Controlled mixed-mode interaction chromatography on membrane adsorbers

Membrane adsorbers (MAs) are used for protein separation in controlled mixed-mode interaction chromatography. The strong anion- and cation-exchange MAs used are made either from a synthetic copolymer or from modified cellulose membranes. The affinity MAs (Cibacron Blue) are also made from the copolymer membranes. Standard protein mixtures, whey proteins, and biotechnological culture supernatants are separated. The influence of the flow-rate, the ratio of cation- and anion-exchange MAs inserted in the stack, and the pattern, i.e. the comparative worth of an alternating vs. a two-consecutive-stacks arrangement, on the separation is investigated. While the flow-rate shows no influence, both the pattern of arrangement and the ratio of the two types of ion exchanger do. Compared to single-mode MA chromatography, a broader range of proteins, e.g. in terms of the isoelectric points, can be separated in a single chromatographic procedure, Whey proteins (beta-lactoglobulin, alpha-lactalbumin, BSA, IgG) are separated at pH 6, using a mixed-mode ion-exchange system. Here however, a two-stack approach is used to allow for module-uncoupling before elution, to prevent IgG and cy-lactalbumin from coeluting. By using a mix of anion-exchange and Cibacron Blue affinity MAs, recombinant human antithrombin III (rh-AT III) can be separated in a single run from the major protein impurities present in the fermenter supernatant, namely transferin and BSA.