New kid on the block: Industrialization of cell-free synthesis for biotherapeutics development

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Building one block at a time toward complex biologics using cell-free protein synthesis process

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Short-term exposure to indoor PM2.5 in office buildings and cognitive performance in adults: An intervention study

Impacts of exposure to particulate matter can be wide-ranging, with some evidence suggesting potential impacts on nervous system, cognition, and productivity. However, most evidence to date addresses ambient exposure and chronic outcomes with limited research on indoor short-term exposure to PM2.5 and cognitive performance. Hence, the aim of this study was to evaluate if there is a relationship between short-term exposure to indoor PM2.5 within the workplace context and cognitive performance in adults. A randomized single-blind cross-over trial was conducted in an urban mixed-mode ventilated office in Beijing (China). Sixty eligible employees participated in the study and fifty-five valid responses were obtained. Cognitive performance was assessed with a validated neurological battery test during intervention and control conditions. Portable air purifiers were placed on the subjects' workstations and used in the intervention condition to control PM2.5 levels at the subjects’ breathing zone whereas in the control condition, the air purifiers were present but switched off. Average PM2.5 levels were respectively 18.0 μg/m³ and 3.7 μg/m³ in the control and intervention condition. In each condition, cognitive performance testing started five to 7 h after arriving in the office. The results showed office workers had significantly better performance for 9 out of the 16 cognitive skills during the intervention, compared to the control condition, with the most consistent effect in the memory domain. This study adds evidence that elevated PM2.5 levels can detrimentally affect cognitive performance even during short-term indoor exposure. Further research is needed on the potential impact of other air pollutants, including ultrafine particles, and on the possible role of sound and air movement from the air purifiers

Making large scale processes transparent – The application of CFD and classical engineering approaches to mitigate risk during cell culture process transfer

The demand for complex therapeutic proteins and especially monoclonal antibodies for treatment of various diseases has increased continuously in the last decades, prompting the continuous development of new processes. For the development of these cell culture processes, equipment knowledge is essential, especially since a process typically is scaled up and transferred numerous times in large pharmaceutical companies. The need for a thorough equipment understanding has been also recognized by the FDA in their PAT-publication. To gain such an understanding at full production scale, Boehringer Ingelheim has put tremendous effort in fully characterizing our multiple bioreactors at different scale across multiple sites. Computational fluid dynamics (CFD) modeling is utilized which allows the evaluation of integral parameters including energy input, mixing time, shear forces and mass transfer coefficient (kLa). The knowledge gained through this tool has been instrumental in understanding the bioreactor characteristics and establish appropriate process scale up and transfer strategies within and across sites. In addition, we have constructed a 15000L acrylic bioreactor model which provides opportunities to validate simulation results with experimental data. For example, local behavior of the reactor regarding the bubble size distributions and dead zones for mixing and gassing can be visualized in the at-scale acrylic bioreactor. One study that was conducted is to understand mixing and mass transfer behavior with respect to agitation rate and superficial gas flow rate. The interactions of these parameters in large scale, however, were in some cases found to be counterintuitive where higher gassing and agitation did not consistently result in higher kLa and better mixing. Although simple modifications and standardization of systems can lead to more similar hydrodynamic conditions, it is difficult to make modification in existing GMP facility due to rigid regulations in the biopharmaceutical sector. Therefore, it is crucial to rely on engineering principles and CFD simulations for transfer between different sites with different bioreactor systems to give additional confidence to ensure successful transfer. Please click Additional Files below to see the full abstract

Marching toward implementation of an ultra-high density dynamic perfusion process

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Product sieving challenges in TFF perfusion cell culture

Integrated continuous biomanufacturing has gained significant interest because of its potential to streamline production by integrating upstream and downstream processes. Combined with an intensified perfusion bioreactor, continuous processing can greatly reduce cost, space requirements, and handling steps, while improving production efficiency.1,2 During perfusion operation, product and spent media are removed while cells are retained within the bioreactor with a cell separation device. In particular hollow fiber membranes, attached externally to the bioreactor, permit tangential or alternating flow filtration (TFF, ATF), as cells are recirculated through the unit and permeate is harvested for downstream processing.3,4 However, membrane fouling and issues with product sieving, especially associated with a TFF setup, have a direct impact on total product yield from the process, and can cause hollow fiber reliability issues which in some cases can result in premature termination of a bioreactor run. It is hypothesized that membrane fouling from host cell proteins, cell debris, or additives such as antifoam can result in decreased product sieving as product transmission through the membrane decreases over time.5,6 Toward addressing issues with product sieving, we aim to identify the underlying causes of membrane fouling, associated with host cell proteins and antifoam, and to develop new methods to lengthen their lifetime during perfusion operation. This presentation will focus on some tools and experiments that we have conducted to address this issue with the goal to identify factors within the bioreactor that lead to reduced product sieving and implementation of new strategies to mitigate the effects of these factors during operation. 1. Konstantinov, K.B., Cooney C.L “White Paper on Continuous Bioprocessing” Journal of Pharmaceutical Sciences 104 (3), 2015, 813-820. 2. Warikoo, V., et al. “Integrated Continuous Production of Recombinant Therapeutic Proteins” Biotechnology and Bioengineering 109 (12), 2012, 3018-3029. 3. Clinke, M.F., et al. “Very High Density of CHO Cells in Perfusion by ATF or TFF in WAVE Bioreactor. Part I.” Biotechnology Progress 29 (3), 2013, 754-767. 4. Karst, D.J., et al. “Characterization and Comparison of ATF and TFF in Stirred Bioreactors for Continuous Mammalian Cell Culture Processes” Biochemical Engineering Journal 110, 2016, 17-26. 5. Van Reis, R., Zydney, A. “Bioprocess Membrane Technology” Journal of Membrane Science 297, 2007, 16-50. 6. Wang, S., et al. “Shear Contributions to Cell Culture Performance and Product Recovery in ATF and TFF Perfusion Systems” Journal of Biotechnology 246, 2017, 52-60

Application of alternative cell separation systems for the harvest of mammalian cell culture processes in a fully disposable single-use facility

In a fully disposable facility where the use of continuous disk-stack centrifuges are not preferred, harvest processes based on conventional depth filtration become more challenging with increasing single-use bioreactor (SUB) size and higher density culture. Lower filterability due to high volume and high cell density diminishes the efficiency of the depth filtration. In addition, the use of larger depth filtration systems is constrained by the facility footprint. To address the challenge described, several alternative single-use harvest technologies were evaluated. A disposable centrifuge with a small foot print, low consumable cost, and ease of operation was tested. In a study using a 1kL SUB for harvest, the disposable centrifuge was compared to a continuous disk-stack centrifuge, both followed by conventional depth filtration. The best operating conditions, as well as the edges of failures, were found for the disposable centrifuge. When compared to the disk-stack centrifuge results showed that the operation can be performed in a wide range without impacting depth filtration area requirements and less depth filter area was needed. In addition to testing a disposable centrifuge, flocculation was evaluated and compared, followed by the depth filtration. This was found to be another low-cost alternative process, while also occupying less facility space. The flocculation technology was tested with different cell lines and results showed significant improvement in filterability and reduction of depth filter area compared to full traditional depth filtration train

Overcoming process intensification challenges to deliver a manufacturable and competitive integrated continuous biomanufacturing platform

Groups in both industry and academia have achieved high densities and productivities in perfusion cell culture processes. At Sanofi, we have demonstrated perfusion densities greater than 100 million cells/mL (with associated high productivities) at a cell-specific perfusion rate of only 20 pL/cell/day. This process intensification reduces the footprint of upstream unit operations as well as capital and operating expenses of manufacturing facilities. The continuous nature of perfusion cell culture also creates opportunities for integration of continuous downstream operations, leading to further process intensifications and volume reductions. In this presentation, we will discuss our work on several upstream challenges that must be overcome to create a manufacturable, continuous bioprocessing platform. These will include (1) mitigation strategies for the large shear forces accompanying the high sparge rates necessary to sustain a high-density culture, (2) efforts to minimize the economic and logistical burden of media cost and consumption in perfusion cell culture, (3) the challenge of maintaining consistent product quality over long durations and (4) scale-up of these intensified processes to 50-L and 500-L manufacturing-scale systems. We can address each of these areas to create an efficient, competitive cell culture platform that generates high cell viabilities and excellent product quality at manufacturing scales. We will demonstrate real-world examples of both enzyme and antibody-producing processes, showing that such a platform can reliably deliver good results across diverse products

Selective acetyl- and butyrylcholinesterase inhibitors reduce amyloid-β ex vivo activation of peripheral chemo-cytokines from Alzheimer's disease subjects: exploring the cholinergic anti-inflammatory pathway

Increasing evidence suggests that elevated production and/or reduced clearance of amyloid-β peptide (Aβ) drives the early pathogenesis of Alzheimer's disease (AD). Aβ soluble oligomers trigger a neurotoxic cascade that leads to neuronal dysfunction, neurodegeneration and, ultimately, clinical dementia. Inflammation, both within brain and systemically, together with a deficiency in the neurotransmitter acetylcholine (ACh) that underpinned the development of anticholinesterases for AD symptomatic treatment, are invariable hallmarks of the disease. The inter-relation between Aβ, inflammation and cholinergic signaling is complex, with each feeding back onto the others to drive disease progression. To elucidate these interactions plasma samples and peripheral blood mononuclear cells (PBMCs) were evaluated from healthy controls (HC) and AD patients. Plasma levels of acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and Aβ were significantly elevated in AD vs. HC subjects, and ACh showed a trend towards reduced levels. Aβ challenge of PBMCs induced a greater release of inflammatory cytokines interleukin-1β (IL-1β), monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-α) from AD vs. HC subjects, with IL-10 being similarly affected. THP-1 monocytic cells, a cell culture counterpart of PBMCs and brain microglial cells, responded similarly to Aβ as well as to phytohaemagglutinin (PHA) challenge, to allow preliminary analysis of the cellular and molecular pathways underpinning Aβ-induced changes in cytokine expression. As amyloid-β precursor protein expression, and hence Aβ, has been reported regulated by particular cytokines and anticholinesterases, the latter were evaluated on Aβ- and PHA-induced chemocytokine expression. Co-incubation with selective AChE/BuChE inhibitors, (-)-phenserine (AChE) and (-)-cymserine analogues (BuChE), mitigated the rise in cytokine levels and suggest that augmentation of the cholinergic anti-inflammatory pathway may prove valuable in AD