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fermentation broth for e.g. monoclonal antibodies and up to more than 20 g/L for e.g. interferon. Together with a range of other methods (e.g. precipitation, ultrafiltration, protein A affinity chromatography), IEX is frequently used for this task, where up to several thousand litres of fermentation broth are being processed. The advantage of using IEX over less discriminating methods, such as ultrafiltration, is that the amount of fermentation impurities (e.g. host cell proteins, unwanted proteins, proteases) present in the captured material can often be reduced significantly
Due to the fast adsorption/desorption mechanism it is possible to process the large volumes of fermentation broth in an acceptable amount of time. Traditionally cross-linked dextran and agarose gels (originally developed by scientists at Pharmacia2
) have been used. New fully
synthetic polymer-based materials, including YMC’s BioPro IEX bulk materials, have favourable properties compared to the traditional media. The newer materials feature higher binding capacities, better pressure stability, lower non-specific binding and higher reproducibility due to their fully synthetic origin.
Because of the large volumes involved in a capture step, the flow rates should be as high as possible in order to get to acceptable process times. As a result, the most important factors impacting on the efficiency of a capturing step are the dynamic binding capacity (DBC) and the maximum flow rate achievable. The latter point is controlled by the fluid dynamic properties of the chromatographic column; DBC will be discussed in the next section.
As for every chromatographic application, particle size and its distribution, together with the mechanical stability of the chromatographic medium, limit the maximum flow. If the mechanical stability is inadequate, the stationary phase will collapse under the backpressure resulting from high flow rates. Modern fully synthetic polymer based materials, are generally sufficiently pressure stable. Obviously, the backpressure generated is influenced by the particle size and distribution. For new synthetic polymer based materials both parameters can be controlled more easily. However, the most efficient particle size for high flow rates will always be a trade off between binding capacity and backpressure. In principle, the binding capacity increases with decreasing particle
size. At the same time backpressure increases. There are big differences between different materials with regards to flow properties and DBC at high flowrates.
Dynamic binding capacity (DBC) and recovery:
The DBC is the capacity to bind the target molecule while the mobile phase is continually flowing through the IEX-column. It is expressed in mg target molecule bound per ml of resin in the column (mg target/ml resin) and depends on the flow rate which is being applied. It is determined at 10% breakthrough and is obviously different for every molecule and resin. For Sepharose FF, a widely used IEX material, a value of 120 mg BSA per ml resin has been reported 3
. For
modern media DBC can be significantly higher. For example, YMC BioPro IEX material has a production specification to achieve 150% of this value and more (i.e. more than 180 mg BSA/mg resin4
).
Theory predicts that increasing the flow rate will have a negative effect on the DBC. Even though the binding process is very fast, at high flows the molecules have less time to diffuse into the porous structure of the stationary phase and to bind the IEX ligands. Because of this and because of the mechanical problems outlined above, flow rates have been fairly limited in the past. However, new synthetic polymeric materials offer high binding capacity even at high flow rates. At the same time, the DBC at these higher flow rates is also higher than with traditional media (at lower flow rates). Increased mechanical stability and improved
concepts of binding the ion exchanger functionalities to the support have made this possible. In Figure 1 the dependency of DBC to linear flow rate is shown for a YMC BioPro cation exchange material, which was tested up to 1000 cm/h without decrease of DBC.
In Figure 1, lysozyme was used as a model/test protein and the DBC was determined at 10% breakthrough. In this experiment, the dynamic binding capacity for the cation exchanger was in the region of 220 mg lysozyme per mg resin material and varied only slightly with increasing flow rates of up to 1000 cm/hr.
For anion exchange materials, several different materials were tested under the same conditions with bovine serum albumin (BSA) as the test substance. It is obvious from Table 2 that there are distinct differences in performance between the different phases. Both the DBC and recovery of the target molecule varies widely for the various media. For BSA, YMC BioPro QA (anion exchanger) shows the highest DBC of 187 mg/ml resin, which is more than 25% higher than some of the high performance materials from competitors.
A similar experiment was performed, using cation exchange materials and lysozyme as the test substance, see Table 3. Again there are distinct differences in performance between the different phases in terms of both the DBC and recovery. For lysozyme, YMC BioPro S (cation exchanger) shows the highest DBC of 186 mg/ml resin of the resins tested.
Figure 1 Dynamic binding capacity for lysozyme measured at different flow rates up to 1000 cm/hr using YMC-BioPro S75
BioPro QA (75um) (YMC)
DBC [mg BSA/mL gel] (10％)
recovery（％） Table 2 187 100
Gigacap Q-650M (Tosoh)
147 93
Super Q 650-M (Tosoh)
149 32 Capto Q GE 102 127 DBC of various anion IEX resins, at a linear flow rate of 180 cm/h and protein concentration of 1.5 mg/ml BSA
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