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In this paper, a fractional Lotka-Volterra mathematical model for a bioreactor is proposed and used to fit the data provided by a bioprocess known as continuous fermentation of Zymomonas mobilis. The model contemplates a time-delay τ due to the dead-time in obtaining the measurement of biomass x(t). A Hopf bifurcation analysis is performed to characterize the inherent self oscillatory experimental bioprocess response. As consequence, stability conditions for the equilibrium point together with conditions for limit cycles using the delay τ as bifurcation parameter are obtained. Under the assumptions that the use of observers, estimators or extra laboratory measurements are avoided to prevent the rise of computational or monetary costs, for the purpose of control, we will only consider the measurement of the biomass. 
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Anchorage-dependent cells, such as Vero cells, are widely used as a platform for viral vaccine production. In perfusion bioprocesses it is possible to constantly add nutrients and to remove byproducts, while retaining the cells in the bioreactor. Therefore higher cell densities can be reached than in conventional batch or fed-batch processes. In this study we tested the suitability of a spin filter as cell retention device.
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In this study, the productivity of Chinese Hamster Ovary (CHO) cells expressing a monoclonal antibody was analyzed over the course of the cultivation process. In addition to calculating the specific cell productivity based on the traditional product titer determined by HPLC analysis, culture productivity of single cells was also analyzed via flow cytometry using a cold capture assay. The cold capture assay is a cell surface labelling technique described by Brezinsky et al., which allows for the visualization of a product on the surface of the producing cell. The cell productivity results obtained via HPLC and the results of cold capture assay remained in great accordance over the whole cultivation process. Accordingly, our study demonstrates that the cold capture assay offers an interesting, comparatively time‐effective, and potentially cheaper alternative for monitoring the productivity of a cell culture.
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Adoptive T‐cell therapy (ACT) has emerged as a promising new way to treat systemic cancers such as acute lymphoblastic leukemia. However, the robustness and reproducibility of the manufacturing process remains a challenge. Here, a single‐use 24‐well microbioreactor (micro‐Matrix) was assessed for its use as a high‐throughput screening tool to investigate the effect and the interaction of different shaking speeds, dissolved oxygen (DO), and pH levels on the growth and differentiation of primary T cells in a perfusion‐mimic process. The full factorial design allowed for the generation of predictive models, which were used to find optimal culture conditions.
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Modern pharmaceuticals frequently rely on cell cultures grown in bioreactors or fermenters as part of large-scale manufacturing lines. This is made possible by the development of prototype processes using benchtop equipment to create controllable environments for research and regulatory approval. At the heart of this equipment is control over the gases that influence fermentation and optimize performance.
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The high costs of and limits on global accessibility of biologics such as monoclonal antibodies (MAbs) are focusing the biopharmaceutical industry’s attention on strategies for rapid, economical development of such therapies. Process intensification is one approach to help shorten manufacturing timelines and reduce cost of goods (CoG). Today, process intensification in upstream cell culture enables biologics manufacturing in facilities with smaller footprints and lower scale-up volumes than was possible before. Intensified processing of Chinese hamster ovary (CHO) clones that produce MAbs is being developed in the seed train of upstream cell cultures to support generation of high–cell-density (HCD) cell banks and processes to reduce plant size, capital investment, and overhead costs while increasing productivity.
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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 for clinical 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 unfavorable 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.
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Chinese hamster ovary (CHO) cells are utilized as primary mammalian expression host cells for the production of biopharmaceuticals. Recombinant CHO cell line development (CLD) has been a crucial step for therapeutic protein production platforms; however, this step remains time-consuming and costly. With the emergence of multiomics data sets of CHO cells and genome editing technology such as CRISPR/Cas9, site-specific integration-based cell line development, and engineering have been successfully implemented in CHO cells for predictable transgene expression and expediting the process of CLD. This review describes the trends in CHO CLD from random to targeted approaches. And we cover the major obstacles faced in rational CHO CLD and the potential strategies employed to overcome its limitations. Finally, we conclude by discussing future directions and challenges for next-generation CHO cell factories.
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Cell therapies constitute one of the most prosperous fields of research, as they advanceing the creation of new companies offering both products and services within a vast range of logical innovations, from autologous cell transplantation in the clinics to cell-printed tissue for research use and validation tests in pharmaceutical development. According to Frost & Sullivan, the cell therapy manufacturing industry is expected to experience a great revolution in terms of products launched and customer niches penetrated in the next five years.  Therefore, costs associated with cycle developmental time around novel solutions become decisive for commercialization success. Flexible manufacturing platforms and smart-based quality control systems embracing a broad spectrum of adjacent technology solutions are poised to lead the way toward world-class, high performance, cost-effective cell therapy manufacturing platforms and facilities.
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With 20 years of experience in the biopharmaceutical industry — at Genentech, Applied Biosystems, Cell Genesys, Cellerant Therapeutics, and Bayer — Yuval Shimoni has written frequently for BioProcess International on a number of production topics. Those have ranged from process improvements and bioreactor scale-down validation, to raw materials management, to addressing variability and virus contamination events. For this featured report, we discussed hardware and instrumentation, quality by design (QbD) and related approaches, and other strategies that can take expediting upstream process development beyond just cell-line development.
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In bioprocess development, the host and the genetic construct for a new biomanufacturing process are selected in the early developmental stages. This decision, made at the screening scale with very limited information about the performance in larger reactors, has a major influence on the efficiency of the final process. To overcome this, scale-down approaches during screenings that show the real cell factory performance at industrial-like conditions are essential. We present a fully automated robotic facility with 24 parallel mini-bioreactors that is operated by a model-based adaptive input design framework for the characterization of clone libraries under scale-down conditions.
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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 during culture can be prevented or inhibited by supplementing media with specific chemicals, synthetic inhibitors, and genetic cell engineering approaches. 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.
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Bioprocess development and optimization is a challenging, costly, and time-consuming effort. In this multidisciplinary task, upstream processing (USP) and downstream processing (DSP) are conventionally considered distinct disciplines. This consideration fosters “one-way” optimization without considering interdependencies between unit operations; thus, the full potential of the process chain cannot be achieved. Therefore, it is necessary to fully integrate USP and DSP process development to provide balanced biotechnological production processes. The aim of the present study was to investigate how different host/leader/antigen binding fragment (Fab) combinations in E. coli expression systems influence USP and primary recovery performance and the final product quality. 
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Modelling turbulence is essential in the chemical and bioprocess industry due to the mixing it creates. In the past, engineers have used two-equation Reynolds Averaged Navier–Stokes (RANS) k –  𝜖 model due to its economical nature; however, it lacks accuracy; whereas direct numerical simulation (DNS) is computationally expensive. Large eddy simulation (LES) turbulence model provides a bridge between the above two models as it resolves the larger scales and models the smaller universal scales of motion. The current work presents a detailed numerical analysis of turbulent flow over a two-dimensional backward-facing step (BFS) using a continuous Galerkin finite element method in an open-source finite element framework—Fluidity, which allows fully unstructured aniostropic adaptive mesh refinement along with the use of distributed parallelism. 
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With the rapid growth of pharmaceutical and biotechnology industry, stirred tank bioreactors have received much attention due to simple design, easy control of operating conditions, and low operating cost. In the development of commercial processes, however, a transition from laboratory to industrial scale faces great challenges because many properties related to size change nonlinearly as a system increases. In this context, along with an understanding of fluid dynamics, application of an efficient method for scale-up is critical for designing successful industrial process. In the present study, the effect of various key operating variables such as agitation rate and aeration rate, impeller diameter, and bioreactor working volume for different impellers on the volumetric mass transfer coefficient (kLa) have been investigated in a stirred tank bioreactor for cultivating Escherichia coli BL21. 
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Disposable orbitally shaken bioreactors have been widely used for mammalian cell culture in suspension. Three kinds of baffle structures: vertical baffle, inclined baffle and horizontal baffle were designed in this work. The flow fields of the shaking bioreactor with different baffle structures were simulated, and the turbulence, dissolved oxygen and shear strain rate of the bioreactor were analyzed. The results showed that the quasi-steady-state flow patterns of the unbaffled shaking bioreactors were broken for the bioreactors with the strengthening effects of baffles. The mixing and the oxygen volumetric mass transfer coefficient (kLa) (simulated results) were improved significantly, and the shear strain rates were also increased greatly for the baffle bioreactors.
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Scalable processes are requisite for the robust biomanufacturing of human pluripotent stem cell (hPSC)‐derived therapeutics. Toward this end, we demonstrate the xeno‐free expansion and directed differentiation of human embryonic (hESCs) and induced pluripotent cells (hiPSCs) to definitive endoderm (DE) in a controlled stirred suspension bioreactor (SSB). Based on previous work on converting hPSCs to insulin‐producing progeny, differentiation of two hPSC lines was optimized in planar cultures yielding up to 87% FOXA2+/SOX17+ cells. Next, hPSCs were propagated in a SSB with controlled pH and dissolved oxygen. This work advances bioprocess development for producing a wide gamut of human DE cell‐derived therapeutics.
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Bioprocesses are often carried out in stirred tank bioreactors. Due to the need of mixing, homogenization and interphase transports, cultures are usually agitated by mechanical, hydraulic or pneumatic means. The increasing in agitation decreases mixing time, improves homogeneity, mass and heat transfer rates and, accordingly, the metabolism and the biochemical reaction rates are also influenced. The primary goal of this work is to describe up to date knowledge on these concepts: fluid dynamic conditions, hydrodynamic stress and oxygen availability influence affecting the microbial processes performance.
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The 3D design and printing process provides a fast, efficient, and cost-effective platform, with which to model and then to test the design and operation of critical reactor features in real world conditions. Impeller design and performance may be matched to and optimized for a particular cell line and performance objective.
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Many shaken bioprocesses progress from a small scale (e.g. microtiter plate) to a relatively large scale (e.g. 2,500L bioreactors). Once a culture has been optimized for a microtiter plate (MTP), it can be scaled into larger vessels using a variety of parameters (e.g. OTRmax); however, the initial shaking conditions for a micro-titer plate are not always known. Here we discuss the key equations and relationships that can be used to determine the initial conditions for a shaken bioprocess in an MTP and illustrate an example in a 96-well plate.
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Guidance to meet pharmaceutical manufacturing regulatory standards is everchanging, as is highlighted in the recent 2019 ASME BPE System Design Standards for Immersion Washers. Two Sani-Matic experts, Solo Yang, sales engineer and BPE contributing member, and Pete Barrie, senior product manager and BPE Systems Design committee member, provide a brief overview of ASME BPE, how standards come to be, and the five must-knows about the recent BPE System Design Standards for Immersion Washers
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In the context of Process Analytical Technologies (PAT) implementation in the biopharmaceutical industry, Quality by Design (QbD) is being developed and widely implemented and used. In upstream processes, one compound of great interest to monitor is glucose, and specifically, being able to control its concentration during the process. Such a monitoring leads to process quality improvement, including glycosylation of the product of interest. In this study, a Raman analyzer has been successfully used to implement a feedback control loop in a CHO cell culture based on glucose concentration. The feedback control loop implied a direct OPC UA connectivity between the analyzer and the bioreactor control system.
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A major obstacle to anti‐viral and ‐tumor cell vaccination and T cell immunotherapy is the ability to produce dendritic cells (DCs) in a suitable clinical setting. It is imperative to develop closed cell culture systems to accelerate the translation of promising DC‐based cell therapy products to the clinic. The objective of this study was to investigate whether viral antigen‐loaded monocyte‐derived DCs (Mo‐DCs) capable of eliciting specific T cell activa‐ tion can be manufactured in fluorinated ethylene propylene (FEP) bags.
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The paper discusses modelling and optimization of multi-component cell culture medium. The specific productivity (Qp) was considered a function of the medium components and possible interactions described by linear factors, two-way interactions and squared terms that results in a high dimensional problem where the number of variables p (represented by the medium components and their interactions) is much larger than the number of observations n. Principal Components Regression (PCR), Partial Least Squares (PLS), Lasso and Elastic Net regressions were compared as modelling tools to deal with a high dimensional 𝑛<𝑝n<p problem. PCR and PLS regression models resulted in better prediction results and were used for robust optimization of the medium composition by a nonlinear optimization. The case studies show that it is possible to formulate new media that result in higher Qp than the ones provided by the initial media experiments available.
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Cell culture media are highly complex solutions of multiple nutrients for mammalian cells and lay the foundation for enhanced quality of the expressed protein and the performance of the bioprocess. Especially for long term, large-volume continuous production processes the constant quality of media without compositional variation is desirable, despite exposure to various stress conditions like (UV)-light or (high)-temperature cannot be represented by a measurable parameter yet. In this study, chemometric analysis of non-enhanced RAMAN spectra was used to identify different cell culture media and to track accelerated degradation conditions by light or temperature in aqueous solutions of glucose and chemically defined media. 
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Shake flasks are a widely used and incredibly useful bioreactor for bioprocess development and scale-up of mammalian and microbial bioprocesses. There are several parameters to consider when optimizing a bioprocess in a shake flask, including the conditions of the shaker platform (e.g. shaking frequency and orbital diameter) and the conditions of the shake flask (e.g. flask size and filling volume). The maximum oxygen transfer capacity (OTRmax) is a measure of the maximum rate at which oxygen transfers between the gas and the liquid phase in the vessel. Since most cells require oxygen to grow, this is generally viewed as a key predictor for the performance of a shaken bioprocess. Here we review how the OTRmax of a shake flask will vary as a function of both shaker conditions and flask conditions.
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Today, the benefits of single-use (SU) technologies for upstream processing are much more prevalent and heavily adopted within biopharmaceutical industry. Pall has developed the Allegro™ STR stirred single-use bioreactor family which is currently available in 4 sizes: STR 50, STR 200, STR 1000, and STR 2000. The direct-bottom mechanically driven impeller allows large range of power inputs from 0.002 W/kg up to 0.3 W/kg, while the macro sparger seated below the three 45-degree elephant ear blades results in high oxygen transfer rates (kLa up to 40 h-1) and short mixing time (minimum tM~10 s).
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This guide describes the detailed procedure of Pichia pastoris fermentation in a BioBLU® 3f Single-Use Bioreactor controlled by a BioFlo® 320 bioprocess control system. We ran a fed-batch fermentation and started feed- ing upon the appearance of a significant dissolved oxygen spike, which indicated carbon source depletion. Finally, based on this fermentation strategy, a reasonably high- density P. pastoris culture was obtained in the single-use vessel. This guide can be very helpful for those who are new to P. pastoris fermentation in bioreactors.
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The production of monoclonal antibodies with a mammalian cell culture process is a complex and challenging step in modern medicine to achieve final pharmaceutical products such as vaccines. Viable cell concentration (VCC) is one of the most important key performance indicator during mammalian cell cultivation mainly measured offline. According to FDA ́s PAT initiative, process monitoring and control should be applied to gain process understanding and improve control of process parameters leading to high product quality. Thus, the implementation of an online capacitance probe in a small scale bioreactor is demonstrated.
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The combined effects of mixed electrolyte species and glucose on oxygen transfer were studied in a bubble column with aqueous solutions. Of particular interest was the presence of electrolytes containing ions which are prone to present solute–solute interactions or to crystallize. Without and at low concentration of glucose (≤ 5 g/L), the increasing concentration of electrolytes (nominal ionic strength: 0–0.43 M), up to a critical value, enhanced the volumetric mass transfer coefficient (kLa) and the availability of specific interfacial area (a), due to the inhibition of bubble coalescence.
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Our "Essential Laboratory Skills" Guide supports lab operators to recognize and avoid potential error sources and ultimately increase laboratory efficiency. Experience shows that modern, well-developed balances and analytical instruments do the most to promote ease of use, operational safety and accurate results. However, even with state-of-the-art solutions, practical know-how is still required for users to do things right and achieve reliability and consistency.
Basic measurement techniques covered in this guide include: Weighing, Pipetting, pH Measurement, Moisture Content, Titration, UV/VIS, Density & Refractive Index, Melting, Dropping, Boiling, Cloud and Slip Melting Point and Thermal Analysis.
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In the past decade, upstream scale-up of mammalian cell culture has changed and so have the challenges associated with it. However, the cost of delaying process transfer remains stubbornly high. As manufacturing scales increase, these soon mount up to hundreds of thousands of dollars, with Matzmorr estimating that for a biologic with $1 billion sales annually, the cost of every month lost during technology transfer is an eye-watering $80 million. With figures like this, it is little wonder that getting scale-up right is what keeps bioprocess scientists awake at night.
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Bioreactors are a space-efficient method of growing cells en masse for industrial operations by suspending the cells in an agitated vessel full of cell culture media. Adherent cell lines can be grown on microcarriers in bioreactors to reduce the space needed for petri dishes or flasks in the lab. One of the important factors in cell culture is changing the cell media to remove cell waste, secreted products, and/or replenish the nutrients available. Bioreactors face unique challenges with media changes, as cells should not be removed from a culture when media is changed. Currently, some labs let the cells settle to the bottom of the bioreactor for 30-45 min before decanting the spent media and refilling the tank with new media. This project provides bioreactor users with three single-use products that separate microcarriers from media in a continuous flow, to shorten the time adherent cells are outside of their ideal environment. Learn More

The recent trend in bioprocess development towards small fermentors and high cell densities presents challenges for accurate on-line monitoring of liquid cell cultures. Traditional on-line optical probes, such as transmission probes (also known as absorbance or optical density probes) and reflectance probes (also known as back-scatter or turbidity probes), typically require a 12 mm or larger diameter port into the bioreactor vessel and are limited in their linear response to about one or two order of magnitude of cell biomass range. In small bioreactors, such as 250 mL capacity vessels, the number of available large diameter ports is necessarily limited. Monitoring microbial cell biomass from inoculation (e.g. < 0.1 g/L dry cell weight) to harvest (e.g. > 100 g/L) frequently requires at least 3 orders of magnitude of cell biomass sensitivity. Learn More

A non-isothermal continuous stirred tank reactor (CSTR) is the most important element of chemical industrial equipment which is characterized by a highly nonlinear behavior. It is a multi-input multi-output (MIMO) nonlinear systems exposed to disturbances. The operation of the non-isothermal CSTR can be disturbed by its uncertain parameter such as variation in heat reaction. Therefore, the two difficult problems in CSTR control are tracking trajectory and disturbance attenuation. In this paper, a robust 𝐻∞ fuzzy tracking control via Takagi-Sugeno (T-S) model is designed to robustly stabilize the non-isothermal CSTR system for both concentration and temperature affected by disturbances. Learn More

Recent studies on biotherapeutic protein production have shown perfusion processes as a superior technology vs. the traditional batch and fed batch approaches. Due to its associated process stability and reducing effect of varying conditions inside the bioreactor, Perfusion can deliver lower production costs and higher titer, especially in the case of low titer or fragile proteins. Learn More

Modern upstream bioprocess development is evolving. The Quality by Design (QbD) directive by the United States FDA expressed how a simple demonstration of bioprocess functionality should no longer be considered a sufficient capstone for process development. Rather, a higher level of process understanding should be obtained. Such understanding should be established through the execution of robust characterization studies and demonstrated through the engineering of strategic process parameters. Such strategic process parameters are essential for the consistent manufacturing of biological drug substances. Therefore, strategic process parameters are necessary to meet quality standards and support final drug product safety and efficacy. Learn More

In this application note we discuss how INOXTORRES has standardized their preparation reactors for oncologic and biosimilar applications with Hamilton Arc pH, conductivity, and dissolved oxygen sensors. The Arc System uses stable and reliable Modbus signal to provide direct communication to the process control system directly from the sensor. In this project, Arc sensors are used in total of 14 preparation tanks, 2 sterile filtration skids and 2 CIP systems, all mobiles and with volumes from 5 to 100 liter, and connected to a standalone automation and control system.
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In this study, a novel tube-fixed-bed bioreactor which consists of a TubeSpin bioreactor 50 tube and 0.44 g macrocarriers was developed as the scale-down model of a fixed-bed bioreactor. The adherent Vero cell–based pseudorabies virus (PRV) production process was tested in this novel model. The Vero cells grew well in the tube-fixed-bed bioreactor, and the cell density reached 5.8 × 106 cells/mL after 7 days of culture. The PRV production parameters (time of infection, multiplicity of infection, and harvest process) were optimized in the tube-fixed-bed bioreactor. The comparable growth curve, metabolism, and PRV production profile of the scaled-up bioreactors confirmed the feasibility and scalability of the tube-fixed-bed bioreactor as a scale-down model of the fixed-bed bioreactor for virus production process development. Learn More

For high-throughput cell culture and associated analytics, droplet-based cultivation systems open up the opportunities for parallelization and rapid data generation. In contrast to microfluidics with continuous flow, sessile droplet approaches enhance the flexibility for fluid manipulation with usually less operational effort. Generating biologically favorable conditions and promoting cell growth in a droplet, however, is particularly challenging due to mass transfer limitations, which has to be solved by implementing an effective mixing technique. Here, capillary waves induced by vertical oscillation are used to mix inside a sessile droplet micro-bioreactor (MBR) system avoiding additional moving parts inside the fluid. Depending on the excitation frequency, different patterns are formed on the oscillating liquid surface, which are described by a model of a vibrated sessile droplet. Learn More

The FDA initiative of Process Analytical Technology (PAT) encourages the monitoring of biopharmaceutical manufacturing processes by innovative solutions. Raman spectroscopy and the chemometric modeling tool partial least squares (PLS) have been applied to this aim for monitoring cell culture process variables. This study compares the chemometric modeling methods of Support Vector Machine radial (SVMr), Random Forests (RF), and Cubist to the commonly used linear PLS model for predicting cell culture components—glucose, lactate, and ammonia. This research is performed to assess whether the use of PLS as standard practice is justified for chemometric modeling of Raman spectroscopy and cell culture data. Learn More

The G-Rex cell culture platform is based on a gas-permeable membrane technology that provides numerous advantages over other systems. Conventional bioreactor platform technologies developed for large scale mammalian cell expansion are typically constrained by the mechanics of delivering oxygen to an expanding cell population. These systems often utilize complex mechanisms to enhance oxygen delivery, such as stirring, rocking, or perfusion, which adds to expense and increases their overall risk of failure. On the other hand, G-Rex gas-permeable membrane-based bioreactors provide a more physiologic environment and avoid the risk and cost associated with more complex systems. The result is a more robust, interacting cell population established through unlimited oxygen and nutrients that are available on demand. Learn More

Biologics represent the fastest growing sector of the pharmaceutical industry, yet their manufacture lags significantly behind that of small molecule drugs. This paper discusses the main product-related and process-related challenges during the development and production of therapeutic proteins, with particular focus on product heterogeneity and process monitoring and analytics. Emphasis is placed on novel contributions from the field of computational research that aim to enable the application of model-based process control strategies or are working towards the development of a digital twin of bioprocesses. Lastly, we review promising developments in the paradigm shift from batch to continuous processing. Learn More

Simulators are well established tools for instructing, design, and development of bioprocesses with the advantages of its portability, safety, user friendliness, and cost-effectiveness. However, commercially simulators commonly work as a black box and consequently, their mathematical models are not displayed and/or being available for its modification. In this work, a prototype database that embeds common limits and values of the parameters of bioprocesses, is presented. This database is integrated inside a software platform called FermProc. FermProc allows the display, reuse and modification of models and it is being developed at the Department of Chemical and Biochemical Engineering of the Technical University of Denmark as a pedagogical software for teaching of bio-manufacturing processes. Learn More

For many therapeutic cellular products, the development of a scalable cell expansion process optimized for quality and costs is a crucial step in the manufacturing process. Ambr® 15 provides the capability to screen different types of media, and optimize process parameters with these cell types under stirred conditions at the small scale and with high throughput operation offering many advantages over static culture. The findings at this scale can be transferred to benchtop or larger scale bioreactor processes. Learn More

Traditional mammalian cell culture is usually anchor-dependent and serum-supplemented. Animal-derived components tend to introduce lot-to-lot variability and blood-borne pathogens. Use of serum also tends to increase immunogenicity and production costs. Because of these issues, suspension cultures in animal-derived component free (ADCF) cell culture media are usually preferred for situations like recombinant protein production or viral vector/virus production due to its suitability to industrial processing and scale up. However, transitioning from adherent to suspension serum-free culture has its own set of challenges and is time consuming. Learn More

Bioreactors of various forms have been widely used in environmental protection, healthcare, industrial biotechnology, and space exploration. Robust demand in the field stimulated the development of novel designs of bioreactor geometries and process control strategies and the evolution of the physical structure of the control system. After the introduction of digital computers to bioreactor process control, a hierarchical structure control system (HSCS) for bioreactors has become the dominant physical structure, having high efficiency and robustness. However, inherent drawbacks of the HSCS for bioreactors have produced a need for a more consolidated solution of the control system. Learn More

To ensure stable product quality of biopharmaceuticals, the U.S. FDA encourages the industry to apply the process analytical technology (PAT) guidelines. These guidelines strongly recommend advancements in sensor monitoring and control technology as the important means for improving performance of pharmaceutical manufacturing. The aim of this thesis is to contribute to this advancement of sensor technology, by proposing alternative ways to apply existing sensors for monitoring and control of upstream bioprocesses. Cutting-edge sensor technologies are evaluated with respect to their suitability for process monitoring of critical process parameters. Learn More

Raman spectroscopy is a robust, well-established tool utilized for measuring important cell culture process variables for example, feed, metabolites, and biomass in real-time. This study further expands the functionality of in-line Raman spectroscopy coupled with partial least squares (PLS) regression modelling to develop a pH measurement tool. Cell line specific models were developed to enhance the robustness for processes with different pH setpoints, deadbands, and cellular metabolism. The modelling strategy further improved robustness by reducing the temporal complexity of pH shifts by splitting data sets into two time zones reflective of major changes in pH. Learn More

Immersible online cell biomass probes have historically suffered from a limited range of linear response to biomass and sensitivity to process variables, such as agitation and gas sparge rate. A new optical probe has been developed that provides linear response over 4 orders of magnitude of biomass (e.g. 0.01 to 200 g/L yeast dry cell weight). The small probe diameter (e.g. 3 mm) and minimal optical penetration depth (e.g. < 3 cm) make it suitable for a wide range of vessel types, including miniature bioreactors (e.g. 250 mL). Interference from bubbles is largely eliminated through a novel measurement technique, making the results nearly insensitive to agitation and aeration rate changes. Learn More

The use of Raman models for glucose and phenylalanine concentrations to provide the signal for a control algorithm to continuously adjust the feed rate of two separate supplemental feeds during the fed-batch culture of a CHOK1SV GS-KO® cell line in a platform process was evaluated. Automated feed rate adjustment of the glucose feed using a Raman model for glucose concentration, maintained the glucose concentration within the desired target. Automated feed rate adjustment of the nutrient feed using a Raman model for phenylalanine concentration, maintained phenylalanine concentrations within the target. Learn More

Currently, there is not a standard means to calculate or measure the oxygen consumption of a cell culture online in a bioreactor. The oxygen transfer rate is directly related to the oxygen uptake rate of the cells. This quantity can be estimated by comparing the input and offgas oxygen concentrations, however, this estimation does not contain high-frequency information about the oxygen transfer rate. An online estimator has been developed that uses measurements of the oxygen concentration in the input gas, in the fluid of the bioreactor, and in the offgas exiting the bioreactor to predict the oxygen transfer rate to the fluid of a bioreactor. Recursive least squares is implemented to fit a model for the mass transfer coefficient, and from this, the oxygen transfer rate can be calculated. Learn More

A key challenge for bioprocess engineering is the identification of the optimum process conditions for the production of biochemical and biopharmaceutical compounds using prokaryotic as well as eukaryotic cell factories. Shake flasks and bench-scale bioreactor systems are still the golden standard in the early stage of bioprocess development, though they are known to be expensive, time-consuming, and labor-intensive as well as lacking the throughput for efficient production optimizations. To bridge the technological gap between bioprocess optimization and upscaling, we have developed a microfluidic bioreactor array to reduce time and costs, and to increase throughput compared with traditional lab-scale culture strategies. Learn More

Single-use technologies enable a flexibility and modularity effectively unattainable with more traditional stainless-steel technologies, particularly in upstream bioprocesses. Single-use bioreactors up to 2,000 L are employed largely in preclinical- and clinical-stage bioprocesses to leverage this flexibility. As products reach commercial maturity, scales larger than 2,000 L frequently become desirable to take advantage of economies of scale. With the typical upper limit of single-use bioreactors at 2,000 L, this has traditionally meant transfer to stainless-steel systems. The introduction of the Thermo Fisher Hyperforma DynaDrive 5,000-L single-use bioreactor opens a new paradigm of operation in terms of volume, while providing unprecedented ergonomics and flexibility. Learn More

We introduce an experimental setup allowing continuous monitoring of bacterial fermentation processes by simultaneous optical density (OD) measurements, long-path FTIR headspace monitoring of CO2, acetaldehyde and ethanol, and liquid Raman spectroscopy of acetate, formate, and phosphate anions, without sampling. We discuss which spectral features are best suited for detection, and how to obtain partial pressures and concentrations by integrations and least squares fitting of spectral features. Noise equivalent detection limits are about 2.6 mM for acetate and 3.6 mM for formate at 5 min integration time, improving to 0.75 mM for acetate and 1.0 mM for formate at 1 h integration. Learn More

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. Despite the advantages, leachable compounds originating from the film's materials of construction present a new challenge. This publication investigates inconsistent cell growth observed in a PER.C6® cell line during bioprocess development. Learn More

The production of biopharmaceuticals in cell culture involves stringent controls to ensure product safety and quality. To meet these requirements, quality by design principles must be applied during the development of cell culture processes so that quality is built into the product by understanding the manufacturing process. One key aspect is process analytical technology, in which comprehensive online monitoring is used to identify and control critical process parameters that affect critical quality attributes such as the product titer and purity. The application of industry-ready technologies such as turbidimetry and dielectric spectroscopy provides a deeper understanding of biological processes within the bioreactor and allows the physiological status of the cells to be monitored on a continuous basis. Learn More

We demonstrate a new micro/nanofluidic system for continuous and automatic monitoring of protein product size and quantity directly from the culture supernatant during a high-cell-concentration CHO cell perfusion culture. A microfluidic device enables clog-free cell retention for a bench-scale (350 mL) perfusion bioreactor that continuously produces the culture supernatant containing monoclonal antibodies (IgG1). A nanofluidic device directly monitors the protein size and quantity in the culture supernatant. The continuous-flow and fully automated operation of this nanofluidic protein analytics reduces design complexity and offers more detailed information on protein products than offline and batch-mode conventional analytics. Learn More

Pipettes are frequently used in laboratories and your results are dependent upon their reliable performance. Are you sure your pipette is dispensing the correct amount? Do you know that your handling also has a great influence on pipette performance? Improve your data quality with Good Pipetting Practice™ - the comprehensive, systematic approach to maximizing pipetting accuracy and reproducibility. This on-demand webinar will give you insights into the world of pipetting. Learn More

Single-use bioreactors (SUBs) and stainless-steel bioreactors (SSBs) differ in their physical properties. This can have an effect on the performed processes. To be able to guarantee flexible planning in a facility, any kind of processes in SUBs and SSBs must be sufficiently similar. To date, assessments of SUBs and SSBs have been exclusively specific for one single product. This study shows a general nonproduct-specific comparison of the production of different types of monoclonal antibodies in different scales of SUBs and SSBs. The study is set up with 72 clinical manufacturing fermentation runs in total by comparing the cell culture performance and quality attributes of these two bioreactor systems. Learn More

Single use bioreactors provide an attractive alternative to traditional deep-tank stainless steel bioreactors in process development and more recently manufacturing process. Wave bag bioreactors, in particular, have shown potential applications for cultivation of shear sensitive human and animal cells. However, the lack of knowledge about the complex fluid flow environment prevailing in wave bag bioreactors has so far hampered the development of a scientific rationale for their scale up. In this study, we use computational fluid dynamics (CFD) to investigate the details of the flow field in a 20-L wave bag bioreactor as a function of rocking angle and rocking speed. Learn More

It is common for biologics manufacturing processes to require hundreds of raw materials, ranging from media and media supplements, buffers, and salts to other process chemicals. Buffers and salts are typically the largest constituents by volume used in the downstream processing steps for manufacturing most biopharmaceutical products. The increase in titers and the trend toward continuous biomanufacturing will lead to increased demand in buffer volumes and prep times. To address this gap, strategies for improving mAb manufacturing productivity by optimizing powder and liquid buffer and media prep process flow are worth considering. Learn More

Turbines for fermenters are rotating devices with a plurality of radially extending blades. The ambition is to reduce non-uniformities in the fluid containing liquids, blend soluble components, disperse gases and solid particles such as cells and promote heat transfer. Fermentation depends vastly on the success of the fermenter design in order to obtain optimum flow field, shear distribution, bubble distribution, Oxygen distribution, mass transfer throughout the entire fermenters liquid volume. The turbine design discussed is relevant for the biotech industry up to 100 liter WV and named after the actual designer, inventor. Learn More

Miniaturization and automation have become increasingly popular in bioprocess development in recent years, enabling rapid high-throughput screening and optimization of process conditions. Monitoring methods, such as fluorometric-based pH sensors, provide elegant solutions for the miniaturization of bioreactors, however, previous research suggests that the intrinsic fluorescence of biomass alters the sigmoidal calibration curve of fluorometric pH sensors, leading to inaccurate pH control. In this article, we present results investigating the impact of biomass on the accuracy of a commercially available fluorometric pH sensor. Learn More

Are you ready to take your therapies from the lab into production and looking for ways to implement a closed system design with single-use technologies?

In this on-demand webinar CPC's Jayanthi Grebin will discuss:

  • Why closing your cell and gene therapy manufacturing is so important
  • How closed systems compare to open systems
  • How aseptic connectors are used to close systems without use of safety cabinets
  • What are your single-use technology options

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Upstream bioprocess characterization and optimization are time and resource-intensive tasks. Regularly in the biopharmaceutical industry, statistical design of experiments (DoE) in combination with response surface models (RSMs) are used, neglecting the process trajectories and dynamics. Generating process understanding with time-resolved, dynamic process models allows to understand the impact of temporal deviations, production dynamics, and provides a better understanding of the process variations that stem from the biological subsystem. The authors propose to use DoE studies in combination with hybrid modeling for process characterization. Learn More

The use of Process Analytical Technology tools coupled with chemometrics has been shown great potential for better understanding and control of mammalian cell cultivations through real-time process monitoring. In-line Raman spectroscopy was utilized to determine the glucose concentration of the complex bioreactor culture medium ensuring real-time information for our process control system.

This work demonstrates a simple and fast method to achieve a robust partial least squares calibration model under laboratory conditions in an early phase of the development utilizing shake flask and bioreactor cultures. Two types of dynamic feeding strategies were accomplished where the multi-component feed medium additions were controlled manually and automatically based on the Raman monitored glucose concentration. Learn More

Impellers are axial fluid directing rotating elements for delivering or absorbing energy. These elements with in general few and large helicoidal shaped blades give very high degree of swirling in a vessel or tube. The flow pattern generated in the fluid resembles helix. Impeller dynamics can be modelled by both Bernoulli's principle and Newton's third law. The impeller (propell) is sometimes colloquially known as a screw or a rotor. Learn More

Spectroscopy techniques are being implemented within the biopharmaceutical industry due to their non-destructive ability to measure multiple analytes simultaneously, however, minimal work has been applied focusing on their application at small scale. Miniature bioreactor systems are being applied across the industry for cell line development as they offer a high-throughput solution for screening and process optimization. The application of small volume, high-throughput, automated analyses to miniature bioreactors has the potential to significantly augment the type and quality of data from these systems and enhance alignment with large scale bioreactors. Learn More

The average shear rate is a parameter used to characterize the shear environment in bioreactors, enabling comparison of the performances of different bioreactor models in terms of microorganism morphology and viability, and consequently bioproduct formation. Based on this approach, pneumatic bioreactors have been classified as low shear devices. However, the shear behavior cannot be generalized over a wide range of operating conditions, suggesting that the maximum shear rate may be more suitable for the purpose of bioreactor performance comparison. Therefore, the aim of this work was to evaluate average and maximum shear rates in pneumatic bioreactors (bubble column and airlift). Learn More

Micro-bioreactors appear frequently in today's biotech industry as screening and process development tools for cell culture applications. The micro-bioreactor's small volume allows for a high throughput, and when compared to other small-scale systems, such as microtiter plates, its measurement and control capabilities offer a much better insight into the bioprocess. Applikon's micro-Matrix is one of the micro-bioreactors that are commercially available today. This chapter describes how the micro-Matrix can be used for fed-batch cultivations of Chinese Hamster Ovary (CHO) cells. Learn More

The platforms for bioprocess development have been developed in parallel to the needs of the manufacturing industry of biopharmaceuticals, aiming to ensure the quality and safety of their products. In this sense, Quality by Design (QbD) and Process Analytical Technology (PAT) have become the pillars for quality control and quality assurance. A new combination of Shake Flask Reader (SFR) and Respiration Activity Monitoring System for online determination of OTR and CTR (RAMOS) allows online monitoring of main culture parameters needed for bioprocess development (pH, pO2, OTR, CTR, and QR) as presented.

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While monitoring and control of inoculum characteristics at the seed fermenter stage are well established in most bioprocesses, the very first steps of culture expansion in shake flasks are usually neglected, even though they are influencing all following process steps. Especially for organisms with complex metabolic regulation or morphological variability, the utilization of suboptimal inocula can severely worsen the bioprocess outcome.
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For the production of biopharmaceuticals, a procedure called seed train or inoculum train is required to generate an adequate number of cells for the inoculation of the production bioreactor. This seed train is time- and cost-intensive but offers potential for optimization. A method and a protocol are described for seed train mapping, directed modeling, and simulation as well as its optimization regarding selected optimization criteria such as optimal points in time for cell passaging. Furthermore, the method can also be applied for the transfer of a seed train to a different production plant or the design of a new seed train, for example, for a new cell line. Learn More

Anaerobic cultivation methods of bacteria are indispensable in microbiology. One methodology is to cultivate the microbes in anaerobic enclosure with oxygen-adsorbing chemicals. Here, we report an electronic extension of such strategy for facultative anaerobic bacteria. The technique is based a bioreactor with entire operation including turbidity measurement, fluidic mixing, and gas delivery in an anaerobic enclosure. Wireless data transmission is employed, and the anaerobic condition is achieved with gas pack. The technique is not meant to completely replace the anaerobic chamber for strict anaerobic bacteria. Learn More

In order to make a quality product by cell culture efficiently, it is important to monitor and control the cell culture environment and the cells as closely as possible. When in-situ sensors are available, their direct use within the cell culture vessel (i.e. the bioreactor) is favored. This is the case of temperature, dissolved oxygen and pH sensors commonly included in the bioreactor, at all scales. However, when analytes and cell attributes cannot be measured directly in-situ, sampling of the cell culture is necessary and external analysis is carried out. For example, cell concentration and cell viability using Trypan Blue, glucose and lactate... Learn More

The volumetric mass transfer coefficient, usually denoted by kLa, is an important parameter for both the design, scale-up and monitoring of aerated bioreactors. Significant research efforts have been expended over the last decades in order to develop reliable and easy to apply methodologies for determining kLa using aeration experiment and fast oxygen (polarographic) probes. These methodologies have been reviewed in this study followed by the presentation of a new and promising methodology for kLa determination, which can be used when data from step experiments are not available... Learn More

A key challenge for bioprocess engineering is the identification of the optimum process conditions for the production of biochemical and biopharmaceutical compounds using prokaryotic as well as eukaryotic cell factories. Shake flasks and bench-scale bioreactor systems are still the golden standard in the early stage of bioprocess development, though they are known to be expensive, time-consuming, and labor-intensive as well as lacking the throughput for efficient production optimizations. To bridge the technological gap between bioprocess optimization and upscaling, we have developed a microfluidic bioreactor array to reduce time and costs, and to increase throughput compared with traditional lab-scale culture strategies. Learn More

The global demand for complex biopharmaceuticals like recombinant proteins, vaccines, or viral vectors is steadily rising. To further improve process productivity and to reduce production costs, process intensification can contribute significantly. The design and optimization of perfusion processes toward very high cell densities require careful selection of strategies for optimal perfusion rate control. In this chapter, various options are discussed to guarantee high cell-specific virus yields and to achieve virus concentrations up to 1010 virions/mL. This includes reactor volume exchange regimes and perfusion rate control based on process variables such as cell concentration and metabolite or by-product concentration. Learn More

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. Learn More

Sensors are essential devices that can be used for most, if not all, typical biopharmaceutical development and manufacturing processes to monitor fundamental process parameters such as flow, temperature, pH, and dissolved oxygen throughout all process stages. As the bioindustry progresses toward automation, digitalization, and other "Manufacturing 4.0" concepts, robust single-use and smart sensors for bioprocess monitoring will be needed. Read this BPI eBook to garner valuable perspectives on both of these types of sensors. Discussions herein focus on smart sensor capabilities, applications, and validation. Learn More

The recent trend in bioprocess development towards small fermentors and high cell densities presents challenges for accurate on-line monitoring of liquid cell cultures. Traditional on-line optical probes, such as transmission probes (also known as absorbance or optical density probes) and reflectance probes (also known as back-scatter or turbidity probes), typically require a 12 mm or larger diameter port into the bioreactor vessel and are limited in their linear response to about one or two order of magnitude of cell biomass range. In small bioreactors, such as 250 mL capacity vessels, the number of available large diameter ports is necessarily limited. Learn More

As biotechnology processes evolve, so too does demand for protocols capable of delivering rich data at high speeds. This new eBook by Brooks Instrument offers clear insight on digital communications devices, such as mass flow controllers (MFCs), equipped for maximum information density in your process system. This biotechnology eBook was created specifically to help biotechnology OEMs and biotechnology end users understand how digital communications bring new capabilities and greater control to their process systems. Learn More

Bioreactor design is a challenging endeavor that aims to provide the most ideal environment in which cells can grow and biological reactions can occur. The emergence of regenerative medicine and stem cell therapies has led to the need for more diverse environmental requirements in the bioreactor design space. The study presented uses an additive manufacturing approach for the initial design phase of a packed/fluidized bed bioreactor for mesenchymal stem cell expansion. Combining 3D-printing with CFD for precision control over the bioreactor flow dynamics. Novel flow distributors were developed to engender swirling particle fluidization. Learn More

As the demand for cell and gene therapies grows, the need for scale up to larger volumes has led to the utilization of single-use technologies with open connections as well as other methods such as tube welding, multi-purpose connectors, quick connects, luers, and luer locks. These approaches are cumbersome and inefficient, though, leading to a greater need for closed manufacturing in cell and gene therapies to protect and provide effective therapies. Learn More

Measurement of dissolved oxygen (DO) has long been known as a critical control parameter for optimizing cell growth in bioprocesses. Much has been written about different strategies for proper DO control. However, little information has been publicly available regarding optimization of the actual measurement accuracy and reducing common sources of process-related error. This paper aims to divulge these details for the first time. Specific consideration will be given to optical dissolved oxygen measurement which has become the predominant measurement technology in bioprocesses. Learn More

This novel Single Use Pressure Sensor was specifically designed for monitoring pressure in flexible bioprocess containers in both gas and liquid applications. The actual pressure sensing technology is the same as PendoTECH's existing inline pressure sensors. The most significant difference with this product is its form factor, which makes it easy to integrate with almost any flexible bioprocess container. Learn More

Today's biopharmaceuticals are revolutionizing healthcare. Pioneering drugs to treat complex cancers, chronic conditions such as rheumatoid arthritis and new gene-based therapies offer new hope to people around the world. Researching and producing these cutting-edge biologics requires some of the most complex manufacturing processes known to man. The newest generation of bioreactors often uses complex botanical or mammalian cell cultures, which are highly sensitive and require a stable, precisely controlled growth environment. To create those environments, bioreactors require accurate, stable gas control to maintain critical process parameters, combined with maximum uptime to reach target yields. Learn More

Biopharmaceutical engineers are frequently tasked with finding new and improved ways to efficiently manufacture at scale. Historically, implementing and validating new systems and processes proved a considerable challenge, making the prospect of upgrading bioproduction equipment daunting, disruptive, and expensive. Today, however, demand for more has them researching ways to scale up and transfer larger volumes of product through the bioproduction process as quickly and easily as possible while maintaining a high-quality standard, sterility, and of course no loss of product. High-flow sterile connectors make upgrading or upscaling bioproduction more reliable, efficient, and rewarding than ever. Learn More

Vaporized Hydrogen Peroxide (VHP) is an increasingly common sterilizing agent in pharmaceutical manufacturing processes, particularly in final fill applications. For this reason, it is important that the single-use materials used in final fill processes are able to withstand exposure to VHP. Through rigorous testing, Saint-Gobain has demonstrated that their 5-layer, single-use bioprocess bags are well-suited for use in applications where VHP is employed for decontamination purposes. Learn More

Antibody and other protein therapeutics are a major focus in drug discovery pipelines today. The overall process for developing protein therapeutics encompasses target selection and validation, library screening to generate early candidates (hits), follow-up characterization for lead selection, lead optimization, and clinical candidate selection. During lead selection, molecules identified as hits are subjected to screening via multiple analytical methods to select a few candidates for progression to the next stage of the development process. This is typically followed by detailed characterization for confirmation of binding and functional activities via biochemical and biophysical analyses. Learn More

The increasing importance of viral vaccine manufacturing has driven the need for high cell density process optimization that allows for higher production levels. Vero cells are one of the more popular adherent cell lines used for viral vaccine production. However, production is limited due to the logistical limitations surrounding adherent cell line processes, such as large equipment footprints, time and labor-intensive processes, and larger costs per dose. We have addressed this limitation with the establishment of a viral vaccine production system utilizing the novel single use scale-X™ carbo bioreactor. Learn More

The objective of this case study was to identify tubing that would minimize the time required for fluid transfer by maximizing flow rate. The capability of the tubing to withstand pumping flow rates between 5 to 50 LPM and resultant changes in pressure drop until point of failure (tubing collapse) at the suction line was studied. Learn More