Overloading ion‐exchange membranes as a purification step for monoclonal antibodies

Abstract

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.

Sample displacement chromatography of plasmid DNA isoforms

“…For successful SDC on particulate chromatographic supports, flow rates must be five times lower to achieve optimal sample displacement compared to standard gradient elution separation [14]. On the contrary, flow rate-independent SDC for purification of monoclonal antibodies has been demonstrated with convective media [15]. Since SD effects on monoliths are independent of column size [10], they can be used for SDC of biomacromolecules at microanalytical, analytical and preparative scales.…”

Selectivity of monolithic supports under overloading conditions and their use for separation of human plasma and isolation of low abundance proteins

“…On the other hand, BaSO 4 precipitate of plasma of Atlantic salmon was a relative complex protein mixture that was used as a starting material for isolation of highly pure and biologically active thrombin by use of SDS as a rapid, simple and cost-effective method [21]. Brown et al [28] recently used ion-exchange membrane chromatography under overloading conditions for purification of monoclonal antibodies. The overloading of membranes results in binding of impurities, and breakthrough of purified antibody.…”

Sample displacement chromatography as a method for purification of proteins and peptides from complex mixtures

“…The use of membranes and monoliths for SDC can be very favorable because the displacement phenomena by sample components with different affinities for the stationary phase occurs at much lower loading than if bulk, porous chromatographic supports are employed [44, 45, 51]. A similar phenomenon is also observed when non-porous bulk supports are used [51, 52].…”

“…After optimization of sample load and application buffers (starting pH value and salt concentration), highly pure ITIp concentrate was isolated from a side-fraction that is produced during clotting factor IX (FIX) preparation. The industrial-scale use of membranes for SDC in ion-exchange mode for the purification of antibodies was demonstrated by Brown et al [45]. …”

“…SDC was first introduced for preparative purification of peptides in reversed-phase mode [41, 42]. Later, it was demonstrated that this kind of chromatography can also be performed in ion-exchange [43, 44], affinity [45] and hydrophobic-interaction modes [46]. We also demonstrated that SDC can be performed on small analytical columns for the effective separation of microgram amounts of proteins from human plasma and can be applied as a sample preparation step for subsequent MS analysis of separated proteins [44,46].…”