0 10 20 30 40 50 60 70 80 90 100 Applied BSA (mg/mL sedimented adsorbent)
0 10 20 30 40 50 60 70 80 90 100 Applied BSA (mg/mL sedimented adsorbent)
Figure 1. Protein breakthrough capacity. Breakthrough curves for bovine serum albumin (BSA) on STREAMLINE® DEAE (15 cm settled bed height at 300 cm/h flow velocity) using STREAMLINE® expanded-bed columns with inner diameters ranging from 25 to 1,200 mm. C0 is the concentration of BSA in the applied feedstock, and C is the concentration in the flowthrough. Source. Copyright Amersham Pharmacia Biotech, Uppsala, Sweden, modified and reproduced with permission.
Because industrial-scale columns have to be automated, these columns are equipped with an adsorbent sensor that locates the position of the expanded bed surface. In response to the signal from the sensor, the adaptor is automatically adjusted to its correct position. Figure 2 shows different scales of expanded-bed columns.
UpFront® Columns (UpFront Chromatography A/S, Denmark) offer another approach to expanded-bed column design (53). The columns are without screens and without distribution plates, and the feedstock is introduced at the bottom of the column, right below a stirring device that distributes the feedstock over the cross-section of the column. The stirrer causes a mixed zone, but gradually above that zone plug flow is obtained. Columns of 20-, 50-, and 200-mm inner diameter are available.
BioProcessing Ltd. (England) uses modified standard chromatography columns for expanded bed adsorption (54). The standard column bottom support net or screen is replaced with a 300-um nylon mesh, and then the column
is packed with a layer (approximately 3 cm in height) of surface-modified solid glass beads (0.4-0.52 or 0.85-1.23 mm in diameter). This bed of beads serves as a distributor of fluid to the bed. The adsorbent particles are then added directly on top of the distributor beads. During operation of the column, the bed of the relatively heavy distributor beads remains static, and the bed of adsorbent particles expands.
STREAMLINE® adsorbents are specially designed for expanded bed adsorption. To ensure that the bed does not expand too much at industrial chromatography flow velocities, a dense core material has been incorporated into the agarose particles to increase the apparent density. The adsorbent has a defined Gaussian-like particle size distribution, which yields a stable classified bed when the adsorbent is expanded. The influence of particle size distribution on protein adsorption performance has been studied using three different particle fractions (120-160 im, 250-300 im, and 120-300 im) (55). The results showed that a wide particle size distribution gives an ex panded bed with low axial dispersion and that small particles give high dynamic protein capacity (due to reduced diffusion length and large surface area). The results also showed that with increasing (settled) bed heights, the dynamic binding capacity increases over a wide range of flow velocities (due to reduced axial dispersion and increased numbers of transfer units for pore and film diffusion).
The STREAMLINE® DEAE, Q XL, SP, and SP XL, che-lating and heparin adsorbents are spherical, macroporous, cross-linked 6% agarose particles with crystalline quartz as core material. The Q XL and SP XL adsorbents have dextran chains coupled to the agarose, and the Q and SP charged groups are attached to the dextran through chemically stable ether bonds. This increases the exposure of the Q and SP groups and results in very high protein binding capacities. The adsorbents can be cleaned with 1 M NaOH. The matrix used for the protein A adsorbent contains highly cross-linked 4% agarose with a metal alloy core. The alloy is stable and composed of 77% Ni, 15.5% Cr, and 7.5% Fe. No leakage of metal was detected in 0.1 M glycine, pH 3.0, a solution commonly used in protein A chromatography. The metal leakage was below the detection limit of the analysis method (0.02 ig/mL eluent using induced coupled plasma atomic-emission spectroscopy). The protein A adsorbent is stable in all aqueous buffers commonly used in protein A chromatography and cleaning. No significant change was observed in chromatographic performance after either 1 week's storage or 100 cycles, normal use, at room temperature with 10 mM HCl (pH 2), 0.1 M sodium citrate (pH 3), 1 mM NaOH (pH 11), 6 M GuHCl, or 20% ethanol. Figure 3 shows micrographs of the adsorbent particles, and Table 2 summarizes the properties of the various STREAMLINE® adsorbents.
Apart from the purpose-designed column and adsorbent, the equipment and the experimental set-up used for expanded bed adsorption is very similar to that used for packed bed chromatography. An example of an industrial-scale set-up with a 600-mm-diameter column is seen in Figure 4.
The strategy for developing an expanded-bed process is in general analogous to that of a conventional packed-bed process. In order to save time, it is advisable to do the first experiments with clarified feedstock, using the expanded-bed adsorbent in a small, packed-bed column, for example, 15 mm in diameter and with 15-cm bed/height. (A bed height below 10 cm of these relatively large particles usually results in a decreased dynamic binding capacity. The increase in dynamic binding capacity with increased bed height is an effect of longer residence time for the sample.) From the packed-bed experiments, the most suitable adsorbent is selected (e.g., ion exchanger or chelating adsorbent), and the conditions for protein binding and elution are determined. It is important to establish that the conditions selected for protein binding are compatible with the properties of the unclarified, cell- or debris-containing feedstock (e.g., low pH might cause aggregation). Such problems are not apparent with clarified feedstock and
should be investigated before the process has progressed too far. Once the most promising method has been selected from the packed-bed experiments, it is then optimized in a lab-scale expanded-bed column using unclarified feedstock. At this stage, the effect of the now present cells or debris on protein binding is evaluated. In theory, if the net surface-charge of the cells and debris is negative, the protein and the cells or debris may compete for binding sites on an anion exchanger. A consequence of this may be that the feedstock load per milliliter adsorbent has to be decreased. When STREAMLINE® DEAE was used to recover insulinase from whole yeast culture, the presence of cells did not cause any reduction in protein capacity (33). However, it has been reported that when the same adsorbent was used to recover glucose-6-phosphate dehydroge-nase from a yeast homogenate, the presence of cell debris did cause a slight reduction in capacity of the adsorbent
(36). Feedstock properties and their effects on the ex-panded-bed process are discussed further in "Feedstocks." Before the process is transferred into larger columns, appropriate cleaning procedures should be determined. The whole process is then verified at pilot scale before scaling up to the final production scale.
Figure 5 is a schematic representation of how an expanded bed is operated—essentially as a packed bed. Avoid getting air into the column; air trapped in the bottom distributor may disturb the bed. If air does enter the column, it can usually be removed with high-flow velocity pulses combined with back-flushes (reversal of flow direction). Before start-up always check that the column tube is positioned vertically; otherwise, flow will be turbulent when the liquid hits the inside walls of the column tube. The height of the settled bed (H0) is noted before start-up.
Bed Expansion and Equilibration. The bed is expanded and equilibrated with buffer. It usually takes 20-30 min at a flow velocity of 300 cm/h before the bed is stable and has stopped expanding. The height of the expanded bed (H) is then noted. At this stage, the bed can be checked empirically, before application of the feedstock, by determining the axial dispersion of a tracer substance. Figure 6 shows the UV signal recording from such a test procedure. Before applying the tracer substance, the adaptor is lowered to a position where the adaptor screen is 0.5-1.0 cm above the expanded-bed surface. When the signal from the UV monitor is stable, the solution is changed to buffertracer substance mixture (e.g., 0.25% acetone v/v), that is, positive step input signal. The solution is changed to buffer when the UV signal is stable at maximum absorbance (100%), that is, negative step input signal. The change is marked on the chart recorder paper, and the UV signal is allowed to stabilize at the baseline level (0%). The number of theoretical plates (N) is calculated from the negative step input signal (57):
where t is the mean residence time, the distance from the mark on the chart recorder paper to 50% of the maximum absorbance; and r is the standard deviation, half the distance between the points 15.85% and 84.15% of maximum absorbance.
The procedure gives the number of theoretical plates not only in the column but also the contribution from pumps, valves, tubing, and so forth. Because different experimental set-ups may vary in their contribution to N, the results should only be compared between runs performed using the same set-up. The negative input signal is recommended for evaluation (at least when acetone is used as tracer) because the reproducibility of the results is higher than that obtained from the positive step input signal (from 0% to 100%).
The relation between N and the axial dispersion coefficient (Da) is
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