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 int racellular enzymes, which could potentially cause proteolytic degradation o f the target protein and may increase the impurities for further chromatogr aphic steps. This especially includes the reduction of host DNA for therape utic proteins. The aim of this investigation was to examine the extent of c ell damage at the bench and pilot scale using a stabilized fluidized bed (e xpanded bed) for direct recovery of IgG from cell culture broth. For this p urpose, Streamline-25 and -200 columns containing 75 mt and 5 L of rProtein A matrix, respectively, were used. The repeated batch cultivations resulte d in high cell viabilities of about 90% prior purification. The pH was gent ly 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 assa ys: microscopic count of the cells using trypan blue dye exclusion to deter mine viability; monitoring of intracellular components (i.e., DNA concentra tion); activity of lactate dehydrogenase (LDH); and, finally, the particle load in the flow through and the eluate. Despite the sensitivity of hybrido ma 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. (C) 19 99 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 114-119, 1999.