Abstract
AbstractBispecific antibodies (bsAbs), though possessing great therapeutic potential, are extremely challenging to obtain at high purity within a limited number of scalable downstream processing steps. 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. 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. 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. 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%.Graphical Abstract
Funder
Cytiva Life Sciences
the Agency for Science, Technology and Research (A*STAR), Singapore.
Publisher
Springer Science and Business Media LLC
Subject
Renewable Energy, Sustainability and the Environment,Biomedical Engineering,Food Science,Biotechnology
Reference60 articles.
1. Allan B, Glasebrook AL, Leung DDM, Lu J, Tang Y, Vendel AC et al. (2014). Anti-tnf-anti-il-17 bispecific antibodies. WO2014137961 A1, 12 September 2014.
2. Andrade C, Arnold L, Motabar D, Aspelund M, Tang A, Hunter A et al (2019) An integrated approach to aggregate control for therapeutic bispecific antibodies using an improved three column mAb platform-like purification process. Biotechnol Prog 35(1):e2720
3. Baehner M, Imhof-Jung S, Kavlie A, Kettenberger H, Klein C, Regula JT et al. (2011). Bispecific, bivalent anti-vegf/anti-ang-2 antibodies. WO2011117329 A1, 29 September 2011.
4. Baeuerle PA, Reinhardt C (2009) Bispecific T-cell engaging antibodies for cancer therapy. Can Res 69(12):4941–4944
5. Bertl S, Duerr H, Schaubmar A. (2015). Separation of bispecific antibodies and bispecific antibody production side products using hydroxyapatite chromatography. WO2015024896 A1, 26 February 2015.
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