Impact of Cell Density and Viability on Primary Clarification of Mammalian Cell Broth An Analysis Using Disc-Stack Centrifugation and Charged Depth Filtration

LEVEL: INTERMEDIATE P roduction of monoclonal antibodies from mammalian cell culture has become ubiquitous in the biotechnology industry as companies continue to identify opportunities to treat diseases with such therapeutic proteins. The first step in recovery of secreted antibodies is to remove most insoluble components of the cell culture from the product stream. These components consist of whole cells, cell debris, colloids, and other such impurities. One industrypreferred method for accomplishing this initial separation is to use continuous disc-stack centrifugation coupled with depth filtration. These primary recovery steps are intended to remove most particulates from cell broth to ease the burden on the subsequent purification steps. A disc-stack centrifuge can remove whole cells and larger cell debris from a cell culture using stacked, inclined conical discs to separate the solids (1– 3). General centrifuge separation theory with computational f luid dynamics (CFD) has been described elsewhere (4, 5). For disc-stack centrifugation, cell culture broth is fed into a rotating bowl, and centrifugal force causes solids to separate in a narrow channel between the discs. Those separated solids slide down the underside of the discs into a solidsholding space, from which they can be discharged regularly. Clarified liquid containing the protein of interest continues up the disc stack and out of the bowl. This technique works extremely well for removing whole mammalian cells, provided that centrifuge conditions cause no shearinduced damage to those cells (4). Cell shearing will increase the amount of submicron particles that cannot be removed by the centrifuge. The minimum particle size that a continuous disc-stack centrifuge can remove is a function of cell culture properties, centrifuge feed rate, and bowl geometry and rotational speed. The rotational speed and geometry are taken into account by the Sigma factor (Σ) for the centrifuge. This factor denotes the area of a gravity-settling tank needed to achieve the same amount of clarification as the centrifuge. For a disc-stack centrifuge, Equation 1 gives this equivalent clarification area (2). The ratio of the feed rate to the centrifuge (Q ) and the equivalent clarification area (Σ) gives the equivalent settling velocity that can be achieved for a given set of operating conditions. Combining this Q/Σ ratio with Stoke’s Law for a gravity-settled spherical particle (assuming a Newtonian f luid and low particle density) allows the minimum particle Disc-stack centrifuge used in biotech and pharmaceutical applications.