We have recently described a non-chromatographic, ligand-free approach for antibody (Ab) purification based on specially designed: [Tween-20:bathophenanthroline:Fe2+] aggregates. To assess the potential generality of this approach, a variety of detergents belonging to four nonionic detergent families (Tween, Brij, Triton and Pluronic) have now been studied. All surfactant aggregates lead to high purity of the recovered Abs (>95%, by gel densitometry). Good overall Ab recovery yields were observed with: Tween-20 (80-83%), Brij-O20 (85-87%) and Triton X-100 (87-90%), while Pluronic F-127 was less efficient (42-53%). Of additional importance is the finding that the process can depend on filtration (rather than centrifugation), thereby allowing a continuous purification mode that leads to the recovery of monomeric IgG’s (by DLS) and preservation of Ab specificity (by ELISA). The amphiphilic chelator, bathophenanthroline (batho) is recycled almost quantitatively (95%) by crystallization. Good IgG recovery yields (~80%) are also observed when Ab concentrations are increased from 1 mg/ml to 3-5 mg/mL. Potential advantages of the purification platform for industrial downstream processing of therapeutic monoclonal antibodies (mAbs), are discussed.

Industrial scale production of therapeutic monoclonal antibodies (mAbs) is commonly achieved with Protein A chromatography, a process that requires exposure of the antibody to strongly acidic conditions during the eluting step. Exposure to acid inactivates virus contaminants but may, in parallel, lead to antibody aggregation that must be eliminated or kept at acceptably low levels. This report seeks to provide a practical method for overcoming a long-standing problem. We show how Brij-O20 detergent micelles, conjugated by the amphiphilic [(bathophenanthroline)3:Fe2+] complex in the presence of amino acid monomers: phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), isoleucine (Ile) or valine (Val), efficiently capture polyclonal human IgG (hIgG) at neutral pH and allow its recovery by extraction either at pH 4 (85-97% yield) or at pH 6.3 (72-84% yield). Of the five amino acid monomers surveyed, Phe or Tyr produced the highest overall process yield at both pH 4 and 6.3. The monomeric state of the purified hIgG’s was confirmed by dynamic light scattering (DLS). Potential advantages of the purification method are discussed.