Arick Brown

Application of high‐performance tangential flow filtration (HPTFF) to the purification of a human pharmaceutical antibody fragment expressed in Escherichia coli

High‐performance tangential flow filtration (HPTFF) is shown to successfully enable concentration, purification and formulation in a single unit operation. This is illustrated with feedstreams comprising recombinant proteins expressed in Escherichia coli (E. coli). Using positively charged cellulosic membranes of 100 kDa molecular weight cut‐off and operating under a selected range of buffer pH and ionic strength at a filtrate flux of 100 L m−2 h−1, a 10‐fold removal of E. coli host cell proteins (HCP) was obtained with an overall process yield of 98%. The HPTFF performance was shown to be robust and reproducible. In addition, the novel charged membrane was regenerated and re‐used seven times without loss of selectivity or throughput. When compared with a conventional purification scheme, the proposed process results in the elimination of one chromatographic step, a 12% yield improvement and a significant reduction in purification cost of goods. Biotechnol. Bioeng. 2008;100: 964–974.

Overloading ion-exchange membranes as a purification step for monoclonal antibodies.

The present study examined the overloading of ion‐exchange membrane adsorbers, a form of frontal chromatography, as the final purification step in the production of mAbs (monoclonal antibodies) produced from CHO (Chinese‐hamster ovary) cells. Preferential binding of impurities over antibody product was exploited using commercially available cation‐ and anion‐exchange membranes. Three different antibody feedstreams previously purified over Protein A and ion‐exchange column chromatography were tested. Feedstream conductivity and pH were adjusted to induce product and impurity adsorption. Membranes were then overloaded in a normal flow mode, resulting in retention of impurities and breakthrough of purified antibody. Although some amount of the product also binds to the membranes (usually ≤30 g mAb/l membrane), yields of ≥99% were achieved by marginalizing the losses, typically by loading more than 3 kg mAb/l membrane. Analyses of the purified pools show consistent removal of impurities despite strong mAb–ligand interactions and high membrane loadings. The clearance of host cell proteins was affected by pH and conductivity, but was unaffected by flow rate, membrane properties or scale. The importance of the present study lies in our demonstration of an alternative use of ion‐exchange membranes for fast, effective and high yielding purification of mAbs.

Increasing parvovirus filter throughput of monoclonal antibodies using ion exchange membrane adsorptive pre-filtration

Pre‐filtration using ion exchange membrane adsorbers can improve parvovirus filter throughput of monoclonal antibodies (mAbs). The membranes work by binding trace foulants, and although some antibody product also binds, yields ≥99% are easily achieved by overloading. Results show that foulant adsorption is dependent on pH and conductivity, but independent of scale and adsorber brand. The ability to use ion exchange membranes as pre‐filters is significant because it provides a clean, well defined, chemically stable option for enhancing throughput. Additionally, ion exchange membranes facilitate characterization of parvovirus filter foulants. Examination of adsorber elution samples using sedimentation velocity analysis and SEC‐MALS/QELS revealed the presence of high molecular weight species ranging from 8 to 13 nm in hydrodynamic radius, which are similar in size to parvoviruses and thus would be expected to plug the pores of a parvovirus filter. A study of two identical membranes in‐series supports the hypothesis that the foulants are soluble, trace level aggregates in the feed. This studys significance lies in a previously undiscovered application of membrane chromatography, leading to a more cost effective and robust approach to parvovirus filtration for the production of monoclonal antibodies. Biotechnol. Bioeng. 2010;106: 627–637.