Perfusion cell culture: Challenges and potentials between lab and manufacturing scale

The maturation of biopharmaceutical process understanding and technical advances have paved the way towards the commercial application of continuous bioprocesses. In particular, mammalian cell perfusion cultures have received manifold attention for cell expansion in the N-1 stage or the continuous production in end-to-end biomanufacturing processes. Perfusion processes uniquely offer both optimal cellular and/or consistent product environment. However, their inherent complexity in equipment and operation demands thorough process characterization and development. In this presentation, development efforts to build on the potential of perfusion processes and challenges during commercial implementation are shared. In a first case study, in depth equipment characterization resulted in a robust and versatile perfusion design1. Stable operation and short bioreactor residence time allowed the continuous harvest of the target protein with constant quality characteristics at lab scale2. Characteristic times for the adaption of cell metabolism and product quality to the constant operation were revealed. The potential of perfusion cultures to distinctively modulate towards a desired quality profile and benefits of their integration to a continuous downstream cascade are demonstrated3. The second part extends on the potential of perfusion to generate an optimal environment for cell growth. Its utilization for the generation of high bioreactor inoculation densities has successfully enabled overall manufacturing process intensification. Challenges of the commercial N-1 perfusion process implementation and associated small scale mitigation strategies are shared. 1. Characterization and comparison of ATF and TFF in stirred bioreactors for the production of therapeutic proteins, D. J. Karst, E. Serra, T. K. Villiger, M. Soos, M. Morbidelli, Biochemical Engineering Journal (2016), 110, 17-26. 2. Process performance and product quality in an integrated continuous antibody production process, D. J. Karst, F. Steinebach, M. Soos, M. Morbidelli, Biotechnology & Bioengineering (2017), 114, 298-307. 3. Modulation and modeling of monoclonal antibody N-linked glycosylation in mammalian cell perfusion reactors, D. J. Karst, E. Scibona, E. Serra, J. M. Bielser, M. Settler, J. Solacoup, H. Broly, M. Soos, M. Morbidelli, T. K

The Dutch national kilometre charge: Impacts on the Dutch car market and environment

The Dutch government has decided to gradually introduce a complex national road pricing system in the years 2012 to 2018. Existing car purchase taxes and the annual road taxes are to be replaced by a kilometre-based charging system. Several appraisal studies have been conducted to examine the impacts of different pricing variants, using the well-known Dutch national model system (NMS) and the national car market model DYNAMO. The car market model has recently been developed and simulates yearly car ownership and car purchase behaviour of households at a detailed level (120 car types * 70 household types), and endogenously models second hand car prices as a pricing mechanism to create an equilibrium in supply and demand. The Dutch road pricing scheme is expected to have major impacts: car ownership is projected to increase by 5-6% in the long run, car use is to reduced by by 10-15% and congestion on the main motorway network in 2020 by about 50%, compared to a reference scenario. Cost-benefit analysis studies, using output from the transport models, show significant positive welfare effects. This paper will review existing appraisal studies on the impacts of the kilometre charge, and describe the Dutch car market model DYNAMO and projections of the impacts of different CO2 pricing schemes. Results from DYNAMO estimations show that abolishing existing car purchase and road taxes by a CO2 differentiated kilometre charge has unintended consequences in the form of rising car ownership and increasing shares of diesel cars and relatively large and heavy vehicle types. Explanations for these unintended effects are that households react more to present one-off fixed costs than to recurrent variable costs, and total car costs are reduced for households with relatively low car mileages. For households with low car usage, the reduction of fixed car taxes is not fully compensated by increases in variable costs, and savings can be used to buy a more expensive and larger car. However, overall environmental impacts of CO2 differentiated kilometre charges are quite positive resulting from the reduction in car travel

Interactions between noradrenaline and corticosteroids in the brain: from electrical activity to cognitive performance

One of the core reactions in response to a stressful situation is the activation of the hypothalamus–pituitary–adrenal axis which increases the release of glucocorticoid hormones from the adrenal glands. In concert with other neuro-modulators, such as (nor)adrenaline, these hormones enable and promote cognitive adaptation to stressful events. Recent studies have demonstrated that glucocorticoid hormones and noradrenaline, via their receptors, can both rapidly and persistently regulate the function of excitatory synapses which are critical for storage of information. Here we will review how glucocorticoids and noradrenaline alone and in synergy dynamically tune these synapses in the hippocampus and amygdala, and discuss how these hormones interact to promote behavioral adaptation to stressful situations

From Fundamentals to Applications: Recent Developments in Atmospheric Pressure Photoionization Mass Spectrometry

Only five years after the first publication on atmospheric pressure photoionization (APPI), this technique has evolved rapidly as a very useful complement to established ionization techniques for liquid chromatography/mass spectrometry (LC/MS). This is reflected in a rapidly increasing number of publications in this field. On the one hand, thorough studies into the photoionization mechanism have provided deep insights into the roles and influences of the solvent, the dopant and other additives. On the other hand, a large number of new and attractive applications have recently been introduced. New instrumental developments have resulted in combined APPI/ESI (PAESI) and APPI/APCI sources and a microfabricated APPI source. In this review, the most important developments within the field are summarized, focusing in particular on the applications of the technique

Chronic Stress Effects on Hippocampal Structure and Synaptic Function: Relevance for Depression and Normalization by Anti-Glucocorticoid Treatment

Exposure of an organism to environmental challenges activates two hormonal systems that help the organism to adapt. As part of this adaptational process, brain processes are changed such that appropriate behavioral strategies are selected that allow optimal performance at the short term, while relevant information is stored for the future. Over the past years it has become evident that chronic uncontrollable and unpredictable stress also exerts profound effects on structure and function of limbic neurons, but the impact of chronic stress is not a mere accumulation of repeated episodes of acute stress exposure. Dendritic trees are reduced in some regions but expanded in others, and cells are generally exposed to a higher calcium load upon depolarization. Synaptic strengthening is largely impaired. Neurotransmitter responses are also changed, e.g., responses to serotonin. We here discuss: (a) the main cellular effects after chronic stress with emphasis on the hippocampus, (b) how such effects could contribute to the development of psychopathology in genetically vulnerable individuals, and (c) their normalization by brief treatment with anti-glucocorticoids

Measuring the Plasticity of Social Approach: A Randomized Controlled Trial of the Effects of the PEERS Intervention on EEG Asymmetry in Adolescents with Autism Spectrum Disorders

This study examined whether the Program for the Education and Enrichment of Relational Skills (PEERS: Social skills for teenagers with developmental and autism spectrum disorders: The PEERS treatment manual, Routledge, New York, 2010a) affected neural function, via EEG asymmetry, in a randomized controlled trial of adolescents with Autism spectrum disorders (ASD) and a group of typically developing adolescents. Adolescents with ASD in PEERS shifted from right-hemisphere gamma-band EEG asymmetry before PEERS to left-hemisphere EEG asymmetry after PEERS, versus a waitlist ASD group. Left-hemisphere EEG asymmetry was associated with more social contacts and knowledge, and fewer symptoms of autism. Adolescents with ASD in PEERS no longer differed from typically developing adolescents in left-dominant EEG asymmetry at post-test. These findings are discussed via the Modifier Model of Autism (Mundy et al. in Res Pract Persons Severe Disabl 32(2):124, 2007), with emphasis on remediating isolation/withdrawal in ASD