Estimation of cell damage in bench- and pilot-scale affinity expanded-bed chromatography for the purification of monoclonal antibodies.
The first step in downstream processing of mammalian cell culture includes the separation of the cells without cell damage to avoid the release of intracellular enzymes, which could potentially cause proteolytic degradation of the target protein and may increase the impurities for further chromatographic steps. This especially includes the reduction of host DNA for therapeutic proteins. The aim of this investigation was to examine the extent of cell damage at the bench and pilot scale using a stabilized fluidized bed (expanded bed) for direct recovery of IgG from cell culture broth. For this purpose, Streamline-25 and -200 columns containing 75 mL and 5 L of rProtein A matrix, respectively, were used. The repeated batch cultivations resulted in high cell viabilities of about 90% prior purification. The pH was gently adjusted to pH 8 before the broth was applied to the gel. In bench scale, 1 to 6 L of unclarified feed was applied to the Streamline-25 column. In pilot scale, up to 95 L was processed using the Streamline-200 column. The antibodies from 95 L of unclarified feed were recovered after approximately 1.5 h. The possible cell damage, caused either by the equipment or by the cells' passage through the expanded bed, was detected by the following assays: microscopic count of the cells using trypan blue dye exclusion to determine viability; monitoring of intracellular components (i.e., DNA concentration); activity of lactate dehydrogenase (LDH); and, finally, the particle load in the flow through and the eluate. Despite the sensitivity of hybridoma cells to shear forces, neither the high flow rate (300 to 450 cm/h) nor the passage of the cells through the expanded bed caused any relevant cell damage or clogging of the gel. Excellent DNA depletion was observed.
Lütkemeyer
D
D
Institute of Cell Culture Technology, Faculty of Technology, University of Bielefeld, D-33501 Bielefeld, Germany. dlu@zellkult.techfak.uni-bielefeld.de
Ameskamp
N
N
Tebbe
H
H
Wittler
J
J
Lehmann
J
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001709:Biotechnology; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000302:isolation & purification; D006825:Hybridomas / Q000166:cytology / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000302:isolation & purification; D008024:Ligands; D051379:Mice; D010865:Pilot Projects; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D008024:Ligands; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D004247:DNA