Depth filtration has been widely used during process scale clarification of cell culture supernatants for the removal of cells and cell debris. However, in addition to their filtration capabilities, depth filters also possess the ability to adsorb soluble species. This aspect of depth filtration has largely not been exploited in process scale separations and is usually ignored during cell culture harvest development. Here, we report on the ability of depth filters to adsorptively remove host cell protein contaminants from a recombinant monoclonal antibody process stream and characterize some of the underlying interactions behind the binding phenomenon. Following centrifugation, filtration through a depth filter prior to Protein A chromatographic capture was shown to significantly reduce the level of turbidity observed in the Protein A column eluate of the monoclonal antibody. The Protein A eluate turbidity was shown to be linked to host cell protein contaminant levels in the Protein A column load and not to the DNA content. Analogous to flowthrough chromatography in which residence time/bed height and column loading are key parameters, both the number of passes through the depth filter and the amount of centrifuge centrate loaded on the filter were seen to be important operational parameters governing the adsorptive removal of host cell protein contaminants. Adsorption of proteins to the depth filter was shown to be due to a combination of electrostatic and hydrophobic adsorptive interactions. These results demonstrate the ability to employ depth filtration as an integrative unit operation combining filtration for particulate removal with adsorptive binding for contaminant removal.

The Drosophila Sprouty (SPRY) protein has been shown to inhibit the actions of epidermal growth factor and fibroblast growth factor. However, the role of mammalian SPRY proteins has not been clearly elucidated. We postulated that human Sprouty2 (hSPRY2) is an inhibitor of cellular migration and proliferation. Indeed, using stably transfected HeLa cells, which expressed hemagglutinin (HA)-tagged hSPRY2 or hSPRY2 tagged at the C terminus with red fluorescent protein, we demonstrated that hSPRY2 inhibits the migration of cells in response to serum, epidermal growth factor, fibroblast growth factor, and platelet-derived growth factor. Additionally, hSPRY2 also inhibited the growth of HeLa cells in response to serum. Previously, two C-terminal domains on hSPRY2, which are necessary for its colocalization with microtubules (residues 123-177) or translocation to membrane ruffles (residues 178 -194), have been identified (Lim, J., Wong, E. S., Ong, S. H., Yusoff, P., Low, B. C., and Guy, G. R. (2000) J. Biol. Chem. 275, 32837-32845). Therefore, using TAT-tagged hSPRY2 and its mutants, we determined the role of these two Cterminal domains in the inhibition of cell migration and proliferation. Our data show that the deletion of either of these two regions in hSPRY2 abrogates its ability to modulate cell migration in response to different growth factors and proliferation in response to serum. Therefore, we conclude that hSPRY2 inhibits the actions of a number of growth factors, and its C terminus, which is homologous among various SPRY isoforms, is important in mediating its biological activity.In Drosophila, Sprouty has been demonstrated to be important in regulating fibroblast growth factor (FGF) 1 and epidermal growth factor (EGF)-mediated cellular actions. Thus, in Drosophila Sprouty mutants, the fibroblast growth factor pathway is overactive, and excessive branching is seen in Drosophila airways (1). Kramer et al. (2) have shown antagonistic actions of Sprouty on both the FGF and EGF signaling pathways in Drosophila, where loss of Sprouty results in supernumerary neurons and glia. In addition, overexpression of Sprouty in wing veins and ovarian follicle cells, tissues where EGF signaling is required for proper patterning, results in phenotypes that resemble the loss-of-function phenotypes of EGF receptors (3, 4). Sprouty has also been shown to interfere with chick embryo development. Hence, infection of the prospective wing territory with a retrovirus containing the Sprouty gene inhibits proper limb growth and formation in the embryonic chick (5). Taken together, these results suggest that Sprouty acts as an antagonist of both FGF and EGF receptor signaling pathways.To date, four isoforms of mammalian Sprouty protein have been described (SPRY1-SPRY4) (1, 4-6). Among these, only partial clones of SPRY1 and SPRY3 are available. The mouse as well as human SPRY2 (hSPRY2) and mouse SPRY4 have been cloned (4, 6, 7), and both these proteins share considerable sequence homology in the C terminus. However, their N termini are...

We report the use of caprylic acid based impurity precipitation as (1) an alternative method to polishing chromatography techniques commonly used for monoclonal antibody purification and (2) an impurity reduction step prior to harvesting the bioreactor. This impurity reduction method was tested with protein A purified antibodies and with cell culture fluid. First, the operational parameters influencing precipitation of host cell proteins and high molecular weight aggregate in protein A pools were investigated. When used as a polishing step, the primary factor affecting purification and yield was determined to be pH. Caprylic acid precipitation was comparable to polishing IEX chromatography in reducing host cell protein and aggregate levels. A virus reduction study showed complete clearance of a model retrovirus during caprylic acid precipitation of protein A purified antibody. Caprylic acid mediated impurity precipitation in cell culture showed that the impurity clearance was generally insensitive to pH and caprylic acid concentration whereas yield was a function of caprylic acid concentration. Protein A purification of caprylic acid precipitated cell culture fluid generated less turbid product pool with reduced levels of host cell proteins and high molecular weight aggregate. The results of this study show caprylic acid precipitation to be an effective purification method that can be incorporated into a production facility with minimal cost as it utilizes existing tanks and process flow. Eliminating flow through chromatography polishing step can provide process intensification by avoiding the process tank volume constraints for high titer processes.

Binding of epidermal growth factor (EGF) to its receptor (EGFR) augments