38 CHROMATOGRAPHY
Fig. 2. Example of sequence table for immunoaffinity separation
detectors to a second six position selector valve, where the output can be directed to reservoirs for waste, eluted material, or material to be reloaded for further processing.
Te second example application is preparative size exclusion chromatography with stacked injections using Clarity. Tis instrument setup is designed to facilitate removal of aggregates and other high molecular weight species from antibodies. Generally, the harshness of the elution buffer used in the affinity step leads to some degree of aggregation in the isolated antibodies.
A six position selector valve is used to switch between a sample reservoir and a running buffer reservoir. Tis is connected to a pump, with backpressure regulator configured as a blow- off valve in order to protect the column from excessive pressure in the event of a clog. Te outlet of the column is connected to a UV absorbance detector. Fractions are collected using a fraction collector. Again, multiple injections are needed to load all the samples to be processed. To reduce the time required to process large samples, a stacked injection technique is
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used. As a run begins, the solvent selection valve loads the first portion of the sample from the sample reservoir then switches back to the running buffer. After the first portion has migrated some distance down the column, the valve switches to the sample reservoir loading a second portion. After the second portion has migrated down the column a third can be loaded, and so on. If the characteristics
of the sample are known, spacing between samples can be reduced significantly versus loading one sample and waiting for it to elute completely. In one example, a single sample takes 165 minutes to elute completely, however eight samples can be processed in 790 minutes, saving almost nine hours by using stacked injections. Multi-gram quantities of material can be processed per day using this technique.
Fig.3. Diagram of the stacked injection SEC system
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