Purification of IgG Using Ion-Exchange HPLC

Conventional ion-exchange chromatography separates molecules by adsorbing proteins onto the ion-exchange resins that are then selectively eluted by slowly increasing the ionic strength (this disrupts ionic interactions between the protein and column matrix competitively) or by altering the pH (the reactive groups on the proteins lose their charge). Anion-exchange groups (such as diethylaminoethyl; [DEAE]) covalently linked to a support matrix (such as Sepharose) can be used to purify IgG in which the pH of the mobile-phase buffer is raised above the pI or IgG, thus allowing most of the antibodies to bind to the DEAE matrix. Compare this method with that described in Chapter 139 in which the IgG passes through the column. The procedure can be carried out using a laboratory-prepared column that is washed and eluted under gravity ( 1 ); however, high-performance liquid chromatography (HPLC) provides improved reproducibility (because the sophisticated pumps and accurate timers), speed (because of the small high-capacity columns), and increased resolution (because of the fine resins and control systems). These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.