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40


November/December 2009


BioPro S (75 µm) Gigacap S-650M (Tosoh) Macrocap SPGE (YMC)


DBC [mg lysozyme /mL


gel](10%) recovery(%)


186 109


The figures in Tables 2 and 3 were obtained using 50 x 4.6 mm 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%.


Selectivity: In chromatography, separation is dictated by the selectivity of the stationary phases


182 108 81 108 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


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 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 50 µm 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.


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.


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)


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


Pharmaceutical Applications Notebook Now Available


The pharmaceutical industry is the largest consumer of HPLC instrumentation. In drug discovery, HPLC and IC systems are used both as stand-alone and front-end tools for mass spectrometers to screen drug candidates. In non-clinical development, the systems are used for analyzing in-vitro and in-vivo samples. In clinical trials, they are used to gather data on a potential drug’s safety and efficacy. The systems are also used in manufacturing for many tasks including QA/QC, and the validation of


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