Purification of Human IgG with Bathophenanthroline Zn²⁺, Fe²⁺ or Cu²⁺ Complexes

• DOI: 10.20944/preprints202503.1332.v1
• Publication Date: 2025-03-20T01:14:06Z
• Authors: Thisara Jayawickr...; Ron Alcalay; Olga Krichevsky; Ellen Wachtel; Ohad Mazor; Guy Patchornik

Pharmaceutical companies are aware of the ongoing effort to satisfy the increasing global demand for therapeutic grade monoclonal antibodies (mAbs), an especially difficult challenge for poor and developing countries. We present a simple, economical, single-step purification approach at neutral pH for polyclonal human IgG (hIgG) that does not require expensive ligands, chromatography columns, polymers, nor membranes. Instead, porous precipitates of commercial, recyclable aromatic [bathophenanthroline:cation] complexes were found to efficiently capture impurity proteins from CHO cells or E. coli lysate while maintaining the majority of the highly concentrated hIgG (5-15 mg/mL) in the supernatant. [(batho)3:Zn2+] complexes were the most promising, resulting in hIgG with purity ≈95%, by SDS-PAGE. This purified hIgG is monomeric (by dynamic light scattering, DLS) and preserves native secondary structure (by far UV circular dichroism spectroscopy, CD). Process yield is >90% (by densitometry) and is maintained after a 100-fold increase in the reaction volume that required only proportional increase in reagents. While Protein A chromatographic columns, the industry gold standard, have limited binding capacity, are costly and require familiarity with column maintenance, we are attempting, by our efforts, to help produce a more efficient, simple and economical purification platform.