperature, making it advantageous to apply the feedstock directly from the fermentor without prior cooling. Since temperature also has a positive effect on protein adsorption (59), expanded-bed adsorption should always be performed at room temperature (or without feedstock cooling), unless the protein of interest is, for example, sensitive to proteases.
To keep aggregation of cells and debris to a minimum, any pH adjustment of the feedstock should be performed immediately before application to the expanded-bed column. Aggregation of cells usually occurs at low pH and is therefore a problem associated with cation exchange, where pH has to be below the isoelectric point of the protein for it to bind to the adsorbent. If aggregation of cells and debris occurs within the expanded bed, when the feedstock is in contact with the adsorbent, and blocks the screen in the adaptor, the expanded bed may be operated without the adaptor screen (provided that the bed has not expanded all the way up to the adaptor) (49). As mentioned previously, cells or debris may, under certain conditions, interact with the adsorbent particles. If this occurs, it is usually with anion exchangers because they are positively charged and may attract the negatively charged cells or debris. A slight change in the binding conditions can help, for example, the addition of some salt and/or lowering the pH.
Alternatively, the feedstock load per milliliter adsorbent may have to be decreased, or it might be necessary to change to an adsorbent with another type of ligand, such as a cation exchanger.
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