GE Healthcare Performance of different Protein A resins with respect to critical attributes Overview Protein A affinity chromatography is the preferred technique for purification of monoclonal antibodies as it enables a platform approach to downstream processing. Currently there are several Protein A resins available on the market. These resins vary in their different properties, including material of base matrix, particle and pore size, and type of Protein A ligand. As a result, their chromatographic performance, such as binding capacities, elution pH, purity of eluted antibody and resin life time, will vary. In this study, we compared five commercially available Protein A resins (MabSelect SuRe™ and MabSelect SuRe LX (both GE Healthcare), TOYOPEARL™ AF–rProtein A-650F (Tosoh Bioscience), ProSep™ Ultra Plus (EMD-Millipore) and AbSolute™ (NovaSep) by looking at typical performance attributes such as dynamic binding capacity (DBC) at different residence times and resin life time. Conclusions • Almost equivalent dynamic binding capacities are obtained for all resins at 2.4 min residence time • At higher residence time, MabSelect SuRe LX shows dynamic binding capacities that are between 15% and 30% higher than other Protein A resins • MabSelect SuRe and MabSelect Sure LX are the most stable Protein A resins maintaining more than 90% of the original binding capacity after 300 cycles with the recommended CIP protocol • MabSelect SuRe and MabSelect SuRe LX are stable for 300 cycles and TOYOPEARL AF–rProtein A-650F for 100 cycles using CIP with 0.1M NaOH, while AbSolute lost 50% of initial DBC after 20 cycles when using 0.05 M NaOH • At the vendor recommended CIP conditions, the following ranking of resin life time was determined (high to low): MabSelect SuRe LX ~ MabSelect SuRe > TOYOPEARL AF–rProtein A-650F >> Prosep Ultra Plus > AbSolute Dynamic binding capacity studies Dynamic binding capacities (DBC) at different levels of breakthrough were determined from frontal analysis. All resins were packed in Tricorn™ 5/100 columns (~1.96 mL). Human IgG (Octapharma) dissolved in PBS buffer pH 7.4 at 1g/l was applied on the columns at different flow rates yielding residence times of: 2.4, 4, 6 and 10 min. All experiments were performed using ÄKTAexplorer™ 10 system. • Dynamic binding capacities, DBC10%, for MabSelect SuRe LX at residence times longer than 2.4 min are between 5% to 50% higher as compared to binding capacities for the other Protein A resins. •A t 2.4 min residence time, DBC10% for MabSelect SuRe LX is 11% lower than DBC10% for ProSep Ultra Plus, and still higher than DBC10% for the other resins. • Total binding capacity (estimated by DBC80%) is the highest for MabSelect SuRe LX (~80 g/L), which is almost 60% higher than for non-agarose based resins. Data obtained in studies performed at GE Healthcare laboratories Lifetime studies Buffer cycling Two types of studies were performed: • Less than 10% change in DBC10% after 300 cycles for MabSelect SuRe and MabSelect SuRe LX at 100 mM NaOH i) Stability data: repeated cycling where each cycle consisted of: equilibration step with PBS-buffer pH 7.4; elution step with 0.1 M citrate pH 3; and, CIP step performed according to supplier data files (contact time 15 min/cycle) and re-equilibration step with PBS-buffer. ii) CHO cell supernatant experiments: one hundred purification cycles, each based on loading of MAb containing feedstock up to 80% of DBC10% measured at 4 min residence time; equilibration, wash and re-equilibration steps with PBS-buffer pH 7.4; elution step with 0.1 M citrate pH 3; and, CIP step performed according to supplier data files (contact time 15 min/cycle). In both cases, changes in resin performance were monitored by measuring the dynamic binding capacity at 6 and 4 min residence time, respectively, every 10th cycle. • More than 20% decrease in DBC10% for TOYOPEARL AF–rProtein A-650F after 300 cycles • More than 50% decrease in DBC10% for AbSolute after 21 cycles at 50 mM NaOH • After 100 CIP cycles with 0.5 M NaOH, MabSelect SuRe resins maintain 80% of initial capacity while capacity of the TOYOPEARL AF–rProtein A-650F decreases by 40% CHO cell supernatant: • Less than 10% decrease in DBC10% breakthrough after 100 cycles for MabSelect SuRe LX and no change in DBC10% for MabSelect SuRe • Around 50% decrease in DBC10% for ProSep Ultra Plus after 100 cycles using the recommended CIP protocol based on 0.15 M Phosphoric acid and 6 M urea For local office contact information, visit: www.gelifesciences.com/contact www.gelifesciences.com/mabs GE Healthcare Bio-Sciences AB Björkgatan 30 751 84 Uppsala Sweden GE and GE Monogram are trademarks of General Electric Company. MabSelect SuRe , ÄKTA and Tricorn are trademarks of GE Healthcare Companies.TOYOPEARL is a trademark of Tosoh Corporation. ProSep is a trademark of Merck KGaA. AbSolute is a trademark of Groupe Novasep SAS. All other third party trademarks are the property of their respective owners. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. © 2012-2014 General Electric Company - All rights reserved. First published Mar. 2012. 29-1148-22 AA 07/14