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Bioprocess Alert | Aspen Xchange

Bioprocess Alert

Advanced control and monitoring of bioprocesses are dependent on accurate state and parameter information. At the same time, bioprocesses are well known for their time-varying behavior and difficulty of obtaining online measurements of the important process states. The selection and the tuning of the estimation algorithms is therefore crucial to the design of reliable monitoring tools. In this work we discuss the design of several Moving Horizon Estimation schemes for a class of bioprocesses. We compare the algorithms in terms of the arrival cost computation and address the implementation of a multi-rate measurement structure. The tuning of the estimators and further details are illustrated using a cell culture case study, where we show that not all the estimators under investigation can cope equally well with process uncertainty and multi-rate measurements.
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Recently Deepmind made big headlines with its AlphaFold success. Did it really "solve" protein folding? What did actually happen? In this video I explain what the protein folding problem is, why it's important, and what the current situation is.
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Regenerative medicine aims to create biological therapies or in vitro substitutes for the replacement or restoration of tissue function in vivo lost due to failure or disease. However, whilst science has revealed the biomedical potential of this approach, and early products have demonstrated the power of such therapies, there is a need for the development of bioprocess technology for the successful transfer of the laboratory-based practice of stem cell and tissue culture to the clinic as therapeutics. This Special Issue of Bioengineering brings together contributions from worldwide experts on stem cell science and engineering, bioreactor design and bioprocess development, scale-up, and the manufacturing of stem cell-based therapies.
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We’ve all heard the saying, ‘time is money’. Now, let’s put it into an industrials perspective: consulting firm McKinsey found that products that hit the market on time but went over their budget by 50% reduced profits by 4%. In comparison, companies on budget but late to market by six months earned 33% less profits. That was in the late ’80s! Now imagine delaying in today’s hypercompetitive market. In this article, we’ll see how an agile data management system enables companies to make decisions faster, encourage collaboration and bring their product to consumers sooner.
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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.
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Multi‐column capture chromatography (MCC) has gained increased attention lately due to the significant economic and process‐related advantages it offers compared to traditional batch mode chromatography. However, for wide adoption of this technology in the clinical and commercial space, it requires scalable models for viral validation. In this study, additional viral validation studies were conducted under cGLP guidelines to assess retro‐(X‐MuLV) and parvo‐virus (minute virus of mice) clearance across twin‐column continuous capture chromatography (CaptureSMB) to supplement work previously performed. A surrogate model was validated using standard batch mode chromatography equipment based on flow path modifications to mimic the loading strategy employed in CaptureSMB.
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The biopharmaceutical industry is transitioning from currently deployed batch-mode bioprocessing to a highly efficient and agile next generation bioprocessing with the adaptation of continuous bioprocessing, which reduces the capital investment and operational costs. Continuous bioprocessing, aligned with FDA’s quality-by-design (QbD) platform, is designed to develop robust processes to deliver safe and effective drugs. With the deployment of knowledge based operations, product quality can be built into the process to achieve desired critical quality attributes (CQAs) with reduced variability. We have integrated an Agilent 2D-LC with a post-flow-splitter in conjunction with the SegFlow automated sampler to the bioreactors. Thereby establishing a platform for online measurements of titer and CQAs of monoclonal antibodies (mAbs) as well as amino acid concentrations of bioreactor cell culture.
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This special issue is devoted to new developments in measurement technologies for upstream and downstream bioprocessing. The recent advances in biotechnology and bioprocessing have generated a number of new biological products that require more qualified analytical technologies for diverse process analytical needs. This includes especially fast and sensitive measurement technology that early in the process train can inform on critical process parameters related to process economy and product quality and that can facilitate ambitions of designing efficient integrated end-to-end bioprocesses. 
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This paper provides an overview of nonlinear state estimation techniques along with a discussion on the challenges and opportunities for future work in the field. Emphasis is given on Bayesian methods such as moving horizon estimation (MHE) and extended Kalman filter (EKF). A discussion on Bayesian, deterministic, and hybrid methods is provided and examples of each of these methods are listed. An approach for nonlinear state estimation design is included to guide the selection of the nonlinear estimator by the user/practitioner. Some of the current challenges in the field are discussed involving covariance estimation, uncertainty quantification, time-scale multiplicity, bioprocess monitoring, and online implementation. 
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Modeling is an active area of research within the biotechnology community, and there is a critical need to assess the current state and opportunities for continued investment to realize the full potential of models, including resource and time savings. Beyond individual presentations and topics of novel interest, a substantial portion of the Workshop was devoted toward group discussions of current states and future directions in modeling fields. All scales of modeling, from biophysical models at the molecular level and up through large scale facility and plant modeling, were considered in these discussions and are summarized in the manuscript. 
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Real‐time monitoring of bioprocesses by the integration of analytics at critical unit operations is one of the paramount necessities for quality by design manufacturing and real‐time release (RTR) of biopharmaceuticals. A well‐defined process analytical technology (PAT) roadmap enables the monitoring of critical process parameters and quality attributes at appropriate unit operations to develop an analytical paradigm that is capable of providing real‐time data. In this review, we discuss a broad spectrum of PAT technologies spanning from vibrational spectroscopy, multivariate data analysis, multiattribute chromatography, mass spectrometry, sensors, and automated‐sampling technologies. 
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Single‐use technologies have brought numerous advantages to the biopharmaceutical industry. In particular, single‐use bags made from multi‐layered polymeric films have been adopted for cell culture and liquid handling operations in place of traditional stainless‐steel systems. This publication investigates inconsistent cell growth observed in a PER.C6® cell line during bioprocess development. The growth inhibition was linked to leachable migration from Bioclear™ 10, a single‐use film from Cytiva (formerly GE Healthcare) that was used for cell expansion. It was shown that the PER.C6® cells displayed a sensitivity to bDtBPP, comparable to that observed in sensitive CHO cell lines. Finally, biocompatibility of PER.C6® with Cytiva's new Fortem film was evaluated, demonstrating that Fortem™ film is a suitable single‐use technology for culturing PER.C6® cells.
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The Chinese hamster ovary (CHO) cell lines that are used to produce commercial quantities of therapeutic proteins commonly exhibit a decrease in productivity over time in culture, a phenomenon termed production instability. Random integration of the transgenes encoding the protein of interest into locations in the CHO genome that are vulnerable to genetic and epigenetic instability often causes production instability through copy number loss and silencing of expression. To this end, the epigenomes and transcriptomes of two distantly related cell lines, an industrially relevant monoclonal antibody‐producing cell line and its parental CHO‐K1 host, were characterized.
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The Biopharmaceutical industry has big ambitions to accelerate the move towards industry 4.0. In this white paper we explore some of the major bottle- necks in the development lifecycle and the current barriers to effective digital transformation. We conclude by exploring the role effective BioPharmaceutical Lifecycle Management BPLM platforms will play in accelerating time to market for vaccines and therapies.
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Unfavorable cell heterogeneity is a frequent risk during bioprocess scale-up and characterized by rising frequencies of low-producing cells. Low-producing cells emerge by both non-genetic and genetic variation and will enrich due to their higher specific growth rate during the extended number of cell divisions of large-scale bioproduction. Here, we discuss recent strategies for synthetic stabilization of fermentation populations and argue for their application to make cell factory designs that better suit industrial needs. 
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Often a lot of time is spent on documentation and turnover packages for components, equipment, and systems - time spent reviewing what is in the package, making sure all the necessary documents are there, locating missing documents, etc.  Having robust documentation and turnover packages helps to realize time and money savings during implementation of new equipment. Join Sani-Matic's Mark Espland, automation and documentation manager and Chris McNulty, director of business development (Bio-Pharm) as they discuss what a robust documentation package means for end users, particularly in critical cleaning and validation process.
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Biological systems are typically composed of highly interconnected subunits and possess an inherent complexity that make monitoring, control and optimization of a bioprocess a challenging task. Today a toolset of modeling techniques can provide guidance in understanding complexity and in meeting those challenges. Over the last four decades, computational performance increased exponentially. This increase in hardware capacity allowed ever more detailed and computationally intensive models approaching a “one-to-one” representation of the biological reality. In this review, past, present and envisioned future of models in biotechnology are compared and discussed with regard to application in process monitoring, control and optimization.
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Scaling up cell & gene therapies from research and development to commercialization is creating a new set of challenges for manufacturers. With the urgent demand of speeding up the market reach for these life-altering innovative therapies a high risk is inherent to implement inefficient workflow processes. This article will demonstrate, how adopting innovative single-use solutions for in-process sampling can have a tremendous effect in efficiency and effectiveness to the workflow.  Single-use solutions continue to provide productive strategies in effectively scaling up these processes while reducing risks and costs.
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Due to sustainability concerns, bio-based production capitalizing on microbes as cell factories is in demand to synthesize valuable products. Nevertheless, the non-homogenous variations of the extracellular environment in bioprocesses often challenge the biomass growth and the bioproduction yield. To enable a more rational bioprocess optimization, we have established a model-driven approach that systematically integrates experiments with modelling, executed from flask to bioreactor scale, using ferulic acid to vanillin bioconversion as a case study. The impacts of mass transfer and aeration on the biomass growth and bioproduction performances were examined using minimal small-scale experiments.
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Vaccines have been around for a long time, but with the advancements in single-use technology there has been a rapid adoption of closed systems in order to reduce the risk of contamination that has effectively improved manufacturing. In order to properly close your process, you need reliable and easy-to-use sterile connectors as well as the assurance of supply to get them when you need them.
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Since the approval of the first biotherapeutic protein produced from CHO cells in 1987, researchers have been studying how to improve protein titer and product quality, mainly through cell line development and bioprocess optimization. With recent advances in genetic editing methods (CRISPR/Cas systems) together with large scale systems biology data, further improvements have been made. Here we outline recent progress in protein production from CHO cells through genetic editing and look to the future of improvements through synthetic biology approaches. We describe new work in the expansion of the genetic parts toolkit, including novel promoters, terminators, transcription factors, and genetic circuits, and how these synthetic parts will be used synergistically to continue improvements to protein production.
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In the bioprocess modeling field, a descriptive methodology that explicity considers the interactions between the environment and cells is lacking. This relationship directly affects all kinetics, which have so far been formulated through empirical relationship. In this research, additional steps were added to a known bioprocess modeling methodology to relate environment and cells to each other. The quantitative validation of the proposed phenomenological-based, semi-physical model was developed for a glucose culture bioprocess carried out by the bacterium Escherichia coli strain BW25113 (a derivative of E. coli K-12 strain BD792). The results indicated that the model obtained through the proposed methodology resulted in more accurate predictions than those in the literature using empirical functions for environment-cell relationship modeling. 
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Cell-free systems utilize a subset of cellular components without intact cell wall and/or membranes. The system was first invented for use in fermentation. Subsequent improvements enabled its application in protein synthesis, which is still the most common use of the system. Lately, attempts have been reported where metabolic engineering concepts and techniques were applied to cell-free systems and/or vice-versa. These attempts and advances led to exciting discoveries about biochemical reactions, as well as properties and/or structures of cellular components that make up complex biological systems. This review will first provide a basic overview and brief history of the cell-free system. Then, explanation on recent advances in the field will be provided, followed by notes on the innovative applications. Future outlook of the field will also be covered with the emphasis on how the emerging data science methods can be applied to improve the system and its applicability.
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Digitization in the pharmaceutical industry, Pharma 4.0, is becoming more important every year. The expected benefits of such systems are highly appreciated and, according to the current state of the industry, also needed, especially in the Quality by Design (QbD), which is applied to guarantee consistent product quality. However, it is difficult to apply the widely used and often applied QbD approach correctly because, from a current perspective, there is a lack of basic QbD interpretation and sufficient statistical knowledge of the process experts. The goal of this thesis is to go back to the roots, such as using process knowledge from models and statistical analysis, by investing more time in data analysis rather than in experimental effort by applying existing data science methods useful within the QbD approach. 
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Recently, a generalized manufacturability-assessment tool with strategies to weigh different aspects of manufacturing has been proposed with numerous similarities to that described herein specific to the field of bioprocess development. Although the specific details and design goals of a DfM approach vary by industry, general principles of manufacturability prevail throughout. They are best captured by Shankar and Jannson, who define manufacturability as “the ability to manufacture a product to obtain the desired quality and rate of production while optimizing cost”.
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At the heart of this long-anticipated fundamental shift is a digital transformation of biomanufacturing processes. A shift that may require the re-examination of several things: the way we deploy bioprocess technologies, how we make best use of data and digital platforms – even the way we think.
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The emergence of cell gene therapy (CGT) as a safe and efficacious treatment for numerous severe inherited and acquired human diseases has led to growing interest and investment in new CGT products. The most successful of these have been autologous viral vector-based treatments. The development of viral vector manufacturing processes and ex vivo patient cell processing capabilities is a pressing issue in the advancement of autologous viral vector-based CGT treatments. A summary of some key CGT manufacturing challenges is provided along with a review of MVDA applications to mammalian and microbial processes, and an exploration of the potential benefits, requirements and pre-requisites of MVDA applications in the development of CGT manufacturing processes.
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Biopharma manufacturing process equipment such as bioreactors, fermenters and process piping are generally cleaned using a strategic CIP system design that meets ASME BPE standards. The most efficient, effective and hygienic results are achieved when the CIP system design is considered during the preliminary stages of the facility’s entire process design.
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As the biopharmaceutical industry sharpens its focus on gene therapy, opportunities for advancements and challenges in manufacturing processes still remain. While the industry hasmade progress to effectively treat and control diseases based on regulatory approvals for novel therapies, the industry needs to continue to find efficiencies and optimize manufacturing processes in order to deploy gene therapies economically and at scale.
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Cell culture processes are used to produce the vast majority of protein therapeutics, valued at over US$180 billion per annum worldwide. For more than a decade now, these processes have become highly productive. To further enhance capital efficiency, there has been an increase in the adoption of disposable apparatus and continuous processing, as well as a greater exploration of in-line sensing, various tools, and cell engineering to enhance process controllability and product quality consistency. These feats in cell culture processing for protein biologics will help accelerate the bioprocess advancements for virus and cell therapy applications.
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The typical manufacturing process for biopharmaceuticals includes a cell culture process that generates the molecule of interest (upstream processing), a process to purify the molecule by removing process and product-related impurities (downstream processing), followed by formulation or lyophilization into the final drug product. Despite the complexity of biomanufacturing processes, a significant amount of research and development has been invested into real time process monitoring to facilitate continuous manufacturing of biologics and real time release (RTR) initiatives.3 A systematic approach is essential for successful development and implementation of technology infrastructure for real time process monitoring.
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New innovative Digital Twins can represent complex bioprocesses, including the biological, physico-chemical, and chemical reaction kinetics, as well as the mechanical and physical characteristics of the reactors and the involved peripherals. Digital Twins are an ideal tool for the rapid and cost-effective development, realization and optimization of control and automation strategies. They may be utilized for the development and implementation of conventional controllers (e.g. temperature, dissolved oxygen, etc.), as well as for advanced control strategies (e.g. control of substrate or metabolite concentrations, multivariable controls), and the development of complete bioprocess control.
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Development of affordable and low carbon biobased manufacturing depends critically on strategies that reduce cost and emission profiles. This paper indicates that efforts around the reduction of capital costs by intensification of process equipment need to be carefully weighed against the inherently fast increasing financial and climate costs of driving forces used for the intensification. The fundamental relation between capital expenditures (CAPEX) and operational expenditures (OPEX) of intensified and non-intensified biobased processes and their financial and climatic impacts are emphasized and provisionally explored for a few industrial processes. General learnings flag the importance in particular of OPEX minimization for sustainable bio-economic development.
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Appropriating Biophorum Operations Group (BPOG) testing on single-use connectors to mitigate the risks of extractables and leachables (E&L), as demonstrated in this article, is crucial to delivering safe and effective drugs. This white paper outlines how the AseptiQuik connectors have been tested to ensure that the levels of extractables that come from the connector is quiet low.
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The world continues to look to the scientific community for solutions to the COVID-19 pandemic. Whether you’re a contract development and manufacturing organization (CDMO) or a vaccine manufacturer, demand for novel therapeutics is higher than ever in today’s crisis. Companies without a clear data strategy will likely at some point block a therapy from entering the market. It’s imperative to have good data management practices for scientists and leaders in all departments to ensure quality data and safety, while keeping production costs down and reducing time-to-market now and in the future. 
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This study was carried out to find the optimum clearance (impeller to bottom distance) for Rushton and pitch-blade turbine impellers in a stirred tank bioreactor for improved substrate mixing time added at interface, taking advantage of computational fluid dynamics. In this regard, the time needed for a thin layer of liquid, resembling substrate-rich or poor part, getting homogenously dispersed within the tank was calculated. The mixing time calculated in this way is called the surface aeration related mixing time (SARMT). SARMT was calculated using two approaches and was compared with each other. For the pitch-blade turbine impeller, a criterion which guarantees accurate mixing time by simulation was not satisfied, so the SARMT profile against clearance was not achieved. For the Rushton impeller, a general descending order of SARMT against impeller–bottom clearance was observed.
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Inspired by recent FDA gene therapy approvals, technological advances and successful results, the Cell Culture Dish have partnered with industry experts Pall Biotech, Cobra Biologics, SCIEX, REGENXBIO Inc., BioTechLogic and Jane Barlow & Associates, LLC to share their perspectives in a new ebook, 'Insights on Successful Gene Therapy Manufacturing and Commercialization'. This publication identifies the key challenges, new developments, recent successes, and areas for improvement facing the industry.
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The term Bioprocessing 4.0 has been tossed around since 2018 and is derived from Industry 4.0, a national strategic initiative from the German government launched in 2010 with the aim of driving manufacturing forward by increasing digitization and the interconnection of products, supply chains and business models. Bioprocessing (or Bioprocess) 4.0 today is defined as a totally end- to-end connected bioprocess, where all systems and equipment in the process are connected digitally, forming the Industrial Internet of Things (IIoT) to run, control and even improve the process via feedback loops and artificial intelligence (AI) or machine learning. 
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Let us take you on a journey to the future of smart sensor technology with an excerpt from our upcoming publication, Sensing the Future. Preview the new book by downloading this free nine-page article, titled The Future of Intelligent Process Sensors, to get an inside look into where we’ve been, where we are now, and most importantly, where we’re going.
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This Special Issue of Processes entitled “Bioprocess Monitoring and Control” presents novel examples of on-line monitoring and closed loop control techniques applied to different bioprocesses. The accepted manuscripts cover a range of important topics in different bioprocess areas, where microorganisms, bird’s eggs, and humans are involved. Different techniques such as those for the construction of sensors, the production of a biocontrol agent, scaling up procedures, the application of observers, closed loop control, and the model-based monitoring of a downstream process are presented. The accepted manuscripts are nine original research papers and three reviews.
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Defining a suitable agitation strategy is essential for the successful development of upstream mammalian bioprocesses. Such a strategy can drive oxygen mass transfer within the bio- reactor system to effectively support the aerobic needs of the expanding culture. An inadequate agitation strategy can result in the emergence of chemical gradients within the system. The existence of such nonuniformity within a bioreactor can result in extracellular conditions which may be unfavorable to the overall growth of the cell culture. Hypoxic conditions in a bioreactor are defined by low dissolved oxygen concentrations.
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When technology groups add consulting services to their portfolio, some customers are initially skeptical. The inevitable question is whether a major corporation like Siemens can offer advice independently of its own products and technological solutions. Siemens Digitalization Consulting has already accompanied numerous decision-makers at pharmaceutical companies on their path to digitalization. Its team of experts with backgrounds in industry, IT, and automation enables consultants to work closely in partnership with customers to develop and define their digitalization roadmap. The roadmap is based on the customers’ existing infrastructure and landscape – IT, IoT (Internet of Things), and OT (operational technology), among others – and is tailored to their business challenges and drivers. 
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In 2019, G-CON provided the cleanroom solution for GenCure’s new multi-product cell therapy facility. The new facility, which anchors the VelocityTX innovation center in San Antonio, Texas, expands GenCure’s manufacturing capabilities, providing state-of-the-art manufacturing services to therapeutic developers. This case study captures the experience from the customer's perspective of the use of prefabricated cleanrooms for cell therapy manufacturing.
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Process control strategies based on the physiological status of cells have recently been used to enhance mammalian cell culture productivity and robustness. In this study, we investigated the feasibility of using full-spectrum dielectric spectroscopy for detecting shifts in cell physiology and as a feedback tool to increase process efficiency. Multi-frequency permittivity spectra were collected from cell culture processes in which apoptosis was induced by glucose depletion, nutrient depletion, or chemical treatment. Meanwhile, key parameters of critical frequency (fc) and Cole-Cole alpha (α) were calculated in real time from the β-dispersion curve and correlated to data from off-line viability measurements. Results show that physiological changes in apoptotic cells were reflected in the on-line parameters earlier than from off-line methods.
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Implementing a personalised feeding strategy for each individual batch of a bioprocess could significantly reduce the unnecessary costs of overfeeding the cells. This paper uses lactate measurements during the cell culture process as an indication of cell growth to adapt the feeding strategy accordingly. For this purpose, a model predictive control is used to follow this a priori determined reference trajectory of cumulative lactate.  Each experimental set-up is performed in triplicate and for each run an individualised model-based predictive control (MPC) controller is developed. All process models exhibit an accuracy of 99.80% ± 0.02%, and all simulations to reproduce each experimental run, using the data as a reference trajectory, reached their target with a 98.64% ± 0.10% accuracy on average. Learn More

Bacteriophages are bionanoparticles with several applications in different biotechnology‐based products. Among them, vaccines have the potential to treat antibiotic‐resistant bacteria and parasitic infections. Traditional methods for their recovery and purification rely on precipitation with polyethylene glycol (PEG) and NaCl. However, the applicability of such an approach is limited, due to large‐scale technical constrains. Recently, our research group developed a bacteriophage M13 recovery and purification strategy using Aqueous Two‐Phase Systems (ATPS), simplifying the methodology and, potentially, reducing costs. This work aims to develop an economic contrast between ATPS and the traditional PEG precipitation method at different operation scales (10 to 1000 L bioreactor volume) to determine the applicability of the ATPS methodology at large scale. Learn More

A process characterization is a regulatory imperative for process validation within the biopharmaceutical industry. Several individual steps must be conducted to achieve the final control strategy. For that purpose, tools from the Quality by Design (QbD) toolbox are often considered. These tools require process knowledge to conduct the associated data analysis. They include cause and effect analysis, multivariate data analysis, risk assessment and design space evaluation. Here, we present a novel workflow that shows how simple data analysis tools can be used to investigate the process holistically. This results in a significant reduction of the experimental effort and in the development of an integrated process control strategy. Learn More

Cell culture is at the heart of the production process for many biopharmaceuticals but finding the optimal conditions to maximize yield can be a complex and time-consuming process. Traditional process development relies on costly and labor-intensive set-ups, significantly limiting throughput and the range of experimental conditions that can be assessed.  Scientists in Roche Pharma Research and Early Development (pRED) have adopted an alternative approach, combining single-use microbioreactors with advanced automation and analytical platforms to streamline the workflow. Learn More

Many companies are working to advance the development of a vaccine in the context of the current COVID-19 crisis. New bioprocess technologies, such as single-use equipment and process automation, open up possibilities for quality control and validation. This is especially important in GMP-regulated environments, such as in the development and manufacturing of new influence vaccines. When facing a pandemic outbreak, the need to smoothly develop new processes and quickly scale up to clinical production volumes is key to efficiently develop new vaccinations. BioPharm International recently spoke with Dr. Jörge Schwinde, Key Segment Manager for Vaccines and Monoclonal Antibodies at the Bioprocess Center of Eppendorf, about the typical developmental process of new vaccines and how he COVID-19 pandemic influences the global vaccine developmental landscape. Learn More

Pseudomonas putida is a micro-organism with great potential for industry due to its stress-endurance traits and easy manipulation of the metabolism. However, optimization is still required to improve production yields. In the last years, manipulation of bacterial small non-coding RNAs (ncRNAs) has been recognized as an effective tool to improve the production of industrial compounds. So far, very few ncRNAs are annotated in P. putida beyond the generally conserved. In the present study, P. putida was cultivated in a two-compartment scale-down bioreactor that simulates large-scale industrial bioreactors. We performed RNA-Seq of samples collected at distinct locations and time-points to predict novel and potentially important ncRNAs for the adaptation of P. putida to bioreactor stress conditions.
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The emergence of cell and gene therapies (CGTs) as a novel therapeutic modality has ushered in significant scope and opportunity to address previously unmet need. The rationale behind such therapies is to use cells, somatic, or modified/reprogrammed, to trigger regeneration of damaged tissues or targeting of cancer cells, for example. With increasing numbers of CGT products receiving regulatory approval, there is now a focus on establishing scalable and robust manufacturing processes for both autologous (patient-specific) and allogeneic (universal donor) which are both cost effective and can meet expected demand. One of the cell types under evaluation in numerous clinical trials is human mesenchymal stem/stromal cells (hMSCs) that possess both immunomodulatory properties and in vitro differentiation capability. Learn More

It was developed a fed-batch bioprocess to produce a recombinant vaccine against Entamoeba histolytica under operational conditions attainable to large scale bioprocesses. We have produced this recombinant protein in shake flask and stirred tank bioreactor. Initial results in shake flask cultures under different methanol concentration of 0.5, 1.5 and 3% (v/v) produced extracellular protein at quantities of 10, 22 and 33 μg/mL, respectively. Then a scale-up process was performed from shake flask to fermenter by keeping similar volumetric power supply (P/V). The operational conditions were set up in fermenter as those used at commercial scale and supply of pure oxygen was avoided to keep the scalability of the bioprocess. Learn More

In this episode of How It's Made they explain how unlike traditional drugs synthesized from chemicals, biologic medicines are proteins made from living cells. Learn More

The success of genetically engineered T-cells modified with a chimeric antigen receptor as an adoptive cell immunotherapy and the subsequent last regulatory approvals of products based on this therapy are leading to a crescent number of both academic and pharmaceutical industry clinical trials testing new approaches of this "living drugs". The aim of this review is to outline the latest developments and regulatory considerations in this field, with a particular emphasis to differences and similarities between academic and industry approaches and the role they should play to coexist and move forward together. Learn More

There are many drivers to intensify the manufacturing of a biological product. Advances are occurring throughout the biomanufacturing arena, from process development techniques to improved manufacturing platforms, equipment and facilities. Many employ the term 'bioprocess intensification' to refer to systems for producing more product per cell, time, volume, footprint or cost. This need is being driven by two emerging priorities: cost control and process efficiency. We are seeing great interest in the power of such disciplines as synthetic biology, process simplification, continuous bioprocessing and digital techniques in the optimization of bioprocess development and manufacturing. Other powerful disciplines here include process automation, improved monitoring and prefabricated facility modularity and podularity. Learn More

In this future paper, we would like to take you on a journey into 2025. After all, we often don't realize what it is we should be doing until we look beyond what is right in front of us. Sluggish innovation and fear of disruption are of course common issues, however, allow us to set out in black and white exactly what we think needs to be done. Learn More

In this application note, we explain the differences between batch, fed-batch, and continuous fermentation and how these influence culture growth. As an example, we look at E. coli fermentation processes at bench scale. We track the biomass and nutrient concentrations during batch, fed-batch, and continuous fermentation runs. We explain different methods to analyze the process, including determination of biomass, growth rate, productivity, yield, and analysis of process costs. The comparisons can help bioprocess engineers to select the most appropriate method to meet their needs. In our examples we studied E. coli fermentation at bench scale. The principles may also apply to bioprocesses using other microbes or mammalian cells, at both smaller and larger scales. Learn More

Bioprocess scale-up is a critical step in process development. However, loss of production performance upon scaling-up, including reduced titer, yield, or productivity, has often been observed, hindering the commercialization of biotech innovations. Recent developments in scale-down studies assisted by computational fluid dynamics (CFD) and powerful stimulus-response metabolic models afford better process prediction and evaluation, enabling faster scale-up with minimal losses. In the future, an ideal bioprocess design would be guided by an in silico model that integrates cellular physiology (spatiotemporal multiscale cellular models) and fluid dynamics (CFD models). Learn More

Embryonic stem cells (ESCs) have almost unlimited proliferation capacity in vitro and can retain the ability to contribute to all cell lineages, making them an ideal platform material for cell-based therapies. ESCs are traditionally cultured in static flasks on a feeder layer of murine embryonic fibroblast cells. Although sufficient to generate cells for research purposes, this approach is impractical to achieve large quantities for clinical applications. In this study, we have developed protocols that address a variety of challenges that currently bottleneck clinical translation of ESCs expanded in stirred suspension bioreactors. We demonstrated that mouse ESCs (mESCs) cryopreserved in the absence of feeder cells could be thawed directly into stirred suspension bioreactors at extremely low inoculation densities. Learn More

The Covid-19 pandemic lead to a strong increase of vaccine development projects and new vaccine manufacturing approaches. In this panel discussion, experts from Eppendorf and ContiVir share their thoughts on how they are "Rethinking Vaccine Development". Learn More

The US FDA has released several guideline documents, which are likely to increase the adoption of digital technologies in pharmaceutical manufacturing. These guidelines provided frameworks for electronic submission of clinical data and manufacturing establishment information, while laying down expectations for maintaining data integrity and compliance with good manufacturing practices (GMP). These guidelines have encouraged pharmaceutical companies to adopt advanced processes such as modular manufacturing, electronic batch record systems and computerized maintenance management systems that employ sensors and advanced analytics. This serves as a great foundation for industry to shift from batch processing to continuous processing. Learn More

The convergence of DCS and PLC technologies has made it more challenging than ever for process manufacturers to select the best technology for their application. A successful evaluation should start with developing a clear picture of the requirements of your application and the needs of your engineering, maintenance, and operations personnel. In this paper we will explore key questions to help select the system that best meets your goals. We will demonstrate why having a clear picture of the application requirements and the needs of your engineering, maintenance, and operations personnel is paramount to finding the right automation technology. Finally, we will provide you with a checklist to help determine your optimal system. Learn More

The use of high-throughput systems in cell culture process optimization offers various opportunities in biopharma process development. Here we describe the potential for acceleration and enhancement of product quality optimization and de novo bioprocess design regarding monoclonal antibody N-glycosylation by using an iterative statistical Design of Experiments (DoE) strategy based on our automated microtiter plate-based system for suspension cell culture. In our example, the combination of an initial screening of trace metal building blocks with a comprehensive DoE-based screening of 13 different trace elemental ions at three concentration levels in one run revealed most effective levers for N-glycan processing and biomass formation. Learn More

How often do you pipette in your cell culture lab every day? Usually, we do it so often that we tend stop thinking about how to pipette right. With this educational webinar we would like you to stop for a moment, re-think what you are doing, and significantly improve your skills and results. We would like to share Eppendorf´s almost 60 years of experience in proper liquid handling with you - for more reproducible cell culture results and better contamination protection in your lab. Learn More

Being different from the continuous process, batch processes in the practical industry have several distinct characteristics, such as the unsteady state, severe nonlinearity, and iterative operation. For tracking a reference trajectory of a batch process, data-driven model predictive controllers have been proposed with the progress of sensors and machine learning. Among them, the latent variable space model-based controllers (LV-MPC) have been applied to the batch processes for decades. We propose to update the local model in the manner of just-in-time learning (JITL) and to use them to the predictive controller design at first. Learn More

Mammalian cell cultures are widely used in the biopharmaceutical industry to produce monoclonal antibodies, vaccines, growth factors, etc. Cell death is an essential biological process for physiological growth and development, but it is a major problem in biopharmaceutical production in bio-industry. Cell death within bioreactor occurs due to various intracellular and extracellular stresses. These stresses negatively affect the culture longevity, overall product quality, and yield. Among all cell death types, apoptosis accounts for most of the cellular death in the bioreactor. In this review, we classified and described different types of cell death and their molecular mechanisms and summarized the cell death inhibition approaches implemented to inhibit cell death for various applications. Learn More

Owing to the increasing demand for large scale and high efficiency in manufacturing processes, computer aided tools for process operation and control are rapidly gaining popularity. An important state variable in aerobic processes is the dissolved oxygen, which can be easily measured online and is an important indicator of the metabolic activity. However, due to the fast kinetics of the oxygen transfer, dynamical models describing aerobic bioprocesses tend to be highly stiff. In this work we use the slow-motion invariant manifold and the quasi steady state assumption methods to eliminate the differential equation describing the dissolved oxygen (the fast mode). Learn More

In recent years, multivariate data analysis (MVDA) and modeling approaches have found increasing applications for upstream bioprocess studies (e.g., monitoring, development, optimization, scale-up, etc.). Many of these studies look at variations in the concentrations of metabolites and cell-based measurements. However, these measures are subject to system inherent variations (e.g., changes in metabolic activity) but also intentional operational changes. It is proposed to perform MVDA and modeling on data representative of the underlying biological system operation, that is, the specific rates, which are per se independent of the scale, operational strategy (e.g., batch, fed-batch), and biomass content. Learn More

The adoption of next generation bioprocessing is challenged as biopharmaceutical companies grapple with the adoption of new manufacturing methods, which some consider to be essential to the industry's continued growth and innovation. Within the next five years, it is estimated that approximately 35 percent of today's biologics will be manufactured using some sort of process intensification methods. Facilities will be smaller but capable of producing higher volumes of multiple molecules.

The key to this advancement will be the use of methods such as continuous bioprocessing technologies, some of which may be single use, which would enable facilities to reduce both their size and cycle times. The complexity of the bioprocessing industry has challenged companies' innovation efforts, due to potential costs required for changes to take place. Learn More

In the biopharmaceutical industry, Raman spectroscopy is now a proven PAT tool that enables in-line simultaneous monitoring of several CPPs and CQAs in real-time. However, as Raman monitoring requires multivariate modeling, variabilities unknown by models can impact the monitoring prediction accuracy. With the widespread use of Raman PAT tools, it is necessary to fix instrumental variability impacts, encountered for instance during a device replacement. In this work, we investigated the impact of instrumental variability between probes inside a multi-channel analyzer and between two analyzers and explored solutions to correct them on model prediction errors in cell cultures. Learn More

A major hurdle during process development and optimization studies is the huge experimental effort in conventional design of experiments (DoE) methods. The combination of DoE with a virtual representation of the bioprocess, called digital twin, in model-assisted DoE (mDoE) can be used as an alternative to decrease the number of experiments significantly. In this chapter, digital twins and their role in mDoE are discussed. First, statistical DoE methods are introduced as the basis of mDoE. Second, the combination of a mathematical process model and DoE into mDoE is examined. This includes mathematical model structures and a selection scheme for the choice of DoE designs. Learn More

Human induced pluripotent stem cells (hiPSCs) hold enormous promise in accelerating breakthroughs in understanding human development, drug screening, disease modeling and cell and gene therapies. Their potential, however, has been bottlenecked in a mostly laboratory setting due to bioprocess challenges in the scale-up of large quantities of high-quality cells forclinical and manufacturing purposes. While several studies have investigated the production of hiPSCs in bioreactors, the use of conventional horizontal-impeller, paddle and rocking-wave mixing mechanisms have demonstrated unfavourable hydrodynamic environments for hiPSC growth and quality maintenance. This study focused on using computational fluid dynamics (CFD) modeling to aid in characterizing and optimizing the use of vertical-wheel bioreactors for hiPSC production. Learn More

Glutathione (GSH) plays a central role in the redox balance maintenance in mammalian cells. Previous studies of industrial Chinese hamster ovary cell lines have demonstrated a relationship between GSH metabolism and clone productivity. However, a thorough investigation is required to understand this relationship and potentially highlight new targets for cell engineering. In this study, we have modulated the GSH intracellular content of an industrial cell line under bioprocess conditions to further elucidate the role of the GSH synthesis pathway. Learn More

Biological molecules are widely produced by fermentation technology using bacteria, fungi or yeast. Fermentation is a biochemical process wherein the rate of bioconversion is governed by the organisms involved. The growth of the organism is mainly limited by mass transfer rates of nutrients and gases that directly affect the product formation in fermentation. The growth rate of microbes can be accelerated by increased mass transfer rates and cell wall permeability with the use of controlled low frequency ultrasound irradiation. The present review provides insights into the application of acoustic cavitation in process intensification of fermentation approaches and the role of various factors involved are highlighted with typical examples. Learn More

Ammonia is an ubiquitous compound. It is used by humans in industrial fertilizers and refrigerants. Furthermore, it is produced by animals as an excretion product as well as a product in amino acid degradation and waste decomposition. Continuous mammalian cell lines (CCLs), which are important hosts for the production of biological pharmaceuticals, generate ammonia during the cell metabolism. This is due to the lack of energy in form of ATP. To close this gap, these cells consume glutamine to produce α-ketoglutarate, which is further degraded in the mitochondria and as a second product poising ammonia. Ammonia inhibits cell growth and further yields to cell death. Learn More

The development of a continuous process for cell separation is growing rapidly due to the current trend of cost-effective manufacturing in biological industries. The continuous cell separation process has a significant reduction in capital equipment costs and facility size compared to the conventional batch process. In the study, a multi-layered microfluidic-based device integrated with the porous membranes was fabricated for continuous size-based isolation of the cells based on the mechanism of restrictive cross-flow filtration, allowing the biological sample entered in a single inlet of the device and separated into two outlet streams. Learn More

Due to its beneficial attributes, silicone is utilized today in countless medical and pharmaceutical applications. It is the preferred material for sealing complex systems as well as for peristaltic and pharmaceutical tubing. Silicone is available in a wide range of durometers, from super soft (000) to relatively hard (90 Shore A), and can withstand a range of temperatures. The silicones readily available in today's market differ not only in their level of approval for medical and pharmaceutical applications, but also in their crosslinking mechanism. There is a clear distinction between platinum-cured and peroxide-cured silicones. Learn More

In the past 15 years, the biopharma industry has seen a steady uptake in the use of single-use technology in bioprocessing. Single-use technology is being developed for nearly every application from upstream mixing and bioreaction to downstream perfusion and chromatography. In the pursuit of personalized medicine and continuous processing, single-use is being hailed as the only way to achieve these goals. Does this mean an end for traditional bioprocesses that employ stainless steel technology? Is stainless steel already being considered a legacy product as we journey towards fast, closed and process-intensified manufacturing systems?

Marc Pelletier, Director of CRB, and Mark Embury from ASEPCO, part of Watson-Marlow Fluid Technology Group (WMFTG), discuss whether stainless steel is a technology of the past. Learn More

This paper presents various software based approaches suitable for the design of knowledge-based monitoring of biotechnological production processes. These processes require special treatment with respect to the complexity in biochemical reactions which make the design and construction of reasonably complex and practically usable mathematical models rather difficult. Additional complexity arises from the lack of industrially viable sensors for on-line measurement of key process variables. Software sensors which often use tools from the field of artificial intelligence represent one of the suitable approaches for the overcoming of the above mentioned limitations for its ability to utilize effectively both quantitative and qualitative knowledge about the monitored bioprocess. Learn More

In the last years, regulatory agencies in biopharmaceutical industry have promoted the design and implementation of Process Analytical Technology (PAT), which aims to develop rapid and high-throughput strategies for real-time monitoring of bioprocesses key variables, in order to improve their quality control lines. In this context, spectroscopic techniques for data generation in combination with chemometrics represent alternative analytical methods for on-line critical process variables prediction. In this work, a novel multivariate calibration strategy for the at-line prediction of etanercept, a recombinant protein produced in a mammalian cells-based perfusion process, is presented. Learn More

What is between Downstream and Fill & Finish? Most of the time, fill and finish services for drug substances are not located at the same venue that the drug substance is produced at. Pharmaceutical and biotechnology companies look to contract manufacturing organizations (CMOs) as a way to meet their fill- and-finish needs and reduce risk. As a result, the shipping of liquid drug substances around the globe is often inevitable and the challenges arise. Learn More

The production of pharmaceuticals, industrial chemicals, and food ingredients from biotechnological processes is a vast and rapidly growing industry. While advances in synthetic biology and metabolic engineering have made it possible to produce thousands of new molecules from cells, few of these molecules have reached the market. The traditional methods of strain and bioprocess development that transform laboratory results to industrial processes are slow and use computers and networks only for data acquisition and storage. Digitalization, machine learning (ML), and artificial intelligence (AI) methods are transforming many fields - how can they be applied to bioprocessing to overcome current bottlenecks? Learn More

The various mammalian cell lines used for diagnostic and therapeutic protein expression exhibit distinct metabolic features. Even clones descending from the same parental cell line and expressing the same product can have individual nutritional requirements. We established a comprehensive platform for fast and robust quantitation of 55 cell culture media constituents and metabolites in spent media. This includes for example an advanced UPLC method developed in-house that allows for the parallel quantitation of more than 30 (non-) proteinogenic amino acids and dipeptides. Learn More

Based on conversations with customers over the past several years, Univercells has identified the major hurdles in largescale virus manufacture, which include expensive manufacturing facilities, lack of expertise, limitations in the number of scalable manufacturing technologies available in the market, and the high cost of good manufacturing practice (GMP) grade reagents including transfection mix, plasmids, and bovine serum. Incorporating design-of-experiment study principles and use of improved bioprocess control and data analysis software are allowing process development/optimization to be conducted at the multiparameter level... Learn More

Large-scale production of induced pluripotent stem cells (iPSCs) is essential for the treatment of a variety of clinical indications. However, culturing enough iPSCs for clinical applications is problematic due to their sensitive pluripotent state and dependence on a supporting matrix. In this review, we present an overview of cell culture media, suspension modalities, and monitoring techniques that preserve the quality and pluripotency of iPSCs during initiation, expansion, and manufacturing. Learn More

A continuous integrated bioprocess available from the earliest stages of process development allows for an easier, more efficient and faster development and characterization of an integrated process as well as production of small-scale drug candidates. The process presented in this article is a proof-of-concept of a continuous end-to-end monoclonal antibody production platform at a very small scale based on a 200ml alternating tangential flow filtration perfusion bioreactor, integrated with the purification process with a model-based design and control. Learn More

A survey of bioprocessing industry professionals asked which method they prefer for their connection needs: sealable and weldable tubing or aseptic connectors. Respondent opinion was almost equally divided with 52 percent choosing aseptic connectors and 48 percent preferring sterile tube welding. So how do you determine the right method to maintain batch integrity and maximize manufacturing flexibility while avoiding product loss, system downtime, and potential for operator error? Learn More

From MIT Video Productions comes "From Controversy to Cure," a 57-minute documentary history of the birth and boom of the molecular biology and biotechnology industries in Cambridge and specifically Kendall Square. Today, the area is home to one of the largest clusters of biotechnology and pharmaceutical companies in the world, and is, according to this film, "on the front lines to defeat COVID-19." Not long ago, Kendall Square was a wasteland of vacant plants and empty space cleared away for a NASA electronics research center project that was killed by the Nixon administration as part of spending cuts demanded by the crippling costs of the Vietnam War. Learn More

In biotechnology, the emergence of high-throughput technologies challenges the interpretation of large datasets. One way to identify meaningful outcomes impacting process and product attributes from large datasets is using systems biology tools such as metabolic models. However, these tools are still not fully exploited for this purpose in industrial context due to gaps in our knowledge and technical limitations. In this paper, key aspects restraining the routine implementation of these tools are highlighted in three research fields: monitoring, network science and hybrid modeling. Advances in these fields could expand the current state of systems biology applications in biopharmaceutical industry. Learn More

The purpose of this book is to present the different approaches most commonly employed in the control of bioprocesses. It aims to develop in some detail the bases and concepts of bioprocesses related to the control theory introduced in basic principles of mathematical modeling in bioprocesses. Learn More

Across the biopharmaceutical industry, the transition from traditional constructs such as large stainless-steel factories, batch manufacturing, and paper-based recording systems to modular facilities, single-use technologies, continuous manufacturing, and electronic systems is picking up pace. It's easy to see why: the benefits of greater agility and responsiveness for capacity planning, significantly reduced manufacturing costs, and improved product quality speak for themselves. Join Eliot Randle and Claire Hill as they discuss what's needed to address the current industry challenges and transform the future of biomanufacturing. Learn More

Process intensification strategies are needed in the field of therapeutic protein production for higher productivities, lower cost of goods and improved facility utilization. This work describes an intensification approach, which connects a tangential-flow- filtration (TFF) based pre-stage perfusion process with a concentrated fed-batch production culture inoculated with an ultra- high seeding density (uHSD). This strategy shifted biomass production towards the pre-stage, reaching up to 45 × 106 cells/ mL in perfusion mode. Subsequently, production in the intensified fed-batch started immediately and the product titer was almost doubled (1.9-fold) in an equivalent runtime. Learn More