High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the development of a new process for affinity purification of monoclonal antibodies (mAbs) from non‐clarified CHO cell broth using a pilot‐scale magnetic separator. The LOABeads had a maximum binding capacity of 65 mg/mL and an adsorption capacity of 25–42 mg IgG/mL bead in suspension for an IgG concentration of 1 to 8 g/L. Pilot‐scale separation was initially tested in a mAb capture step from 26 L clarified harvest. Small‐scale experiments showed that similar mAb adsorptions were obtained in cell broth containing 40 × 106 cells/mL as in clarified supernatant. Two pilot‐scale purification runs were then performed on non‐clarified cell broth from fed‐batch runs of 16 L, where a rapid mAb adsorption ≥96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single protein A capture step, the mAb purity was similar to the one obtained by column chromatography, while the host cell protein content was very low, <10 ppm. Our results showed that this magnetic bead mAb purification process, using a dedicated pilot‐scale separation device, was a highly efficient single step, which directly connected the culture to the downstream process without cell clarification. Purification of mAb directly from non‐clarified cell broth without cell separation can provide significant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2775, 2019.
Cell clarification represents a major challenge for the intensification through very high cell density in the production of biopharmaceuticals such as monoclonal antibodies (mAbs). The present report proposes a solution to this challenge in a streamlined process where cell clarification and mAb capture are performed in a single step using magnetic beads coupled with protein A. Capture of mAb from non‐clarified CHO cell suspension showed promising results; however, it has not been demonstrated that it can handle the challenge of very high cell density as observed in intensified fed‐batch cultures. The performances of magnetic bead‐based mAb capture on non‐clarified cell suspension from intensified fed‐batch culture were studied. Capture from a culture at density larger than 100 × 106 cells/ml provided an adsorption efficiency of 99% and an overall yield of 93% with a logarithmic host cell protein (HCP) clearance of ≈2–3 and a resulting HCP concentration ≤≈5 ppm. These results show that direct capture from very high cell density cell suspension is possible without prior processing. This technology, which brings significant benefits in terms of operational cost reduction and performance improvements such as low HCP, can be a powerful tool alleviating the challenge of process intensification.
The safety of the cells used for Advanced Therapy Medicinal Products is crucial for patients. Reliable methods for the cell purification are very important for the commercialization of those new therapies. With the large production scale envisioned for commercialization, the cell isolation methods need to be efficient, robust, operationally simple and generic while ensuring cell biological functionality and safety. In this study, we used high magnetized magnetic agarose-based beads conjugated with protein A to develop a new method for cell separation. A high separation efficiency of 91% yield and consistent isolation performances were demonstrated using population mixtures of human mesenchymal stem cells and HER2+ SKBR3 cells (80:20, 70:30 and 30:70). Additionally, high robustness against mechanical stress and minimal unspecific binding obtained with the protein A base conjugated magnetic beads were significant advantages in comparison with the same magnetic microparticles where the antibodies were covalently conjugated. This study provided insights on features of large high magnetized microparticles, which is promising for the large-scale application of cell purification.
The cover image is based on the Article Antibody capture process based on magnetic beads from very high cell density suspension by Nils A. Brechmann et al., https://doi.org/10.1002/bit.27776.
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