Development of a scale down toolbox for perfusion process development

Background and novelty Merck KGaA has a legacy of perfusion manufacturing processes with several commercial products that were developed in the 90s. These processes were developed for adherent cell systems. Since then, with the advances in media design and the monoclonal antibody driven portfolio, development efforts have been focusing on fed-batch with suspended cells. Recently and as many players in the biomanufacturing industry, Merck is working on the development of an integrated continuous process. To support this effort a process development platform for perfusion cell culture needs to be developed. Process development for fed-batch is clearly defined and scale down tools are well defined. Similar capabilities are needed for the development of perfusion processes but technical limitations (continuous flow, cell retention) complicate the scale-down process. Because of the duration of perfusion runs and the lack of small scale models, the experimental throughput for perfusion process development is generally considered as very low. The present study proposes a scale down toolbox for perfusion process development and optimization. Experimental approach To support perfusion process development, an experimental toolbox is proposed to address the need for higher experimental throughput. In the presented study, existing small scale systems were used to mimic perfusion conditions. Ideas on how to use these systems for media development and/or clone screening will be presented here. The generated data will help to understand how the proposed scale down experiments for perfusion are aligned to a bench scale continuous perfusion model data set. This work should also help to define clone selection criteria specific for perfusion process development. Results and discussion First a methodology for the screening of 470 clones in fed-batch mode was established. From this initial screening, 12 clones were selected and tested in a series of small scale models ranging from microliters to several liters. This extensive screening was performed to asses relative clone performance in each model by comparing the ranking obtained in the different scale down systems based on a number of process criteria and/or quality attributes [1]. A selection of the clones mentioned above with different growth and productivity characteristics were tested in 3.5L continuous perfusion cell cultures. The proposed scale down toolbox and screening methodology was then applied to the same clones. The benefit of this experimental approach is two-fold. First, the generated data helped to understand how the toolbox can be used to predict different process conditions (CSPR, productivity…) and thus evaluate the performance of different media and/or cell lines. Second, this study should help to define the key clone specific parameters needed to assess the ranking during clone screening for perfusion. [1] A. Y. Rouiller, J-M. Bielser, D. Brühlmann, M. Jordan, and H. Broly, “Screening and assessment of performance and molecule quality attributes of industrial cell lines across different fed-batch systems,” Biotechnol. Prog., vol. 32, no. 1, pp. 160–170, 201

Upstream perfusion process: Back to the future

At an early stage of bioprocess science, continuous operations were the workhorse in the industry. Therefore, EMD-Serono has legacy with continuous upstream processes as many of its molecules such as Rebif are being produced in perfusion mode. These processes from the 90’s consume large volumes of commercial media and demonstrate low productivities. Cells are mainly attached because no performant cell retention device existed for cells in suspension at that time. Around 10-15 years ago, the industry decided to move on fed-batch operations. EMD-Serono also re-oriented towards fed-batch operations. Molecules such as Erbitux are now being produced in 15,000 L stainless steel bioreactors. Merck Serono scientists developed a strong fed-batch platform defining specific process parameters, feed strategies and last but not least a chemically defined proprietary media (and feeds). The production process development of most mAbs in the pipeline is now based on this fed-batch platform. Today, rapid technology evolution such as inexpensive culture media or robust cell retention system brings the interest of the industry in perfusion back to life. EMD-Serono combines its experience on legacy perfusion processed with the well-developed fed-batch platform knowledge. Exploring perfusion capabilities in a modern way (performant retention devices, chemically defined and proprietary media, single-use technology, link to continuous DSP), this presentation will describe the approach to reach a high cell density, high specific productivity process with a rational design. The impact of process parameter and some media component concentrations on metabolic stoichiometry and product quality will also be discussed. Results are promising but raise a huge amount of questions. How far can we push the limits of productivity in regards to the bioreactor volume? How can quality be impacted or modulated in such systems? If manufacturing of mAbs switches to perfusion, how will this impact the equipment scale, costs and flexibility? How can process development adapt to this new challenge? These are the questions that will be addressed during this talk

Does my brain want what my eyes like? - How food liking and choice influence spatio-temporal brain dynamics of food viewing.

How food valuation and decision-making influence the perception of food is of major interest to better understand food intake behavior and, by extension, body weight management. Our study investigated behavioral responses and spatio-temporal brain dynamics by means of visual evoked potentials (VEPs) in twenty-two normal-weight participants when viewing pairs of food photographs. Participants rated how much they liked each food item (valuation) and subsequently chose between the two alternative food images. Unsurprisingly, strongly liked foods were also chosen most often. Foods were rated faster as strongly liked than as mildly liked or disliked irrespective of whether they were subsequently chosen over an alternative. Moreover, strongly liked foods were subsequently also chosen faster than the less liked alternatives. Response times during valuation and choice were positively correlated, but only when foods were liked; the faster participants rated foods as strongly liked, the faster they were in choosing the food item over an alternative. VEP modulations by the level of liking attributed as well as the subsequent choice were found as early as 135-180ms after food image onset. Analyses of neural source activity patterns over this time interval revealed an interaction between liking and the subsequent choice within the insula, dorsal frontal and superior parietal regions. The neural responses to food viewing were found to be modulated by the attributed level of liking only when foods were chosen, not when they were dismissed for an alternative. Therein, the responses to disliked foods were generally greater than those to foods that were liked more. Moreover, the responses to disliked but chosen foods were greater than responses to disliked foods which were subsequently dismissed for an alternative offer. Our findings show that the spatio-temporal brain dynamics to food viewing are immediately influenced both by how much foods are liked and by choices taken on them. These valuation and choice processes are subserved by brain regions involved in salience and reward attribution as well as in decision-making processes, which are likely to influence prospective dietary choices in everyday life

Development of the PAT toolkit for continuous bioprocessing

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Performance consistency of fed-batch cultures across multiple systems used in upstream process development

Each stage of cell culture process development requires fit for purpose tools. The selection of a fed-batch cultivation system is often based on throughput and cost. However, the process knowledge derived from different systems and scales is not necessarily identical. Hence, a careful evaluation of systems which are already established or newly implemented is essential. We recently introduced a novel high throughput fed-batch screening system (1) and the objective of this study was to provide data on how it compares with other systems used in early and late stage cell culture process development. We describe the performance of 12 different recombinant CHO cell lines expressing the same antibody in fed-batch culture systems ranging from a few hundred microliters to lab scale. The 12 cell lines were selected based on distinct phenotypes covering a range which can be expected in typical industrial process development projects. The cell lines were cultivated using the same expansion and fed-batch protocol (proprietary fed-batch system). The following cultivation systems were evaluated: shaking 96-deepwell plates, 50 mL vented shake tubes, micro-scale bioreactors (ambr15TM system) and lab-scale bioreactors (3L). The results of this study show both the limitations and the potential of each cultivation system and their suitability for process development, process characterization and scale-up. The shaking systems offer unprecedented parallel throughput but are limited with respect to culture control (e. g. lack of pH and pO2 control). Despite their limitations, they are expected to be used in the future as important tools for early process development and for the improvement of fed-batch platform processes. On the other hand, the data obtained from this study show that micro- and lab-scale bioreactors represent ideal tools for the confirmation of process consistency. Both micro- and lab-scale systems will be extensively used in the future to support tech transfers and perform process characterization studies

Energized soft tissue dissection in surgery simulation

With the development of virtual reality technology, surgery simulation has become an effective way to train the operation skills for surgeons. Soft tissue dissection, as one of the most frequently performed operations in surgery, is indispensable to an immersive and high-fidelity surgery simulator. Energized dissection tools are much more commonly used than the traditional sharp scalpels for patient safety. Unfortunately, the interaction of such tools with the soft tissues has been largely ignored in the research of surgical simulators. In this paper, we have proposed an energized soft tissue dissection model. We categorize the soft tissues into three types (fascia, membrane, and fat) and simulate their physical property accordingly. The dissection algorithm we propose employs an edge-based structure, which offers an effective mechanism for the generation of incisions dissected with energized tools. The mesh topology will not be changed when it is dissected by an energized tool, rather it is controlled by the heat transfer model. Our dissection method is highly compatible and efficient to the physically based simulation resolved by a pre-factorized linear system. We have proposed an energized soft tissue dissection model. We categorize the soft tissues into three types (fascia, membrane, and fat) and simulate their physical property accordingly. The dissection algorithm employs an edge-based structure, which offers an effective mechanism for the generation of incisions dissected with energized tools. Our dissection method is highly compatible and efficient to the physically based simulation resolved by a pre-factorized linear system

Brain dynamics of meal size selection in humans.

Although neuroimaging research has evidenced specific responses to visual food stimuli based on their nutritional quality (e.g., energy density, fat content), brain processes underlying portion size selection remain largely unexplored. We identified spatio-temporal brain dynamics in response to meal images varying in portion size during a task of ideal portion selection for prospective lunch intake and expected satiety. Brain responses to meal portions judged by the participants as 'too small', 'ideal' and 'too big' were measured by means of electro-encephalographic (EEG) recordings in 21 normal-weight women. During an early stage of meal viewing (105-145ms), data showed an incremental increase of the head-surface global electric field strength (quantified via global field power; GFP) as portion judgments ranged from 'too small' to 'too big'. Estimations of neural source activity revealed that brain regions underlying this effect were located in the insula, middle frontal gyrus and middle temporal gyrus, and are similar to those reported in previous studies investigating responses to changes in food nutritional content. In contrast, during a later stage (230-270ms), GFP was maximal for the 'ideal' relative to the 'non-ideal' portion sizes. Greater neural source activity to 'ideal' vs. 'non-ideal' portion sizes was observed in the inferior parietal lobule, superior temporal gyrus and mid-posterior cingulate gyrus. Collectively, our results provide evidence that several brain regions involved in attention and adaptive behavior track 'ideal' meal portion sizes as early as 230ms during visual encounter. That is, responses do not show an increase paralleling the amount of food viewed (and, in extension, the amount of reward), but are shaped by regulatory mechanisms

Time-dependent genetic effects on gene expression implicate aging processes

Gene expression is dependent on genetic and environmental factors. In the last decade, a large body of research has significantly improved our understanding of the genetic architecture of gene expression. However, it remains unclear whether genetic effects on gene expression remain stable over time. Here, we show, using longitudinal whole-blood gene expression data from a twin cohort, that the genetic architecture of a subset of genes is unstable over time. In addition, we identified 2213 genes differentially expressed across time points that we linked with aging within and across studies. Interestingly, we discovered that most differentially expressed genes were affected by a subset of 77 putative causal genes. Finally, we observed that putative causal genes and down-regulated genes were affected by a loss of genetic control between time points. Taken together, our data suggest that instability in the genetic architecture of a subset of genes could lead to widespread effects on the transcriptome with an aging signature

The impact of replacing sugar- by artificially-sweetened beverages on brain and behavioral responses to food viewing - An exploratory study.

Several studies indicate that the outcome of nutritional and lifestyle interventions can be linked to brain 'signatures' in terms of neural reactivity to food cues. However, 'dieting' is often considered in a rather broad sense, and no study so far investigated modulations in brain responses to food cues occurring over an intervention specifically aiming to reduce sugar intake. We studied neural activity and liking in response to visual food cues in 14 intensive consumers of sugar-sweetened beverages before and after a 3-month replacement period by artificially-sweetened equivalents. Each time, participants were presented with images of solid foods differing in fat content and taste quality while high-density electroencephalography was recorded. Contrary to our hypotheses, there was no significant weight loss over the intervention period and no changes were observed in food liking or in neural activity in regions subserving salience and reward attribution. However, neural activity in response to high-fat, sweet foods was significantly reduced from pre-to post-intervention in prefrontal regions often linked to impulse control. This decrease in activity was associated with weight loss failure, suggesting an impairment in individuals' ability to exert control and adjust their solid food intake over the intervention period. Our findings highlight the need to implement multidisciplinary approaches when aiming to help individuals lose body weight

Colloques interprofessionnels en milieu hospitalier ::rapport final

Comment la collaboration interprofessionnelle est-elle vécue actuellement en contexte stationnaire ? Comment cela influence-t-il l’efficacité et la qualité des soins ? Quelles opportunités et quels défis les colloques interprofessionnels présentent-ils ? Ces trois questions constituent le sujet de recherche principal de ce projet. Les résultats de l’étude permettent de mieux comprendre les processus de groupe lors de colloques interprofessionnels et d’identifier les défis à relever lors de la constitution et de la conduite d’une équipe. Le projet de recherche fournit des résultats pour deux contextes différents dans le domaine des soins (réadaptation et médecine interne) et pour deux régions linguistiques (Suisse alémanique et Suisse romande) ; il permet ainsi de formuler des recommandations pour toute la Suisse. Question de recherche, objectif et démarche méthodologique La principale question de recherche de ce projet est la suivante : comment la collaboration interprofessionnelle en contexte stationnaire s’organise-t-elle actuellement pour améliorer la qualité et l’efficacité des soins ? Où est-il nécessaire d’agir, et qui doit en assumer la responsabilité (principale) ? L’étude visait à mieux comprendre l’impact des différentes manières de faire (pratiques, processus, organisation, etc.) sur l’efficacité et le bon déroulement des colloques interprofessionnels. Il s’agissait d’identifier les défis présentés par leur réalisation, en particulier via l’analyse des structures d’organisation et des pratiques interprofessionnelles. En outre, le projet cherchait à déterminer quels colloques sont perçus comme efficaces, ainsi qu’à lister les défis liés à la formation et à la conduite d’une équipe interprofessionnelle. Deux approches analytiques complémentaires ont été choisies : d’une part, l’enregistrement vidéo de colloques interprofessionnels et l’analyse systématique de leurs conversations ; d’autre part, l’évaluation d’entretiens semi-directifs effectués avec des responsables hiérarchiques et des responsables d’équipe, sur la base des enregistrements vidéo. Le projet de recherche a identifié au total trois facteurs principaux pour l’efficacité des colloques interprofessionnels : (1) les relations interprofessionnelles, (2) les compétences collaboratives et (3) l’organisation des colloques. La formation ciblée à l’organisation de colloques interprofessionnels semble très importante pour les équipes, car elle permet de développer les compétences requises lors de ceux-ci (p. ex. communication efficace, capacité d’adaptation etc.)