| 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. |
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| 6. |
- 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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| 7. |
- 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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| 8. |
- Colin, Kevin, et al.
(författare)
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Gaussian process modeling of macroscopic kinetics : a better-tailored kernel for Monod-type kinetics
- 2022
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Ingår i: 10th Vienna International Conference on Mathematical Modelling MATHMOD 2022 Vienna Austria, 27–29 July 2022. - : Elsevier BV. ; , s. 397-402
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Konferensbidrag (refereegranskat)abstract
- In bioprocesses, it is important to model the kinetics of the macroscopic rates of reactions since these are required to catch the dynamical aspects of a process. In [Wang et al. 2020], a modeling method involving Gaussian processes has been developed, using a kernel especially designed for the modeling of Monod-type kinetics (activation, inhibition, double component, neutral effect). However, as will be illustrated in this paper, when the number of training data is limited or the metabolite concentration data do not have large variations (which is generally the case for real-life data), this kernel can yield inaccurate models for the kinetics. In this paper, we develop a new kernel better tailored for the modeling of Monod-type kinetics and we show that it has good modeling performances in the case of a limited number of data. The idea is to use the particular structure of Monod-type functions in the design of the kernel, i.e., we incorporate prior knowledge in the modeling.
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| 9. |
- Dewasme, Laurent, et al.
(författare)
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Multivariable robust tube-based nonlinear model predictive control of mammalian cell cultures
- 2024
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Ingår i: Computers and Chemical Engineering. - : Elsevier BV. - 0098-1354 .- 1873-4375. ; 183
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Tidskriftsartikel (refereegranskat)abstract
- In this paper, the application of a robust nonlinear model predictive control (NMPC) framework to mammalian cell cultures is proposed, dealing with possible large kinetic parameter uncertainties. Industrial constraints formulated in view of good manufacturing practice and quality-by-design approach are also considered, namely the assurance that all state trajectories are contained within a corridor defined by lower and upper safety bounds. The latter are assimilated to the well-known tube-based paradigm which is used to formulate the corresponding robust NMPC problem. Both classical and tube-based NMPC performances are assessed in numerical simulations where specific key-species are regulated while dealing with an uncertain plant model. The capability of the tube-based method to reduce the impact of the parameter variations on the state trajectories and the violation of the constraints is highlighted, suggesting the transfer of the method on a real pharmaceutical process.
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| 10. |
- Dewasme, L., et al.
(författare)
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Practical data-driven modeling and robust predictive control of mammalian cell fed-batch process
- 2023
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Ingår i: Computers and Chemical Engineering. - : Elsevier BV. - 0098-1354 .- 1873-4375. ; 171
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Tidskriftsartikel (refereegranskat)abstract
- Even if the performances of bioprocesses can be significantly improved by model-based control, there often remains a tradeoff between model complexity and control robustness. This paper proposes an original data -driven strategy for fast design of dynamic bioprocess models with minimal complexity (i.e., minimal number of bioreactions). Maximum likelihood principal component analysis (MLPCA) is applied to infer the minimal reaction scheme from a 25-state mammalian cell culture database. Then, a systematic algorithm is used to provide a continuous kinetic model formulation assuming all rates to occur simultaneously, which may be far from true cell metabolic conditions sometimes presenting discontinuous metabolic switches. A robust model predictive formulation is therefore adopted to reduce the impact of model structural uncertainty on the process performances. Additional numerical results show that the proposed strategy presents excellent performances in presence of unexpected metabolic switches.
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