240 200 160 120 0 .


Column size: Sample:


50 x 5.0 mm I.D. 1.0 mg/mL lysozyme in equilibration buffer


Equilibration buffer: 20mM glycine-NaOH (pH 9.0) Elution buffer: 20mM glycine-NaOH (pH 9.0) containing 0.5 M NaCl Detection:


UV at 300 nm SELECTIVITY: 20


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 120mg 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 180mg BSA/mg resin [4]).


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


40 60 Linear flow rate(cm/hr) Figure 1: Dynamic binding capacity for lysozyme measured at different flow rates up to 1000 cm/hr using YMC-BioPro S75.


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 220mg lysozyme per mg resin material and varied only slightly


80 100


In chromatography, separation is dictated by the selectivity of the stationary phases for the given molecules. Because of the electrostatic nature of the separation principle and the resulting ‘on/off’ mechanism, the selectivity is very similar for media from different manufacturers. Generally speaking, under otherwise identical conditions, the elution order is the same, only the peak shape can change. Figure 2 shows a comparison of three cation exchange materials. In the example shown, a mixture of three


Table 2: DBC of various anion IEX resins, at a linear flow rate of 180 cm/h and protein concentration of 1.5 mg/ml BSA. BioPro


QA (75um) (YMC)


DBC


(10%) recovery (%)


[mg BSA/mL gel] 187 100


with increasing flow rates of up to 1000cm/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 187mg/ml resin, which is more than 25% higher than some of the high


Table 3: DBC of various cation IEX resins, at a linear flow rate of 180 cm/h and protein concentration of 1.5 mg/ml lysozyme. BioPro S (75 µm)


DBC


[mg lysozyme /mL gel] (10％）


recovery（％） 109


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 1000cm/h without decrease of DBC.


108 108 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


(YMC) 186


Gigacap S-650M (Tosoh)


182


Macrocap SP (GE）


81


147 93


149 32


102 127


proteins was separated with a generic salt gradient.


The elution order is the same and also the retention times are similar for all three materials. Only the peak shape varies between the different materials due to differences in particle and pore size. Obviously, decreasing particle size increases the number of theoretical plates and this, in turn, yields sharper peaks. As can be seen from the chromatogram for GE’s Macrocap resin (particle size of 50µm as compared to 75µm for the Tosoh and the YMC materials), particle size has a strong influence on the peak shape, with smaller particles giving sharper peaks. On the other hand, smaller particles will normally generate higher backpressures.


SUMMARY


Using the optimal IEX material for a specific application can results in a significant decrease in the costs of the process. Due to the charge-based mechanism, selectivity is similar with different media. However, there are big differences in dynamic binding capacity, recovery and backpressure. As shown above, new media exhibit DBCs of 150% and more compared to traditionally used media. Also new synthetic media can be engineered to show less non-specific binding of the target molecule to the media, which increases the recovery. These increases are highly dependent on the molecule used, but can also be in the region of 10 to 50% higher recoveries. By improving particle size distribution and homogeneous pore size distribution, sharper peaks and lower backpressure have been obtained. This not only aids in more selective isolation of the target molecule but together with high DBC at high flow rates, enables faster processes. As a result columns will be smaller and lower buffer volumes are required which will have a great impact on the overall processing costs.


Gigacap Q- 650M (Tosoh)


Super Q 650-M Capto Q (Tosoh)


GE


highest DBC of 186mg/ml resin of the resins tested.


The figures in Tables 2 and 3 were obtained using 50 x 4.6mm ID columns filled with the corresponding bulk material.


It is obvious that a high DBC is only useful if the recovery is high at the same time. As the table shows, modern materials generally show both. Non-specific binding which causes losses in recovery has been reduced with newer media. For YMC BioPro non- specific binding reaches a very low level, which warrants the high recoveries close to 100%.


REFERENCES


[1] Curling, J, The development of antibody purification, in: Process Scale Purification of Antibodies, Edited by Uwe Gottschalk 2009 John Wiley & Sons, Inc.


[2] Porath, J, Flodin P. ( 1959 ). Gel filtration: A method for desalting and group separation. Nature 183, 1657.


[3] Data file: Sepharose Fast Flow ion exchangers, Code number: 18-1020-66 AB, 2003-09, GE Life sciences, Upsala, Sweden.


[4] Product information (2009) YMC BioPro 75 µm, YMC Japan.


Figure 2: A comparison of three cation exchange materials used to separate a mixture of three proteins using a generic salt gradient. (1) ribonuclease A (MW：13,700）(2) cytochrome C (MW：12,400）(3) lysozyme（MW：14,300）


DBC (mg/mL


Chromatography Focus


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