Competing climate feedbacks of ice sheet freshwater discharge in a warming world.

Freshwater discharge from ice sheets induces surface atmospheric cooling and subsurface ocean warming, which are associated with negative and positive feedbacks respectively. However, uncertainties persist regarding these feedbacks' relative strength and combined effect. Here we assess associated feedbacks in a coupled ice sheet-climate model, and show that for the Antarctic Ice Sheet the positive feedback dominates in moderate future warming scenarios and in the early stage of ice sheet retreat, but is overwhelmed by the negative feedback in intensive warming scenarios when the West Antarctic Ice Sheet undergoes catastrophic collapse. The Atlantic Meridional Overturning Circulation is affected by freshwater discharge from both the Greenland and the Antarctic ice sheets and, as an interhemispheric teleconnection bridge, exacerbates the opposing ice sheet's retreat via the Bipolar Seesaw. These results highlight the crucial role of ice sheet-climate interactions via freshwater flux in future ice sheet retreat and associated sea-level rise.

Vaccine process technology-A decade of progress.

In the past decade, new approaches to the discovery and development of vaccines have transformed the field. Advances during the COVID-19 pandemic allowed the production of billions of vaccine doses per year using novel platforms such as messenger RNA and viral vectors. Improvements in the analytical toolbox, equipment, and bioprocess technology have made it possible to achieve both unprecedented speed in vaccine development and scale of vaccine manufacturing. Macromolecular structure-function characterization technologies, combined with improved modeling and data analysis, enable quantitative evaluation of vaccine formulations at single-particle resolution and guided design of vaccine drug substances and drug products. These advances play a major role in precise assessment of critical quality attributes of vaccines delivered by newer platforms. Innovations in label-free and immunoassay technologies aid in the characterization of antigenic sites and the development of robust in vitro potency assays. These methods, along with molecular techniques such as next-generation sequencing, will accelerate characterization and release of vaccines delivered by all platforms. Process analytical technologies for real-time monitoring and optimization of process steps enable the implementation of quality-by-design principles and faster release of vaccine products. In the next decade, the field of vaccine discovery and development will continue to advance, bringing together new technologies, methods, and platforms to improve human health.

Comparing an Integrated Amphiphilic Surfactant to Traditional Hydrophilic Coatings for the Reduction of Catheter-Associated Urethral Microtrauma.

Hydrophilic-coated intermittent catheters have improved the experience of intermittent urinary catheterization for patients compared to conventional gel-lubricated uncoated catheters. However, the incorporation of polyvinylpyrrolidone (PVP) within hydrophilic coatings can lead to significant issues with coating dry-out. Consequently, increased force on catheter withdrawal may cause complications, including urethral microtrauma and pain. Standard methods of evaluating catheter lubricity lack physiological relevance and an understanding of the surface interaction with the urethra. The tribological performance and urethral interaction of commercially available hydrophilic PVP-coated catheters and a coating-free integrated amphiphilic surfactant (IAS) catheter were evaluated by using a biomimetic urethral model designed from a modified coefficient of friction (CoF) assay. T24 human urothelial cells were cultured on customized silicone sheets as an alternate countersurface for CoF testing. Hydrophilic PVP-coated and coating-free IAS catheters were hydrated and the CoF obtained immediately following hydration, or after 2 min, mimicking in vivo indwell time for urine drainage. The model was observed for urethral epithelial cell damage postcatheterization. The majority of hydrophilic PVP-coated catheters caused significantly greater removal of cells from the monolayer after 2 min indwell time, compared to the IAS catheter. Hydrophilic PVP-coated catheters were shown to cause more cell damage than the coating-free IAS catheter. A biomimetic urethral model provides a more physiologically relevant model for understanding the factors that govern the frictional interface between a catheter surface and urethral tissue. From these findings, the use of coating-free IAS catheters instead of hydrophilic PVP-coated catheters may help reduce urethral microtrauma experienced during catheter withdrawal from the bladder, which may lead to a lower risk of infection.

Cell-based shear stress sensor for bioprocessing.

Shear stress during bioreactor cultivation has significant impact on cell health, growth, and fate. Mammalian cells, such as T cells and stem cells, in next-generation cell therapies are especially more sensitive to shear stress present in their culture environment than bacteria. Therefore, a base knowledge about the shear stress imposed by the bioprocesses is needed to optimize the process parameters and enhance cell growth and yield. However, typical computational flow dynamics modeling or PCR-based assays have several limitations. Implementing and interpreting computational modeling often requires technical specialties and also relies on many simplifications in modeling. PCR-based assays evaluating changes in gene expression involve cumbersome sample preparation with the use of advanced lab equipment and technicians, hampering rapid and straightforward assessment of shear stress. Here, we developed a simple, cell-based shear stress sensor for measuring shear stress levels in different bioreactor types and operating conditions. We engineered a CHO-DG44 cell line to make its stress sensitive promoter EGR-1 control GFP expression. Subsequently, the stressed CHO cells were transferred into a 96 well plate, and their GFP levels (population mean fluorescence) were monitored using a cell analysis instrument (Incucyte®, Sartorius Stedim Biotech) over 24 hours. After conducting sensor characterization, which included chemical induced stress and fluid shear stress, and stability investigation, we tested the shear stress sensor in the Ambr® 250 bioreactor vessels (Sartorius Stedim Biotech) with different impeller and vessel designs. The results showed that the CHO cell-based shear stress sensors expressed higher GFP levels in response to higher shear stress magnitude or exposure time. These sensors are useful tools to assess shear stress imposed by bioreactor conditions and can facilitate the design of various bioreactor vessels with a low shear stress profile.

Advanced control strategies for continuous capture of monoclonal antibodies based upon biolayer interferometry.

The semi and fully continuous production of monoclonal antibodies (mAbs) has been gaining traction as a lower cost, and efficient production of mAbs to broaden patient access. To be truly flexible and adaptive to process demands, the industry has lacked sufficient advanced control strategies. The variation of the upstream product concentration typically cannot be handled by the downstream capture step, which is configured for a constant feed concentration and fixed binding capacity. This inflexibility leads to losses of efficiency and product yield. This study shows that these challenges can be overcome by a novel advanced control strategy concept that includes dynamic control throughout a perfusion bioreactor, with cell retention by alternating tangential flow, integrated with simulated moving bed (SMB) multi-column chromatography. The automation workflow and advanced control strategy were implemented through the use of a visual programming development environment. This enabled dynamic flow control across the upstream and downstream process integrated with a dynamic column loading of the SMB. A sensor prototype, based on continuous biolayer interferometry measurements was applied to detect mAb breakthrough within the last column flow-through to manage column switching. This novel approach provided higher specificity and lower background signal compared to commonly used spectroscopy methods, resulting in an optimized resin utilization while simultaneously avoiding product loss. The dynamic loading was found to provide a twofold increase of the mAb concentration in the eluate compared to a conservative approach with a predefined recipe with similar impurity removal. This concept shows that advanced control strategies can lead to significant process efficiency and yield improvement.

Cost-efficient cell clarification using an intensified fluidized bed centrifugation platform approach.

The intensification of industrial biopharmaceutical production and the integration of process steps pave the way for patients to access affordable treatments. The predominantly batchwise biomanufacturing of established cell clarification technologies, stainless steel disc stack centrifugation (DSC) and single-use (SU) depth filtration (DF), pose technological and economical bottlenecks, that include low biomass loading capacities and low product recoveries. Therefore, a novel SU-based clarification platform was developed combining fluidized bed centrifugation (FBC) with integrated filtration. The feasibility of this approach was investigated for high cell concentration with more than 100E6 cells/mL. Furthermore, scalability to 200 L bioreactor scale was tested for moderate cell concentrations. In both trials, low harvest turbidities (4 NTU) and superior antibody recoveries (95%) were achieved. The impact on the overall economics of industrial SU biomanufacturing using an up-scaled FBC approach was compared with DSC and DF technologies for different process parameters. As a result, the FBC showed to be the most cost-effective alternative for annual mAb production below 500 kg. In addition, the FBC clarification of increasing cell concentrations was found to have minimal impact on overall process costs, in contrast to established technologies, demonstrating that the FBC approach is particularly suitable for intensified processes.

Climate model differences contribute deep uncertainty in future Antarctic ice loss.

Future projections of ice sheets in response to different climate scenarios and their associated contributions to sea level changes are subject to deep uncertainty due to ice sheet instability processes, hampering a proper risk assessment of sea level rise and enaction of mitigation/adaptation strategies. For a systematic evaluation of the uncertainty due to climate model fields used as input to the ice sheet models, we drive a three-dimensional model of the Antarctic Ice Sheet (AIS) with the output from 36 climate models to simulate past and future changes in the AIS. Simulations show that a few climate models result in partial collapse of the West AIS under modeled preindustrial climates, and the spread in future changes in the AIS's volume is comparable to the structural uncertainty originating from differing ice sheet models. These results highlight the need for improved representations of physical processes important for polar climate in climate models.

Non-invasive real-time monitoring of cell concentration and viability using Doppler ultrasound.

Bioprocess optimization towards higher productivity and better quality control relies on real-time process monitoring tools to measure process and culture parameters. Cell concentration and viability are among the most important parameters to be monitored during bioreactor operations that are typically determined using optical methods on an extracted sample. In this paper, we have developed an online non-invasive sensor to measure cell concentration and viability based on Doppler ultrasound. An ultrasound transducer is mounted outside the bioreactor vessel and emits a high frequency tone burst (15 MHz) through the vessel wall. Acoustic backscatter from cells in the bioreactor depends on cell concentration and viability. The backscattered signal is collected through the same transducer and analyzed using multivariate data analysis (MVDA) to characterize and predict the cell culture properties. We have developed accurate MVDA models to predict the Chinese hamster ovary (CHO) cell concentration in a broad range from 0.1 × 106 cells/mL to 100 × 106 cells/mL, and cell viability from 3% to 99%. The non-invasive monitoring is ideal for single use bioreactor and the in-situ measurements removes the burden for offline sampling and dilution steps. This method can be similarly applied to other suspension cell culture modalities.

Retrospective assessment of clonality of a legacy cell line by analytical subcloning of the master cell bank.

In recent years, assurance of clonality of the production cell line has been emphasized by health authorities during review of regulatory submissions. When insufficient assurance of clonality is provided, augmented control strategies may be required for a commercial production process. In this study, we conducted a retrospective assessment of clonality of a legacy cell line through analysis of subclones from the master cell bank (MCB). Twenty-four subclones were randomly selected based on a predetermined acceptance sampling plan. All these subclones share a conserved integration junction, thus providing a high level of assurance that the cell population in the MCB was derived from a single progenitor cell. However, Southern blot analysis indicates that at least four subpopulations possibly exist in the MCB. Additional characterization of these four subpopulations demonstrated that the resulting changes in product quality attributes of some subclones are not related to the genetic heterogeneity observed in Southern blot hybridization. Furthermore, process consistency, process comparability, and analytical comparability have been demonstrated in batches produced across varying manufacturing processes, scales, facilities, cell banks, and cell ages. Finally, process and product consistency together with a high level of assurance of clonal origin of the MCB helped clear the hurdle for regulatory approval without requirement of additional control strategies.

Automation of high CHO cell density seed intensification via online control of the cell specific perfusion rate and its impact on the N-stage inoculum quality.

Current CHO cell production processes require an optimized space-time-yield. Process intensification can support achieving this by enhancing the productivity and improving facility utilization. The use of perfusion at the last stage of the seed train (N-1) for high cell density inoculation of the fed-batch N-stage production culture is a relatively new approach with few industry applicable examples. Within this work, the impact of the cell-specific perfusion rate (CSPR) of the N-1 perfusion and the relevance of its control for the quality of generated inoculation cells was evaluated using an automated perfusion rate (PR) control based on online biomass measurements. Precise correlations (R² = 0.99) between permittivity and viable cell counts were found up to the high densities of 100⋅106 c·mL-1. Cells from N-1 perfusion were cultivated at a high and low CSPR with 50 and 20 pL·(c·d)-1, respectively. Lowered cell growth and an increased apoptotic reaction was found as a consequence of the latter due to nutrient limitations and reduced uptake rates. Subsequently, batch cultivations (N-stage) from the different N-1 sources were inoculated to evaluate the physiological state of the inoculum. Successive responses resulting from the respective N-1 condition were uncovered. While cell growth and productivity of approaches inoculated from high CSPR and a conventional seed were comparable, low CSPR inoculation suffered significantly in terms of reduced initial cell growth and impaired viability. This study underlines the importance to determine the CSPR for the design and implementation of an N-1 perfusion process in order to achieve the desired performance at the crucial production stage.

The Paris Climate Agreement and future sea-level rise from Antarctica.

The Paris Agreement aims to limit global mean warming in the twenty-first century to less than 2 degrees Celsius above preindustrial levels, and to promote further efforts to limit warming to 1.5 degrees Celsius1. The amount of greenhouse gas emissions in coming decades will be consequential for global mean sea level (GMSL) on century and longer timescales through a combination of ocean thermal expansion and loss of land ice2. The Antarctic Ice Sheet (AIS) is Earth's largest land ice reservoir (equivalent to 57.9 metres of GMSL)3, and its ice loss is accelerating4. Extensive regions of the AIS are grounded below sea level and susceptible to dynamical instabilities5-8 that are capable of producing very rapid retreat8. Yet the potential for the implementation of the Paris Agreement temperature targets to slow or stop the onset of these instabilities has not been directly tested with physics-based models. Here we use an observationally calibrated ice sheet-shelf model to show that with global warming limited to 2 degrees Celsius or less, Antarctic ice loss will continue at a pace similar to today's throughout the twenty-first century. However, scenarios more consistent with current policies (allowing 3 degrees Celsius of warming) give an abrupt jump in the pace of Antarctic ice loss after around 2060, contributing about 0.5 centimetres GMSL rise per year by 2100-an order of magnitude faster than today4. More fossil-fuel-intensive scenarios9 result in even greater acceleration. Ice-sheet retreat initiated by the thinning and loss of buttressing ice shelves continues for centuries, regardless of bedrock and sea-level feedback mechanisms10-12 or geoengineered carbon dioxide reduction. These results demonstrate the possibility that rapid and unstoppable sea-level rise from Antarctica will be triggered if Paris Agreement targets are exceeded.

A Machine Vision Approach for Bioreactor Foam Sensing.

Machine vision is a powerful technology that has become increasingly popular and accurate during the last decade due to rapid advances in the field of machine learning. The majority of machine vision applications are currently found in consumer electronics, automotive applications, and quality control, yet the potential for bioprocessing applications is tremendous. For instance, detecting and controlling foam emergence is important for all upstream bioprocesses, but the lack of robust foam sensing often leads to batch failures from foam-outs or overaddition of antifoam agents. Here, we report a new low-cost, flexible, and reliable foam sensor concept for bioreactor applications. The concept applies convolutional neural networks (CNNs), a state-of-the-art machine learning system for image processing. The implemented method shows high accuracy for both binary foam detection (foam/no foam) and fine-grained classification of foam levels.

End-to-end collaboration to transform biopharmaceutical development and manufacturing.

An ambitious 10-year collaborative program is described to invent, design, demonstrate, and support commercialization of integrated biopharmaceutical manufacturing technology intended to transform the industry. Our goal is to enable improved control, robustness, and security of supply, dramatically reduced capital and operating cost, flexibility to supply an extremely diverse and changing portfolio of products in the face of uncertainty and changing demand, and faster product development and supply chain velocity, with sustainable raw materials, components, and energy use. The program is organized into workstreams focused on end-to-end control strategy, equipment flexibility, next generation technology, sustainability, and a physical test bed to evaluate and demonstrate the technologies that are developed. The elements of the program are synergistic. For example, process intensification results in cost reduction as well as increased sustainability. Improved robustness leads to less inventory, which improves costs and supply chain velocity. Flexibility allows more products to be consolidated into fewer factories, reduces the need for new facilities, simplifies the acquisition of additional capacity if needed, and reduces changeover time, which improves cost and velocity. The program incorporates both drug substance and drug product manufacturing, but this paper will focus on the drug substance elements of the program.

A common framework for integrated and continuous biomanufacturing.

There is a growing application of integrated and continuous bioprocessing (ICB) for manufacturing recombinant protein therapeutics produced from mammalian cells. At first glance, the newly evolved ICB has created a vast diversity of platforms. A closer inspection reveals convergent evolution: nearly all of the major ICB methods have a common framework that could allow manufacturing across a global ecosystem of manufacturers using simple, yet effective, equipment designs. The framework is capable of supporting the manufacturing of most major biopharmaceutical ICB and legacy processes without major changes in the regulatory license. This article reviews the ICB that are being used, or are soon to be used, in a GMP manufacturing setting for recombinant protein production from mammalian cells. The adaptation of the various ICB modes to the common ICB framework will be discussed, along with the pros and cons of such adaptation. The equipment used in the common framework is generally described. This review is presented in sufficient detail to enable discussions of IBC implementation strategy in biopharmaceutical companies and contract manufacturers, and to provide a road map for vendors equipment design. An example plant built on the common framework will be discussed. The flexibility of the plant is demonstrated with batches as small as 0.5 kg or as large as 500 kg. The yearly output of the plant is as much as 8 tons.

An Emerging Fluorescence-Based Technique for Quantification and Protein Profiling of Extracellular Vesicles.

Robust and well-established techniques for the quantification and characterization of extracellular vesicles (EVs) are a crucial need for the utilization of EVs as potential diagnostic and therapeutic tools. Current bulk analysis techniques such as proteomics and Western blot suffer from low resolution in the detection of small changes in target marker expression levels, exemplified by the heterogeneity of EVs. Microscopy-based techniques can provide valuable information from individual EVs; however, they are time-consuming and statistically less powerful than other techniques. Flow cytometry has been successfully employed for the quantification and characterization of individual EVs within larger populations. However, traditional flow cytometry is not highly suited for the examination of smaller, submicron particles. Here we demonstrate the accurate and precise quantification of nanoparticles such as EVs using the Virus Counter 3100 (VC3100) platform, a fluorescence-based technique that uses the principles of flow cytometry with critical enhancements to enable the effective detection of smaller particles. This approach can detect nanoparticles precisely with no evidence of inaccurate concentration measurement from masking effects associated with traditional nanoparticle tracking analysis (NTA). Fluorescently labeled EVs from different sources were successfully quantified using the VC3100 without a postlabeling washing step. Moreover, protein profiling and characterization of individual EVs were achieved and have been shown to determine the expression level of target protein markers.

QCM Sensor Arrays, Electroanalytical Techniques and NIR Spectroscopy Coupled to Multivariate Analysis for Quality Assessment of Food Products, Raw Materials, Ingredients and Foodborne Pathogen Detection: Challenges and Breakthroughs.

Quality checks, assessments, and the assurance of food products, raw materials, and food ingredients is critically important to ensure the safeguard of foods of high quality for safety and public health. Nevertheless, quality checks, assessments, and the assurance of food products along distribution and supply chains is impacted by various challenges. For instance, the development of portable, sensitive, low-cost, and robust instrumentation that is capable of real-time, accurate, and sensitive analysis, quality checks, assessments, and the assurance of food products in the field and/or in the production line in a food manufacturing industry is a major technological and analytical challenge. Other significant challenges include analytical method development, method validation strategies, and the non-availability of reference materials and/or standards for emerging food contaminants. The simplicity, portability, non-invasive, non-destructive properties, and low-cost of NIR spectrometers, make them appealing and desirable instruments of choice for rapid quality checks, assessments and assurances of food products, raw materials, and ingredients. This review article surveys literature and examines current challenges and breakthroughs in quality checks and the assessment of a variety of food products, raw materials, and ingredients. Specifically, recent technological innovations and notable advances in quartz crystal microbalances (QCM), electroanalytical techniques, and near infrared (NIR) spectroscopic instrument development in the quality assessment of selected food products, and the analysis of food raw materials and ingredients for foodborne pathogen detection between January 2019 and July 2020 are highlighted. In addition, chemometric approaches and multivariate analyses of spectral data for NIR instrumental calibration and sample analyses for quality assessments and assurances of selected food products and electrochemical methods for foodborne pathogen detection are discussed. Moreover, this review provides insight into the future trajectory of innovative technological developments in QCM, electroanalytical techniques, NIR spectroscopy, and multivariate analyses relating to general applications for the quality assessment of food products.

Future climate response to Antarctic Ice Sheet melt caused by anthropogenic warming.

Meltwater and ice discharge from a retreating Antarctic Ice Sheet could have important impacts on future global climate. Here, we report on multi-century (present-2250) climate simulations performed using a coupled numerical model integrated under future greenhouse-gas emission scenarios IPCC RCP4.5 and RCP8.5, with meltwater and ice discharge provided by a dynamic-thermodynamic ice sheet model. Accounting for Antarctic discharge raises subsurface ocean temperatures by >1°C at the ice margin relative to simulations ignoring discharge. In contrast, expanded sea ice and 2° to 10°C cooler surface air and surface ocean temperatures in the Southern Ocean delay the increase of projected global mean anthropogenic warming through 2250. In addition, the projected loss of Arctic winter sea ice and weakening of the Atlantic Meridional Overturning Circulation are delayed by several decades. Our results demonstrate a need to accurately account for meltwater input from ice sheets in order to make confident climate predictions.

Development of Conjugate Addition of Lithium Dialkylcuprates to Thiochromones: Synthesis of 2-Alkylthiochroman-4-ones and Additional Synthetic Applications.

Lithium dialkylcuprates undergo conjugate addition to thiochromones to afford 2-alkylthiochroman-4-ones in good yields. This approach provide an efficient and general synthetic approach to privileged sulfur-containing structural motifs and valuable precursors for many pharmaceuticals, starting from common substrates-thiochromones. Good yields of 2-alkyl-substituted thiochroman-4-ones are attained with lithium dialkylcuprates, lithium alkylcyanocuprates or substoichiometric amount of copper salts. The use of commercially available inexpensive alkyllithium reagents will expedite the synthesis of a large library of 2-alkyl substituted thiochroman-4-ones for additional synthetic applications.

Numerical simulations of a kilometre-thick Arctic ice shelf consistent with ice grounding observations.

Recently obtained geophysical data show sets of parallel erosional features on the Lomonosov Ridge in the central Arctic Basin, indicative of ice grounding in water depths up to 1280 m. These features have been interpreted as being formed by an ice shelf-either restricted to the Amerasian Basin (the "minimum model") or extending across the entire Arctic Basin. Here, we use a numerical ice sheet-shelf model to explore how such an ice shelf could form. We rule out the "minimum model" and suggest that grounding on the Lomonosov Ridge requires complete Arctic ice shelf cover; this places a minimum estimate on its volume, which would have exceeded that of the modern Greenland Ice Sheet. Buttressing provided by an Arctic ice shelf would have increased volumes of the peripheral terrestrial ice sheets. An Arctic ice shelf could have formed even in the absence of a hypothesised East Siberian Ice Sheet.

The Orbiting Carbon Observatory (OCO-2) tracks 2-3 peta-gram increase in carbon release to the atmosphere during the 2014-2016 El Niño.

The powerful El Niño event of 2015-2016 - the third most intense since the 1950s - has exerted a large impact on the Earth's natural climate system. The column-averaged CO2 dry-air mole fraction (XCO2) observations from satellites and ground-based networks are analyzed together with in situ observations for the period of September 2014 to October 2016. From the differences between satellite (OCO-2) observations and simulations using an atmospheric chemistry-transport model, we estimate that, relative to the mean annual fluxes for 2014, the most recent El Niño has contributed to an excess CO2 emission from the Earth's surface (land + ocean) to the atmosphere in the range of 2.4 ± 0.2 PgC (1 Pg = 1015 g) over the period of July 2015 to June 2016. The excess CO2 flux is resulted primarily from reduction in vegetation uptake due to drought, and to a lesser degree from increased biomass burning. It is about the half of the CO2 flux anomaly (range: 4.4-6.7 PgC) estimated for the 1997/1998 El Niño. The annual total sink is estimated to be 3.9 ± 0.2 PgC for the assumed fossil fuel emission of 10.1 PgC. The major uncertainty in attribution arise from error in anthropogenic emission trends, satellite data and atmospheric transport.