431648 Antibody Purification Via an Affinity Membrane Chromatography Method Utilizing Nucleotide Binding Site Targeting Small Molecule

Monday, November 9, 2015: 3:35 PM
150D/E (Salt Palace Convention Center)
Nur Mustafaoglu1, Peter Deak1, Tanyel Kiziltepe2 and Basar Bilgicer1, (1)Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, IN, (2)Advanced Diagnostics and Therapeutics / Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, IN

Antibodies have extraordinary specificity and affinity to antigens, which in turn makes them important candidates to be used in numerous applications including detection, diagnosis, and therapy. Over last two decades, therapeutic antibodies have continued to be evaluated extensively for treatment of many diseases including cancer and autoimmune diseases. Even though antibody therapies are very efficacious for patients, monoclonal antibody-based treatments are expensive; therefore many patients cannot afford these treatments. A major contributor to the cost of antibody treatments are mostly linked to the downstream production process, or more specifically the usage of Protein A (or G) affinity columns for purification of antibodies. These columns are expensive and have short life cycles with several obstacles that preventing them to be used repeatedly. Here, we design a novel affinity membrane chromatography technique for purification of monoclonal and polyclonal antibodies from cell culture media and ascites fluids. This method utilizes the nucleotide-binding site (NBS) that is located on variable domain of Fab region nearly all antibodies to facilitate capturing antibodies on a membrane affinity column. Regenerated cellulose membranes has been choose as a matrix in this method due to their major advantages over traditionally used resin-based affinity systems. Rituximab was used for proof of concept experiments. A small molecule was attached to the membrane in order to target the NBS for capturing antibodies on the column. Antibody capture was accomplished by injection of samples while running equilibration buffer (50 mM sodium phosphate pH 7.0) and elution of the antibody was achieved by running a gradient of mild elution buffer (3M NaCl in 50 mM phosphate pH 7.0). The results indicate that the tryptamine column efficiency for selected antibodies was >98%, with a purity level of >98%. This small molecule affinity purification method is further tested for a number of necessary parameters such as: injection concentrations, volumes, wash/bind time, elution gradient, antibody/protein-contaminant combinations, effects of injection buffer, post-purification antigen binding activity of antibodies, and column reusability and stability. This method provides a superior alternative to the protein-A affinity purification method that is widely used for purification of humanized and chimeric antibodies. Results from these findings will help to increase the availability of antibody based treatment and diagnostic systems to patients.

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See more of this Session: Bioseparations and Downstream Processing
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division