Abstract
Protein A affinity chromatography is a central part of most commercial monoclonal antibody and Fc-fusion protein purification processes. In the last couple years an increasing number of new Protein A technologies have emerged. One of these new Protein A technologies consists of a novel, alkaline-tolerant, Protein A ligand coupled to a macroporous polymethacrylate base matrix that has been optimized for immunoglobulin (Ig) G capture. The resin is interesting from a technology perspective because the particle size and pore distribution of the base beads are reported to have been optimized for high IgG binding and fast mass transfer, while the Protein A ligand has been engineered for enhanced alkaline tolerance. This resin was subjected to a number of technical studies including evaluating dynamic and static binding capacities, alkaline stability, Protein A leachate propensity, impurity clearance, and pressure–flow behavior. The results demonstrated similar static binding capacities as those achieved with industry standard agarose Protein A resins, but marginally lower dynamic binding capacities. Removal of impurities from the process stream, particularly host cell proteins, was molecule dependent, but in most instances matched the performance of the agarose resins. This resin was stable in 0.1 M NaOH for at least 100 h with little loss in binding capacity, with Protein A ligand leakage levels comparable to values for the agarose resins. Pressure–flow experiments in lab-scale chromatography columns demonstrated minimal resin compression at typical manufacturing flow rates. Prediction of resin compression in manufacturing scale columns did not suggest any pressure limitations upon scale up. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1125–1136, 2014
Highlights
Affinity chromatography employing immobilized Protein A is a well-established technology that is used extensively for industrial scale monoclonal antibody and Fc-fusion protein purification.[1,2,3,4,5] Due to the strong binding interaction between the Fc moiety and Protein A, high purity is achieved in a relatively simple purification step
We have investigated the performance of Amsphere Protein A affinity resin, a recent addition to the growing repertoire of Protein A resins available for monoclonal antibodies (mAbs) and Fc-fusion protein purification, and compared its performance to industry leading agarose-based Protein A technology
Equilibrium binding capacities were comparable between Amsphere and the agarose resins (MabSelect and MabSelect SuRe) for all molecules tested
Summary
Affinity chromatography employing immobilized Protein A is a well-established technology that is used extensively for industrial scale monoclonal antibody and Fc-fusion protein purification.[1,2,3,4,5] Due to the strong binding interaction between the Fc moiety and Protein A, high purity is achieved in a relatively simple purification step. Clarified cell culture fluid containing the target protein of interest is applied to Protein A resin at near neutral pH, after which one or more wash steps are employed in order to reduce the levels of product and process related impurities such as host cell proteins (HCPs).[6] Product desorption from the resin is achieved by a reduction of the mobile phase pH. The resin is regenerated and subjected to a clean-in-place protocol. Due to the simple nature of this bind-and-elute operation, Protein A resins are very amenable for use in a platform process.[7,8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
