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 separations, that allows the continuous separation of multicomponent mixtures. The potential of the method for continuous bioseparation has been discussed for some time, however, we demonstrate for the first time the processing of a complex feed (cell culture supernatant) taken from an actual (bio)process. Moreover, while previously published applications of annular chromatography concentrated on noninteractive (gel filtration) or nonspecific (ion exchange) chromatography, we show the possibility of continuous annular affinity chromatography. In particular, a commercially available preparative continuous annular chromatography (P-CAC) system was used to purify a recombinant antibody (human IgG(1)-kappa) from CHO cell culture supernatants by (pseudo)affinity chromatography on hydroxyapatite (HA) and rProtein A. Methods developed using small (2 mL) batch columns could be directly transferred to the P-CAC, where they yielded similar results in terms of final product 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 total protein content was raised by one order of magnitude in HA and by a factor of 50 by rProtein A chromatography. In addition, a novel HA material (particle diameter -120 microm) was investigated, which was compatible with expanded-bed applications. However, the final purity of the antibody thus obtained and also the yields (<70%) were less than satisfactory.
Giovannini
R
R
Center of Biotechnology, Department of Chemistry, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland.
Freitag
R
R
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D016466:CHO Cells; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D006224:Cricetinae; D017886:Durapatite / Q000737:chemistry; D011994:Recombinant Proteins / Q000302:isolation & purification*; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000378:metabolism
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D017886:Durapatite