| 1. |
- Brechmann, Nils A., et al.
(författare)
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Antibody capture process based on magnetic beads from very high cell density suspension
- 2021
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Ingår i: Biotechnology and Bioengineering. - : John Wiley and Sons Inc. - 0006-3592 .- 1097-0290. ; 118:9, s. 3499-3510
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Brechmann, Nils A.
(författare)
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Magnetic bead-based isolation of biological therapeutic modalities
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biopharmaceutical modalities, such as monoclonal antibodies or the less established cell therapies, are nowadays very important for the treatment of severe or incurable diseases. The manufacturing of such modalities is complex and costly, including the downstream processing, which is highly essential to ensure the safety and quality of the product.Currently, monoclonal antibody downstream processes are heavily based on column chromatography, such as Protein A affinity capture, and highly depended on clarified liquid. This leads to a step intensive process, which is not only costly but also generates significant reduction of yield for every additional step. The cell clarification, in particular, for high cell density cultures can be insufficient and result in clogging of the following step due to remaining particles in the liquid. Alternatively, the clarification can lead to a higher contamination of product variants and process related impurities, such as antibody aggregations and Host Cell Proteins (HCPs). On the other hand, for large scale commercialization of allogenic cell therapy approaches based on human induced pluripotent stem cell (hiPSC) cell lines, efficient and reliable methods to ensure safety and quality of the cell product are needed. The presence of undifferentiated cells in a cell product derived from hiPSCs represent a risk of tumour and teratoma formation in the patient. The removal of undifferentiated cells in the cell therapy product is critical, and reliable and scalable methods are needed to support off-the-shelf production.The work in this thesis aimed to develop an alternative downstream operational step based on magnetic beads linked with Protein A or Protein G and a magnetic separator system suitable for the purification of monoclonal antibodies or cell therapy products. Efforts were made to develop an efficient monoclonal antibody capture step, based on magnetic bead separation, directly applied on the harvest of monoclonal antibodies producing Chinese Hamster Ovary (CHO) cell cultures at different cell densities up to very high cell density (> 100 x 106 cells/mL) and scales ranging from small-scale to pilot-scale (up to 16 L). The system proved to be highly gentle towards the cell, minimizing aggregation and the release of HCPs (< 10 ppm) already complying with the regulatory constraint after only one downstream operational step. Furthermore, the magnetic bead-based separation was applied for the negative isolation of cell subpopulations based on unique surface marker expression. Here a flexible isolation system was developed based on Protein A or based on Protein G magnetic beads providing high variability towards the surface receptor recognizing antibody. The magnetic beads were substantially larger compared to a cell resulting in a binding process where a bead is being covered by several cells. The system was evaluated towards different surface receptors, i.e. HER2, TRA2-49 and SSEA-4. The magnetic beads showed to be non-toxic towards the delicate human mesenchymal stem cells and iPSCs. The system also provided excellent negative selection of HER2+ SKBR3 cells, taken as model, and TRA2- 49+/SSEA-4+ iPSCs from different heterogenous model cell populations.In conclusion, the present downstream strategies based on magnetic bead separation for the capture of monoclonal antibodies or for the negative selection of cell subpopulations showed great alternatives to resolve the challenges provided by intensified cultures in mAb manufacturing, and could provide a viable solution for cell therapy.
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| 4. |
- Brechmann, Nils A., et al.
(författare)
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Proof-of-Concept of a Novel Cell Separation Technology Using Magnetic Agarose-Based Beads
- 2022
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Ingår i: MAGNETOCHEMISTRY. - : MDPI AG. - 2312-7481. ; 8:3, s. 34-
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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