Chromatographic separation techniques ensure purification quality of biotechnically produced proteins. Most of the time, proteins are eluted with salt gradients. Purification with pH gradients is usually difficult to control, however it is appealing because subsequent salt removal steps are unnecessary. Mixed-bed chromatography columns have shown to be an effective way of performing pH gradient-based protein separation. The approach of present work was to create a linear pH gradient, generated by a step change in pH. A separation of protein charge variants with the mixed-bed concept was demonstrated for a monoclonal antibody mixture. A selection of appropriate ion exchange resins has given a mixed-bed column with a dual function: a small-pore weak base resin serves for intracolumn generation of pH gradients, and a large-pore strong base resin serves for interacting with proteins, gaining high binding capacity. A combination of different mobile phases has shown that concentration and pH value of the buffer can regulate the duration of the induced pH gradient. Weak (AG® 4-x4) and strong (Fractogel® EMD TMAE Hicap (M)) anion exchanger have turned to be good candidates in our experiments, however, intermediate anion exchanger (Bio-RexTM 5) has not given us positive results. The separation of monoclonal antibody isoforms has been partially achieved. In future, one would have to define an optimal ratio of weak and strong ion exchange resins, and a suitable buffer system with a higher chromatographic sensitivity for the separation of isoforms.
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