| 1. |
- Malm, Magdalena, 1983-, et al.
(författare)
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Evolution from adherent to suspension: systems biology of HEK293 cell line development
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.
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| 2. |
- Saghaleyni, Rasool, 1987, et al.
(författare)
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Enhanced metabolism and negative regulation of ER stress support higher erythropoietin production in HEK293 cells
- 2022
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 39:11
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant protein production can cause severe stress on cellular metabolism, resulting in limited titer and product quality. To investigate cellular and metabolic characteristics associated with these limitations, we compare HEK293 clones producing either erythropoietin (EPO) (secretory) or GFP (non-secretory) protein at different rates. Transcriptomic and functional analyses indicate significantly higher metabolism and oxidative phosphorylation in EPO producers compared with parental and GFP cells. In addition, ribosomal genes exhibit specific expression patterns depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased EPO secretion, we detect higher gene expression related to negative regulation of endoplasmic reticulum (ER) stress, including upregulation of ATF6B, which aids EPO production in a subset of clones by overexpression or small interfering RNA (siRNA) knockdown. Our results offer potential target pathways and genes for further development of the secretory power in mammalian cell factories.
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| 3. |
- Bastin, G., et al.
(författare)
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Metabolic flux analysis of VERO cells under various culture conditions
- 2021
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Ingår i: Processes. - : MDPI AG. - 2227-9717. ; 9:12
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Tidskriftsartikel (refereegranskat)abstract
- Although the culture of VERO cells in bioreactors is an important industrial bioprocess for the production of viruses and vaccines, surprisingly few reports on the analysis of the flux distribution in the cell metabolism have been published. In this study, an attempt is made to fill this gap by providing an analysis of relatively simple metabolic networks, which are constructed to describe the cell behavior in different culture conditions, e.g., the exponential growth phase (availability of glucose and glutamine), cell growth without glutamine, and cell growth without glucose and glutamine. The metabolic networks are kept as simple as possible in order to avoid underdeterminacy linked to the lack of extracellular measurements, and a unique flux distribution is computed in each case based on a mild assumption that the macromolecular composition of the cell is known. The result of this computation provides some insight into the metabolic changes triggered by the culture conditions, which could support the design of feedback control strategies in fed batch or perfusion bioreactors where the lactate concentration is measured online and regulated by controlling the delivery rates of glucose and, possibly, of some essential amino acids.
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| 4. |
- 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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| 5. |
- 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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| 6. |
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| 7. |
- Brechmann, Nils Arnold, et al.
(författare)
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Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture
- 2019
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Ingår i: Biotechnology progress (Print). - : AIChE. - 8756-7938 .- 1520-6033.
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Tidskriftsartikel (refereegranskat)abstract
- High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the developmentof a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarifiedCHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum bindingcapacity of 65 mg/mL and an adsorption capacity of 25–42 mg IgG/mL bead in suspension for anIgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture stepfrom 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions wereobtained in cell broth containing 40 Å~ 106 cells/mL as in clarified supernatant. Two pilot-scalepurification 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 proteinA capture step, the mAb purity was similar to the one obtained by column chromatography, whilethe host cell protein content was very low, <10 ppm. Our results showed that this magnetic beadmAb purification process, using a dedicated pilot-scale separation device, was a highly efficientsingle 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 providesignificant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step.
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| 8. |
- 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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| 9. |
- Chotteau, Veronique, 1963-, et al.
(författare)
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Comparison of cultivation in Techne spinner, Bellco spinner, shake flask and T-flask of human embryonic stem cells
- 2010
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Ingår i: Proceedings of the SBE's Second International Conference on Stem Cell Engineering.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The recent progress in regenerative medicine indicates that pluripotent human embryonic stem cells (hESCs) may hold great potential providing cellular models for drug development and screening, modelling diseases as well as aid in the development of future cell-based therapies for neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease. Crucial to the success of generating specialized cell populations, is an understanding of the mechanisms, which influence the control of cell growth and differentiation by extrinsic and intrinsic factors. Nowadays, a limitation for the use of hESCs is the lack of proliferation methods in large scale. The purpose of the present work was to study several cultivation systems, which could potentially provide large-scale cultivation processes suitable for human therapy applications. Pluripotent human embryonic stem cells (hESCs), isolated from the inner cell mass of the blastocyst, were cultivated undifferentiated as embryoids bodies, i.e. large spherical aggregates of cells, in absence of serum and feeder layer. The cell growth and culture behavior in T-falsk, orbitally agitated shake flask, Bellco stirred spinner and Techne stirred spinner were observed. In Bellco spinner, the cells were agitated by a rotating impeller providing a movement comparable to stirred bioreactors. In Techne spinner, a slow and gentle orbital movement provided by a rotating bulb-ended stirrer maintained the cells in suspension. The design of this latter spinner allowed lower shear stress in comparison to Bellco spinner and shake flask. It was observed that the cell growth was fastest in Techne spinner followed by cultivation in T-flask and then cultivation in shake flask. Cultivating in Bellco spinner resulted in embryoid dissociation and viability decrease after 14 days. A larger number of single cells, i.e. cells not growing in aggregates, was observed in the static T-flask culture compared to the agitated systems, i.e. shake flask, Bellco spinner or Techne spinner. Probably the agitation promoted the spontaneous aggregation of the cells in spheres. In particular the Techne spinner allowed the most perfect spherical form among the different compared systems. Finally it was observed that hypoxia with 4 % oxygen concentration improved significantly the growth in Techne spinner or T-flask in comparison with normoxia with 21 % oxygen concentration. It was concluded that cultivation in Techne spinner under hypoxia was the most favorable condition among the ones studied here. The agitation provided by Techne spinner improved the cell growth in comparison with static system (T-flask). However using the other agitated systems, shake flask and Bellco spinner, was not comparably beneficial to the cell growth and viability, probably due to the higher shear stress of these systems compared to Techne spinner.
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| 10. |
- Chotteau, Veronique, 1963-, et al.
(författare)
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Development of a fed-batch process for the production of a recombinant protein X in CHO-GS system : Case study from the cell to reactor process ready for pilot scale cultivation
- 2010
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Ingår i: Cells and Culture. - Dordrecht : Springer Science+Business Media B.V.. - 9789048134182 ; , s. 723-725
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- A new cell line was created using CHO-GS system. The most promising clones were adapted to different base cultivation media leading to the selection of one medium. The fed-batch process development was performed in spinner, shake flask and bioreactor scale. It included the selection of a feed medium, the choice of the feed strategy and the optimisation of the glucose feeding. The process was then simplified by using a single feed including the feed medium and the glucose feed. Finally up-scaling parameters like aeration and CO2 stripping were studied in 3 L and 15 L bioreactors in preparation for pilot scale operation. This process proved to be robust, reproducible and suitable for large and commercial scale operation.
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