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Purification of Murine IgG1 Using UNOsphere S and CHT ,,, Ceramic Hydroxyapatite Chromatography, Rev A

3- ions in its spherical and ceramic structure, provides excellent resolution and selectivity for protein isolation.


Methods and Results
The BioLogic DuoFlow workstation was used to develop the processes. An analytical column (0.7 x 5 cm) was loaded with UNOsphere S support for small-scale process development. A preparative column (2.2 x 20 cm) was used in process scale-up. All SDS-PAGE was performed using Ready Gel precast gels, the Criterion cell, and the PowerPac 3000 power supply. Ceramic hydroxyapatite (Type I, 10 m and 20 m) media were used for this study.

Small-Scale Process Development
UNOsphere S chromatography was used as the capture step for murine IgG1 isolation. The binding capacity of UNOsphere S support for murine IgG1 was optimized at pH values from 7.0 to 4.0. Decreasing the pH in the cell culture increased the binding capacity of UNOsphere S support for murine IgG1 (data not shown). The murine IgG1 was stable for at least 6 hr in buffer at pH 4.0 (data not shown), so the cell culture was adjusted to pH 4.0. The cell culture was then loaded onto the UNOsphere S column as described in Figure 1. The murine IgG1 was eluted in the NaCl gradient. The fractions were analyzed by SDS-PAGE (Figure 2). It was found that Phenol Red and some contaminating proteins were removed in the flow-through pool by UNOsphere S chromatography.

For ceramic hydroxyapatite chromatography, the murine IgG1 fractions were pooled and diluted 5-fold with 1 mM sodium phosphate buffer, pH 6.8. The murine IgG1 elution pool from the UNOsphere S column could not be loaded directly on the CHT supports bec
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