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

Abstract

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.

Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography

“…In principle, HA can be integrated at all stages of a typical downstream process, from initial capture (due to the pseudo-affinity nature of the material) to the final polishing step, where the ability of HA to distinguish between multimers and monomers, as well as between native and denatured forms of the same molecule, while "bleeding" only biocompatible compounds and concentrating rather than diluting the substance zones, are considerable advantages. Direct antibody capture from cell culture supernatant benefits from the relative insensitivity of the antibody retention to the conductivity of the feed and may result in purities equal to those obtainable with Protein A or G [25,[56][57][58][59]. However, in the case of cell culture supernatants buffered by phosphate, quantitative binding may not be possible.…”

“…HA has also been used for the separation of ssDNA and ds-DNA and in 2010 was proposed for the efficient fractionation of dsDNA and ssDNA genomes of bacteriophages [73]. The largest scale pDNA isolation using HA, to date, was proposed by Giovannini and Freitag [59]. They described the transfer of the separation of pDNA (pEGFP-N1, 4.7 kb) from a clarified E. coli lysate on a 2 mL HA batch column to a 500 mL continuously operated annular column.…”

Performance and characterization of a nanophased porous hydroxyapatite for protein chromatography

“…Besides purification of nucleic acids (Bachvarov and Ivanov, 1983;Colman et al, 1978;Freitag, 2000, 2002;Johnson and Ilan, 1983;Kuiper et al, 2002;Pakroppa et al, 1975;Shoyab and Sen, 1979;Udvardy et al, 1979), a prime application of hydroxyapatite adsorbents is the purification of antibodies at small scale as well as industrial scale (Aoyama and Chiba, 1993;Bowles et al, 1988;Giovannini and Freitag, 2001;Jungbauer et al, 1989;Luellau et al, 1998;Pavlu et al, 1986;Poiesi et al, 1989;Zola and Neoh, 1989). Another standard adsorbent for large-scale separation of antibodies in the biopharmaceutical industry is staphylococcal Protein A affinity chromatography (Boschetti and Jungbauer, 2000;Jungbauer and Wenisch, 1989).…”

“…Ligand-leakage and the price of the adsorbents may sometimes be prohibitive for large-scale applications (Boschetti and Jungbauer, 2000). Thus, alternative process schemes consisting of ion-exchange chromatography, hydroxyapatite chromatography, and size exclusion chromatography have been developed to avoid staphylococcal Protein A affinity chromatography (Boschetti and Jungbauer, 2000;Giovannini and Freitag, 2001;Guerrier et al, 2001;Jungbauer et al, 1989).…”

PEG enhances viral clearance on ceramic hydroxyapatite

“…The original eluant used almost exclusively for hydroxyapatite was phosphate. However, sodium chloride as an eluant has also been used for some 50 years [10], with improvements noted for antibody purity [11,12] and aggregate removal [9,13]. In more recent years, a variety of other additives, such as poly(ethylene glycol) (PEG) [14], have been employed as elution modifiers.…”