![]() ![]() Development of effective strategies for their downstream processing is therefore important to keep in step with these breakthroughs in upstream development, preventing any potential bottleneck in their subsequent industrial manufacturing process as conventional downstream processing methods may be ineffective at the removal of specific product and process-related impurities associated with this important class of biotherapeutics. This is exemplified by the four FDA approved bsAbs currently in the market, namely blinatumomab, emicizumab, amivantamab and faricimab, along with a wide variety of reported bsAb formats and large numbers in clinical development (Kontermann 2005 Baeuerle and Reinhardt 2009 Chames and Baty, 2009 Kontermann 2012 Brinkmann and Kontermann 2017 Labrijn et al. Graphical Abstractīispecific antibodies (bsAbs) represent a particularly promising class of biotherapeutics due to their ability to bind to two different antigens, opening up a wide range of potential therapeutic applications (Kontermann 2005 Baeuerle and Reinhardt 2009 Chames and Baty, 2009 Kontermann 2012 Brinkmann and Kontermann 2017 Labrijn et al. Overall, we demonstrate that through the employment of (1) Protein A chromatography step and (2) flow-through polishing steps, a final product containing < 1% HMW species, < 1% LMW species and < 100 ppm HCP can be obtained with an overall process recovery of 56–87%. This complete flow-through polishing strategy, guided by Design of Experiments (DoE), eliminates undesirable aggregation problems associated with the higher aggregation propensity of scFv containing bsAbs that may occur in the bind and elute mode, offering an improved ease of overall process operation without additional elution buffer preparation and consumption, thus aligning well with process intensification efforts. Here, we demonstrate using two knob-into-hole (KiH) bsAb constructs that two flow-through polishing steps utilising Capto Butyl ImpRes and Capto adhere resins, performed after an optimal Protein A affinity chromatography step can further reduce the HCP by 17- to 35-fold as well as HMW and LMW species with respect to monomer by ~ 4–6% and ~ 1%, respectively, to meet therapeutical requirement at 30–60 mg/mL-resin (R) load. Complementary to Protein A chromatography, polishing strategies play a critical role at removing the remaining high molecular weight (HMW) and low molecular weight (LMW) species, as well as host cell proteins (HCP) in order to achieve a final product of high purity. Capto Blue medium's excellent chemical stability also ensures tolerance for sodium hydroxide, enabling repeated cleaning-in-place (CIP) procedures to remove tightly bound impurities and maintain capacity.Ĭapto Blue medium, an excellent choice for the removal or purification of proteins at both laboratory and process scales, is available as an easy-to use prepacked HiScreen column for fast small-scale purification and process development, as well as in laboratory and process bulk pack sizes.Bispecific antibodies (bsAbs), though possessing great therapeutic potential, are extremely challenging to obtain at high purity within a limited number of scalable downstream processing steps. These improvements allow faster flow rates and larger sample volumes, leading to higher throughput and improved process economy.ĭepending on the target molecule, the ligand functionality of Capto Blue may be modified through the use of appropriate buffer salts and buffer conductivity to increase selectivity for desired targets. Developed from Blue Sepharose 6 Fast Flow, Capto Blue medium is more chemically stable and has a more rigid agarose base matrix than its predecessor. GE Healthcare's Capto Blue affinity chromatography medium is for the capture of human serum albumin (HSA), as well as purification of HSA fusion proteins, blood coagulation factors, enzymes, and recombinant proteins at laboratory and process scales. ![]()
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