Calcium-dependent magnetic separation: A novel approach for the integrated processing of high-quality mAbs

Current monoclonal antibody (mAb) biomanufacturing typically involves expression in mammalian cells during upstream processing (USP), followed by purification during downstream processing (DSP). DSP is a major bottleneck, primarily caused by the prevalent Protein A affinity chromatography capture step. Key drawbacks include the extensive clarification required to prevent clogging of the chromatography column, limited productivity due to diffusional mass transport, and the risk of mAb denaturation under acidic elution conditions. To address these challenges, we introduce the novel concept of calcium-dependent magnetic separation using iron oxide nanoparticles with covalent immobilization of an engineered calcium-dependent affinity ligand (Z<inf>Ca</inf>). The Z<inf>Ca</inf> ligand is employed for mild elution conditions, while the dispersed particle adsorbent promises fast mAb interaction and processing of non-clarified feeds. Using Trastuzumab as a model mAb, we confirmed the intended rapid capture directly from Chinese hamster ovary (CHO) culture and achieved high mAb purities, with reductions in DNA and host cell protein (HCP) comparable to state-of-the-art Protein A chromatography. The demonstrated potential of integrating the clarification and the capture steps is promising for improving productivity and simultaneously reducing costs. Additionally, we developed a mild and sustainable elution protocol based on citrate/NaCl buffers, reaching recoveries above 90% at pH 6.0. The functionality of Trastuzumab after the novel separation approach was proven by high physical integrity and binding functionality to the human epidermal growth factor receptor 2 (HER2). Our findings underline the high potential of calcium-dependent magnetic separation for integrated, gentle, and sustainable bioseparation of mAbs.