Continuous precipitation of IgG from CHO cell culture supernatant in a tubular reactor

Abstract

We successfully transferred a two-stage batch precipitation-based antibody capture step to continuous mode using continuous tubular reactors. The precipitation process solely employs a cheap mineral salt (CaCl2 ) and an organic solvent (ethanol) and could replace the costly protein A capture step in the purification of recombinant antibodies from cell culture supernatant. The time from startup untill attaining steady state conditions was reached in less than 15 minutes and both reactors were operated for several hours at steady state without manual intervention, delivering antibody at a constant yield and purity. An overall yield of > 90 percent, with a host cell protein reduction from 42 777 to 9000 ppm and a DNA reduction from 359 ppm to 7 ppm, could be achieved for the antibody investigated. The precipitated antibody can be dissolved at very high concentrations (> 40 g/L) in numerous buffer systems of various pH and high and low ionic strength, thereby rendering a subsequent concentration or buffer exchange step redundant. This system enables cell culture supernatants with low or high antibody titer to be processed with constant reactor size and without changing any parameters or increasing precipitant consumption. Aggregate levels were below 1% under all conditions tested. Purification by precipitation did not affect binding to CD16a or the isoform distribution of the antibody.

Microheterogeneity of therapeutic monoclonal antibodies is governed by changes in the surface charge of the protein

“…Correspondence: Prof. Alois Jungbauer, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria E-mail: alois.jungbauer@boku.ac.at Abbreviations: ADCC, antibody dependent cellular cytotoxicity; ApHGE, analytical ionexchange chromatography with pH gradient elution; CEX, Cation exchange chromatography; EGFR, epidermal growth factor receptor; mAb, monoclonal antibody; DSC, differential scanning calorimetry; SPR, surface plasmon resonance ants it is still difficult to decide, how precisely a product produced by different methods must match [14][15][16][17][18]. Cation exchange chromatography (CEX) with pH gradient elution has repeatedly been reported as a suitable method for high-resolution separation of antibody variants exhibiting different surface charge characteristics [19][20][21][22].…”

“…Either a batch process is directly converted into a continuous process or new unit operations are used in continuous manufacturing. Without knowing the biological significance of the different vari-ants it is still difficult to decide, how precisely a product produced by different methods must match [14][15][16][17][18].…”

Continuous desalting of refolded protein solution improves capturing in ion exchange chromatography: A seamless process

“…Not only FDA has addressed this topic [12,13], it also raised the interest of numerous researchers in both academia and industry [14,15]. Even it is not realized on a large production scale yet, various different strategies for continuous biomanufacturing have been developed in recent years [8,[16][17][18][19][20][21].…”

“…Not only FDA has addressed this topic [12,13], it also raised the interest of numerous researchers in both academia and industry [14,15]. Even it is not realized on a large production scale yet, various different strategies for continuous biomanufacturing have been developed in recent years [8,[16][17][18][19][20][21].In this work, we describe a novel alternative desalting process using microporous ion exchange resins, which are typically employed for water demineralization and are relatively inexpensive [22]. In the current work, this type of resin is referred to as "micropore resin" due to its small pore sizes of approximately 1-1.5 nm according to manufacturer information.…”

End-to-end integrated fully continuous production of recombinant monoclonal antibodies

“…2). Fully continuous downstream processing can be achieved by any combination of technologies capable of facilitating continuous flow such as SMB in its various modes of operation, membrane adsorbers, precipitation, etc (Hammerschmidt et al, 2015;Sá Gomes and Rodrigues, 2011;Warikoo et al, 2012;Xie et al, 2002). In this study, to develop an integrated and end-to-end continuous process for the production Fig.…”