The [(bathophenanthroline)3:Fe2+] complex as an aromatic non-polymeric
medium for purification of human lactoferrin

We describe a non-chromatographic, ligand-free platform for the efficient purification of recombinant human lactoferrin. The platform consists of a [metal:chelator] complex precipitate in the presence of osmotically active polyethylene glycol 6000 . Purification is achieved in three stages. Following formation of the complex, LF is captured under neutral conditions by the aggregated complexes (Step I), a washing step follows (Step II) and then, (Step III) LF is extracted in pure form with 100 mM tribasic Na citrate buffer (pH 7). Of the four complexes investigated, [bathophenanthroline (batho)3:Fe2+] was determined to be the most efficient. LF is recovered with high yield (~90%) and purity (≥97%, by SDS polyacrylamide gel electrophoresis ) from an artificial contamination background comprising E. coli lysate proteins. Purified LF is demonstrated to be monomeric by dynamic light scattering ; to preserve its native secondary structure by circular dichroism spectroscopy; and, as apo-LF, to efficiently inhibit bacterial growth. Process yield is not
affected by a 45-fold increase in LF concentration from 0.2 to 9 mg/mL. We provide evidence that protein
capture relies on [cation:π] interactions between the lysine and arginine residues of LF with the fully aromatic
[(batho)3:Fe2+] complexes. The use of [metal:chelator] complex aggregates is demonstrated to provide an
economical and efficient avenue for LF purification.