Isolation of a recombinant antibody from cell culture supernatant: Continuous annular versus batch and expanded-bed chromatography

Annular chromatography represents a crossflow approach to chromatographic s eparations, that allows the continuous separation of multicomponent mixture s. The potential of the method for continuous bioseparation has been discus sed for some time, however, we demonstrate for the first time the processin g of a complex feed (cell culture supernatant) taken from an actual (bio)pr ocess. Moreover, while previously published applications of annular chromat ography concentrated on noninteractive (gel filtration) or nonspecific (ion exchange) chromatography, we show the possibility of continuous annular af finity chromatography. In particular, a commercially available preparative continuous annular chromatography (P-CAC) system was used to purify a recom binant antibody (human IgG(1)-kappa) from CHO cell culture supernatants by (pseudo)affinity chromatography on-hydroxyapatite (HA) and rProtein A. Meth ods developed using small (2 mL) batch columns could be directly transferre d to the P-CAC, where they yielded similar results in terms of final produc t quality. Yields were between 87% and 92% in the case of HA and between 77 % and 82% in the case of rProtein A chromatography. DNA removal was nearly quantitative in all cases. Concomitantly, the antibody fraction of the tota l protein content was raised by one order of magnitude in HA and by a facto r of 50 by rProtein A chromatography. In addition, a novel HA material (par ticle diameter -120 mum) was investigated, which was compatible with expand ed-bed applications. However, the final purity of the antibody thus obtaine d and also the yields (<70%) were less than satisfactory. (C) 2001 John Wil ey & Sons, Inc.