Proof-of-concept of a novel scalable magnetic bead-based cell separation technology

Advanced Therapy Medicinal Products (ATMPs) are gaining great interest for the treatment of severe, currently consider incurable, diseases. Therapies based on stem cells have an enormous potential in applications such as cardiac cells and neurons to name a few. However, the production of these cell systems is expensive, complex and lack, nowadays, scalability both for their cultivation and the purification. The lack of scalability is a major bottleneck to bring these therapies to patient at commercial scale. Magnetic beads are well-established for sorting of cells, e.g. magnetic activated cell sorting. However, today´s systems size is still limited in terms of scale-up potential. We have developed a new scalable separation process based on the magnetic bead MAG for the isolation of receptor positive cell subpopulations. We have previously published that our new magnetic bead system MAG is extremely gentle towards cells1 and can easily be scaled up at pilot-scale for the separation of monoclonal antibody from a cell suspension2. In the present study, this magnetic bead system has been further developed for cell separation. In a model system with a mixture of hMSC and HER2+ SK BR3 cells (20:80), a proof-of-concept was demonstrated showing exceptional elimination of the HER2+ cells. Different ligand densities were evaluated, showing that the largest cell removals were achieved with the lowest ligand densities. Furthermore, in a study of mechanical and chemical stress conditions, the MAG separation system showed robustness of sorting performances. From our previous knowledge about the scalability of the MAG magnetic beads separation, this provides promising potential for the production of therapeutic stem cells at larger scale. 1. Brechmann, N. A.; Schwarz, H.; Eriksson, P.-O.; Eriksson, K.; Shokri, A.; Chotteau, V., Antibody capture process based on magnetic beads from very high cell density suspension. Biotechnology and Bioengineering 2021, n/a, (n/a). 2. Brechmann, N. A.; Eriksson, P.-O.; Eriksson, K.; Oscarsson, S.; Buijs, J.; Shokri, A.; Hjälm, G.; Chotteau, V., Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture. Biotechnology Progress 2019, 35, (3), e2775

Data-driven Bayesian estimation of Monod kinetics

In this paper, we consider the well known problem of non-linear identification of the rates of the reactions involved in cells with Monod functions. In bioprocesses, generating data is very expensive and long and so it is important to incorporate prior knowledge on the Monod kinetic parameters. Bayesian estimation is an elegant estimation technique which deals with parameter estimation with prior knowledge modeled as probability density functions. However, we might not have an accurate knowledge of the kinetic parameters such as interval bounds, especially for newly developed cell lines. Hence, we consider the case when there is no accurate prior information on the kinetic parameters except qualitative knowledge such that their non-negativity. A log-Gaussian prior distribution is considered for the parameters and the mean and variances of these distribution are tuned using the Expectation Maximization algorithm. The algorithm requires to use Metropolis Hastings within Gibbs sampling which can be computationally expensive. We develop a novel variant of the Metropolis-Hastings within Gibbs sampling sampling scheme in order to accelerate and improve on the hyperparameter tuning. We show that it can give better modeling performances on a relatively large-scale simulation example compared to available methods in the literature.Comment: Preprint submitted to Automatic

Proof-of-concept of a novel scalable magnetic bead-based cell separation technology

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Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor

AbstractRecombinant Chinese Hamster Ovary (CHO) cells producing IgG monoclonal antibody were cultivated in a novel perfusion culture system CellTank, integrating the bioreactor and the cell retention function. In this system, the cells were harbored in a non-woven polyester matrix perfused by the culture medium and immersed in a reservoir. Although adapted to suspension, the CHO cells stayed entrapped in the matrix. The cell-free medium was efficiently circulated from the reservoir into- and through the matrix by a centrifugal pump placed at the bottom of the bioreactor resulting in highly homogenous concentrations of the nutrients and metabolites in the whole system as confirmed by measurements from different sampling locations. A real-time biomass sensor using the dielectric properties of living cells was used to measure the cell density. The performances of the CellTank were studied in three perfusion runs. A very high cell density measured as 200pF/cm (where 1pF/cm is equivalent to 1×106viable cells/mL) was achieved at a perfusion rate of 10 reactor volumes per day (RV/day) in the first run. In the second run, the effect of cell growth arrest by hypothermia at temperatures lowered gradually from 37°C to 29°C was studied during 13 days at cell densities above 100pF/cm. Finally a production run was performed at high cell densities, where a temperature shift to 31°C was applied at cell density 100pF/cm during a production period of 14 days in minimized feeding conditions. The IgG concentrations were comparable in the matrix and in the harvest line in all the runs, indicating no retention of the product of interest. The cell specific productivity was comparable or higher than in Erlenmeyer flask batch culture. During the production run, the final harvested IgG production was 35 times higher in the CellTank compared to a repeated batch culture in the same vessel volume during the same time period

High cell density perfusion process for rAAV9 production based on transient transfection of HEK293 cells

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Continuous antibody capture step based on Magnetic Beads

The integration of a perfusion culture process with periodic counter chromatography currently draws a lot of attention in industry, with the goal of generating more efficient and flexible processes at reduced footprint. Purification process based on magnetic beads has been used for decades for analytical purposes. It shows interesting potential and begins to gain interest in the biopharmaceutical industry for fed-batch processes. This type of separation has characteristics that can facilitate continuous process integrated to perfusion operation. However integrated mAb capture using magnetic beads has not yet been published. In this study we have designed novel system and process for the affinity purification of monoclonal antibodies (mAbs) using magnetic beads based on work we performed in a pilot-scale set-up1. High capacity magnetic protein A agarose beads (LOABeads PrtA) were used with a prototype separation system for this development. In a proof-of-concept, the prototype set-up was tested with supernatant generated from perfusion process. As shown in Figure 1, the integrated process had a rapid adsorption of the mAb within 2 hours. It provided an overall adsorption of 94% and a yield of 87% (Figure 2). The unique use of high capacity magnetic beads together with this novel prototype system for continuous separation showed promising results, which can provide an efficient way for the integration of up-stream and down-stream process. Please click Additional Files below to see the full abstract

Architecture et bibliothèque : Rénover, réaménager : repenser les espaces pour de nouveaux usages

La bibliothèque municipale de Lyon et l’enssib organisaient le 3 décembre 2013 une journée d’étude sur le thème « Rénover, réaménager : repenser les espaces pour de nouveaux usages ». Cette journée s’inscrivait dans la prolongation des journées « l’Avenir des bibliothèques publiques » de décembre 2012 et « Architecture et bibliothèque » de mai 2013. Suite à la vague de constructions de bibliothèque des années 70 et 80, les chantiers de rénovation vont se multiplier en France dans les années à venir. Pour les bibliothécaires, il s’agit d’une part de rendre en compte les contraintes juridiques et de bâti spécifiques à ce contexte de maîtrise d’ouvrage, et d’autre part d’intégrer les nouveaux usages dans la réorganisation des espaces. La première partie de la journée donnera un éclairage juridique et un regard l’architecte sur la manière d’aborder une rénovation. Une rapide introduction au nouveau rapport de l’IFLA sur les tendances mondiales des bibliothèques permettra de situer les enjeux au niveau international. Les interventions suivantes présenteront des cas concrets de rénovation et de réaménagement de bibliothèques publiques et universitaires en France et à Cologne en Allemagne