Exploitation of the Adsorptive Properties of Depth Filters for Host Cell Protein Removal during Monoclonal Antibody Purification

Abstract

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.

Recovery and purification process development for monoclonal antibody production

“…23 The removal of host cell proteins through depth filtration prior to a Protein A affinity chromatography column was shown to significantly reduce precipitation during the pH adjustment of the Protein A pool.…”

Development of adsorptive hybrid filters to enable two-step purification of biologics

“…In general, two distinct mechanisms are involved in impurity removal by depth filters, namely mechanical entrapment and adsorption via either electrostatic or hydrophobic interactions. [28][29][30][31] Diatomaceous earth-based depth filters have been demonstrated to remove impurities through hydrophobic interactions. 29 We hypothesized that the decrease of HMW1 levels in Zeta Plus 90ZB05A filtrate could be related to differences in hydrophobicity, as determined by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC).…”

“…27 Several groups have demonstrated the ability of adsorptive filters to lower HCP and DNA levels in a Protein A pool. [28][29][30][31] In a recent study, Iskra et al demonstrated increased robustness of xenotropic murine leukemia virus (xMuLV) clearance across a downstream anionexchange (AEX) chromatography step due to enhanced impurity removal from adsorptive filters after Protein A pool. 32 Adsorptive filters have also been shown to provide viral clearance capability by either electrostatic adsorption or a combination of adsorption and mechanical entrapment.…”

PDADMAC flocculation of Chinese hamster ovary cells: Enabling a centrifuge-less harvest process for monoclonal antibodies

“…Protein A purification Clarified harvested cell culture fluid was purified using the Protein A chromatography method described by Yigzaw et al 36 with the following modifications: 1) the Protein A column loading was between 25 and 35 mg per mL of resin, and 2) the Protein A eluate was not neutralized prior to product quality determination, since the mAb product quality attributes were stable in the Protein A eluate.…”

“…Protein A eluate was analyzed for size variants as measured by SEC-HPLC, 37 HCP as determined by ELISA, 36 and chargedbased variants as measured by CEX-HPLC. 38 Host cell DNA was determined by quantitative polymerase chain reaction as described below.…”