Heparinization of anodized aluminum oxide (AAO) membranes for affinity separations of biocolloids.

Affinity membranes are developed by the immobilization of heparin onto anodized aluminum oxide (AAO) supports via amine grafting followed by amide coupling. Optimized procedures give heparinized AAO (HEP-AAO) membranes with a stable layer of 0.832-1.33 mg heparin per m2 pore area in 224 nm diameter pores. Following the heparinization of the membrane, the bioavailability of heparin is demonstrated through the capture and release of fibroblast growth factor 2 (FGF2), a protein for which heparin acts as a ligand. The capping of unreacted amines using citraconic anhydride reduces nonspecific protein binding without loss of heparin activity. The pore accessibility of colloidal particles is confirmed through the electrostatically-driven binding and release of 20 nm fluorescent amine-grafted silica nanoparticles, which are used as a model system to investigate the capture and release of biocolloids. Application of heparinized AAO membranes for the affinity capture and release of adeno associated virus 2 (AAV-2) is demonstrated in tangential flow filtration. Preliminary results for the affinity capture of AAV-2 pre-filtered culture medium capture significant AAV2 with negligible retention of host protein and DNA, paving a path for further optimization of viral vector purification.