Articles

Small-scale models that simulate large-scale freezing of bulk drug substance of biopharmaceuticals are highly needed to define freezing and formulation parameters based on process understanding. We evaluated a novel scale-down device (SDD), which is based on a specially designed insulation cover, with respect to changes in concentration after freezing, referred to as cryoconcentration, and 3D temperature profiles. Furthermore, the effect of the initial monoclonal antibody (mAb) concentration on cryoconcentration was addressed.
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Isolating membrane proteins from their native cells while maintaining structural and functional integrity is challenging. Many detergents have been developed over the years that interact favorably with membrane proteins and mimic the physical properties of the lipid bilayer. Choosing the appropriate detergent is crucial for the successful extraction of a protein in its properly folded, active conformation.
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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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Developing ultrafiltration (UF) membranes for protein purification requires a critical understanding of the pore engineering and active-layer morphological control. In this study, we systematically explored the use of polyethylene glycol (PEG) with molecular weights (MW, 600–6000 g⋅mol−1) and concentration (3–7.5 wt%) as a pore modifier to prepare polyvinyl difluoride (PVDF) membranes via the non-solvent phase-inversion process. The membranes demonstrated a high degree of control of the structure-property-performance relationship for improving the water flux, bovine serum albumin (BSA) rejection and antifouling properties.
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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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The handbook describes how to design a protein purification procedure, how to select and combine chromatography methods and purification formats, and how to monitor and evaluate protein purification. It gives an overview of the available methods and provides advice on how to avoid pitfalls in all operations, from initial sample preparation to final analysis of the purified protein. The handbook is a complement to the series of handbooks from Cytiva that describe the different chromatographic purification methods in more detail.
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At YMC America, we receive quite a few inquiries regarding what is the best approach for developing analytical IEX methods that utilize salt gradients for charge variant analysis of mAbs. A relatively easy way to approach this is to use a quaternary pump HPLC and the dial-a-mix method. This enables the lab scientist to utilize all four mobile phase bottles/lines to very easily (and quickly) screen various pH conditions and salt gradients. Once the ideal conditions are determined, the method can be fine-tuned for optimal performance. The method can then be transferred to a binary HPLC system if desired, for simplicity and better reliability.
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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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The various GMP guidelines and International ISO standards around GMP cleanrooms are complex and often appear to give conflicting advice, leading to confusion and sometimes incorrect interpretation. In the case of Routine Environmental Monitoring, there is very little prescriptive advice and the onus is on the cleanroom owner to devise an appropriate monitoring plan and, in the frequently prescriptive GMP industry, the lack of direct guidance leaves users struggling to know what to do and the temptation is to either create over-burdensome monitoring programs or to try to simply use the same monitoring plan as is used for classification. This paper discusses the differences between GMP Cleanroom Classification and Routine Environmental Monitoring and explains how Beckman Coulter can help.
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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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After over a quarter century in the industry — including downstream processing (DSP) and manufacturing directorships at Boehringer Ingelheim and leadership roles in technology development, quality, and manufacturing at Novasep — European consultant Margit Holzer is a recognized expert in downstream processing of biopharmaceutical products. Holding a doctorate in biotechnology from the University of Natural Resources and Applied Life Sciences in Austria, Holzer is familiar to BPI readers as both an author and conference participant. And in May she taught her first BPI Academy course on bioprocess characterization, qualification, and validation. Here we discuss membrane adsorbers, high-throughput screening, modeling and analytics, and monoclonal antibodies (MAbs) as vanguard products for advancing tools and technologies.
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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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Ion exchange chromatography is a powerful and ubiquitous unit operation in the purification of therapeutic proteins. However, the performance of an ion‐exchange process depends on a complex interrelationship between several parameters, such as protein properties, mobile phase conditions, and chromatographic resin characteristics. Consequently, batch variations of ion exchange resins play a significant role in the robustness of these downstream processing steps. Ligand density is known to be one of the main lot‐to‐lot variations, affecting protein adsorption and separation performance. The use of a model‐based approach can be an effective tool for comprehending the impact of parameter variations (e.g., ligand density) and their influence on the process. The objective of this work was to apply mechanistic modeling to gain a deeper understanding of the influence of ligand density variations in anion exchange chromatography. 
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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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Cell harvesting and clarifications are both crucial steps that can have a significant impact on the design of downstream processes. Briefly, the primary goals for harvest and clarification (H & C) operations are to remove cells and cell debris from mammalian cell culture and further purify and clarify the resulting product to capture the mAbs via chromatography downstream. As processes change upstream in the pharmaceutical manufacturing workflow, causing variability of cell culture fluid, downstream purification unit operations will be impacted. By mapping these different process inter-dependencies, H & C operations can be sized to build an optimized scheme without wasted capacity and without incurring additional costs.
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The accurate assessment of antibody glycosylation during bioprocessing requires the high-throughput generation of large amounts of glycomics data. This allows bioprocess engineers to identify critical process parameters that control the glycosylation critical quality attributes. The advances made in protocols for capillary electrophoresis-laser-induced fluorescence (CE-LIF) measurements of antibody N-glycans have increased the potential for generating large datasets of N-glycosylation values for assessment. With large cohorts of CE-LIF data, peak picking and peak area calculations still remain a problem for fast and accurate quantitation, despite the presence of internal and external standards to reduce misalignment for the qualitative analysis. Here, we describe an approach to quantitatively and qualitatively curate large cohort CE-LIF glycomics data.
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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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Selection of chromatography adsorbents and optimization of chromatography conditions by screening methods has always been an integral part of protein purification process development. Traditionally, this was carried out in small laboratory scale columns which is a time, sample and buffer consuming process. Incremental improvements in these methods have resulted in the emergence of high-throughput screening tools providing fast, cost-effective methods for evaluating several parameters in a single experiment with reduced requirement for material and sample volumes. When this approach is combined with design of experiment (DoE), it provides a rapid means of identifying the optimal chromatography adsorbent and operating conditions which can be quickly verified and scaled up.
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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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Technology to increase yield by 2 to 3-fold while reducing solvent usage by up to 70% while maintaining purity targets is presented in this 30-minute presentation. Animations illustrate the technique and quickly provide the viewer of how the process mimics conventional HPLC purification - but with internal, automated recycling of side fractions. Economics of the process as applied to oligonucleotides are illustrated by the impact of incorporating the patented MCSGP process on plant productivity and cost-out (and we explain what the heck "MCSGP" stands for!). This same MCSGP process can be applied to peptides and other difficult to purify or highly potent molecules such as ADC's.
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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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Technologies capable of monitoring product quality attributes and process parameters in real time are becoming popular due to the endorsement of regulatory agencies and also to support the agile development of biotherapeutic pipelines. The utility of vibrational spectroscopic techniques such as Fourier transform mid‐infrared (Mid‐IR) and multivariate data analysis (MVDA) models allows the prediction of multiple critical attributes simultaneously in real time. This study reports the use of Mid‐IR and MVDA model sensors for monitoring of multiple attributes (excipients and protein concentrations) in real time (measurement frequency of every 40 s) at ultrafiltration and diafiltration (UF/DF) unit operation of biologics manufacturing. 
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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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The switch towards continuous/intensified downstream processes requires optimal control through dedicated processes and analytical tools. We developed a Downstream Process (DSP) control strategy to enable a complete and integrated process line with a single skid. The BioSC™ technology allows for a closely integrated DSP, from the initial capture right up to the final polishing step. Powerful sensor technologies and highly adaptable control strategies lead to extremely efficient processes and, ultimately, to consistent product quality. Once process ranges or design spaces have been defined, the system can detect process deviations and trigger the most appropriate actions accordingly.
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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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Computer modeling is an area of broad multidisciplinary knowledge that includes the study of various biological systems. This chapter will describe the molecular aspects of viral infections and molecular modeling techniques applied to drug discovery with examples of applications in protein activity inhibition in several pathologies. The first part will cover topics of computational chemistry methods, DNA technologies, structural modeling of virus proteins, molecular biology, viral vectors, virus-like particles, and pharmaceutical bioprocess with application in some specific viruses such as papillomavirus, hepatitis B virus, hepatitis C virus, Coronavirus, and Zika Virus. The second part will deal with methods in Virtual Screening for the drug design based on ligands and on the structure of target macromolecules. Molecular docking in drug design, its search algorithms, and scoring functions will be covered in the third part. Finally, a protocol of the Molecular Dynamics technique for studies of protein-ligand complexes and analysis of free energy of binding will be exposed in the last part.
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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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According to the IDF Diabetes Atlas, diabetes is one fastest growing health challenges in the 21st century. Over the past 20 years, the number of adults living with diabetes has more than tripled. As a result, the global insulin market is growing and expected to reach US$43.6 billion by 2021, according to Zion Market Research. Insulin is generally purified by a combination of several chromatographic purification techniques. The packing material used in the purification process must have a long lifetime, good separation, and high loadability. To discuss packing materials and stationary phases used to purify insulin, LCGC sat down with Takashi Sato, product specialist in the sales and marketing department at YMC Co., Ltd., to shed light on the challenges that arise when separating large molecules such as insulin, proteins, and peptides; the high price of insulin purification; and solutions to reducing processing times and labor/material costs.
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Biomedical research advances over the past two decades in bioseparation science and engineering have led to the development of new adsorbent systems called monoliths, mostly as stationary supports for liquid chromatography (LC) applications. They are acknowledged to offer better mass transfer hydrodynamics than their particulate counterparts. Monoliths are categorized under three main groups based on the monomer composition of their synthesis; organic, inorganic and hybridized organic- inorganic monoliths. This review article discusses in-process analytical process conditions, functionalization chemistries and ligands relevant to establish the characteristics of polymer monoliths in order to facilitate a wide range of enhanced bioscreening applications.
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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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Chromatography is an important and ubiquitous unit operation in downstream bioprocessing. Currently, there are mainly three different ways to pack process chromatography columns: Flow Packing, which uses constant pressure or flow Pack-in-Place, which incorporates packing valves; and Dynamic axial packing (DAP), achieved by axially compressing the media slurry. Novasep recently developed Elemental Packing on its Prochrom®-Bio columns combining pack-in-place and dynamic axial packing advantages.
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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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The term “optimize” is often applied to complex manufacturing, automation and business processes and implies that the most efficient function of all the elements of a process – technologies, sequences and procedures – has not been achieved. While no method can be perfect, every process can benefit from a careful consideration where new thinking, new techniques and new technology can yield significant improvements. The dramatic growth in the use of biologics across multiple therapeutic applications and categories will only continue to increase. As the demand for these drugs accelerates, there are growing concerns about their cost and availability. Biologics manufacturers are investigating ways to address these concerns – and downstream production in bioprocessing operations is one such area.
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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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The bottleneck for real-time control and real-time release is the lack of product-specific in-line sensors or fast at-line methods suitable for model-based prediction of process outcome. There are multiple sensor-based parameters that help monitor a purification process, but which ones serve as predictors for protein concentration?
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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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Host cell proteins (HCPs) are a major class of bioprocess-related impurities generated by the host organism and are generally present at low levels in purified biopharmaceutical products. The monitoring of these impurities is identified as an important critical quality attribute of monoclonal antibody (mAb) formulations not only due to the potential risk for the product stability and efficacy but also concerns linked to the immunogenicity of some of them. While overall HCP levels are usually monitored by enzyme-linked immunosorbent assay (ELISA), mass spectrometry (MS)-based approaches have been emerging as powerful and promising alternatives providing qualitative and quantitative information. However, a major challenge for liquid chromatography (LC)-MS-based methods is to deal with the wide dynamic range of drug products and the extreme sensitivity required to detect trace-level HCPs. In this study, we developed powerful and reproducible MS-based analytical workflows coupling optimized and efficient sample preparations, the library-free data-independent acquisition (DIA) method, and stringent validation criteria. 
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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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The advantages of single-use systems have been well documented in biomanufacturing for over a decade and are frequently used in the manufacture of monoclonal antibodies. While certainly beneficial to monoclonal antibody production, there is even a more compelling case for using single-use technology in viral production, specifically for viral vectors and vaccine manufacturing, where even a small cross-contamination could be detrimental.Within viral production, one area where single-use can provide a significant benefit is in tangential flow filtration (TFF). TFF is frequently used during viral vector manufacturing to concentrate, exchange buffer and in final formulation. The compact system design also allows for low minimum recirculation volume for high concentration factors and product recovery.
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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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Mass spectrometry (MS) is arguably the most important analytical tool in the biopharma developer’s tool chest but its historic size, cost and complexity have limited its use. To address this industry dynamic, Waters has been working to make mass spectrometry instruments smaller, easier to use and more accessible to analytical scientists throughout the organization. This SelectScience application eBook is intended as a free resource for anyone in biopharmaceutical development, manufacturing and QC who wishes to make greater use of mass data, and who seeks to accelerate time-to-market and fuel greater productivity within their respective organizations.
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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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Biomolecules can interact with LPS either by binding to specific sites or by electrostatic/hydrophobic interactions making the separation of endotoxins from proteins difficult to achieve. These interactions can result in interference with endotoxin assays masking the presence or removal of LPS leading to inefficient removal. Many methods have been adopted for the removal of endotoxin based on adsorption, two-phase portioning and chromatography, however these methods often lead to loss of the target protein or introduction of toxic ligand components, such as polymyxin B which has been used for this purpose. This paper addresses the issue of removal of endotoxin from biological preparations. Specific reference will be made to a novel synthetic ligand affinity adsorbent, EtoxiClear™.
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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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Here we present our miniaturized TFF system, one of the smallest device of its kind available, intended specifically for lab-scale purifications. It has the completely recoverable dead volume under one milliliter and can accommodate volumes up to 50 mL if performing diafiltration in the same run, or up to 100 mL if only concentrating. The PULSE TFF System is capable of ultrafiltration rates up to 5 times faster compared to a 15 mL centrifugal device and is completely automated with non-contact liquid sensing, bringing the retentate volume down to the specified setpoint.
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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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This new case study collection highlights innovative weighing solutions that can help improve processes and product quality in pharmaceutical manufacturing, It shows how having a dependable and proficient partner that offers complete and innovative weighing solutions can help you achieve a higher level of productivity, compliance and safety than ever before. In this case study collection, you will find pharmaceutical weighing solutions and pharmaceutical best practices to help you.
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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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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.
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Raman spectroscopy has the potential to revolutionise many aspects of biopharmaceutical process development. The widespread adoption of this promising technology has been hindered by the high cost associated with individual probes and the challenge of measuring low sample volumes. To address these issues, this paper investigates the potential of an emerging new high-throughput (HT) Raman spectroscopy microscope combined with a novel data analysis workflow to replace off-line analytics for upstream and downstream operations. On the downstream front, the HT Raman device was incorporated into the development of a cation exchange chromatography step for an Fc-fusion protein to compare different elution conditions.
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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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In the development of therapeutic peptides and proteins, the characterization of product-related impurities is required by ICH Q6B and ICH Q3A (R2) guidelines. The preparation of analytical standards and preparative isolation of product-related impurities is a laborious task. N-Rich is a preparative twin-column chromatography process for fast automated enrichment and isolation of minor compounds from complex samples operated by the Contichrom system. This presentation provides an introduction to the process principle of N-Rich and shows application examples for monoclonal antibody charge isoform isolation and peptide impurity isolation.
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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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Ongoing efforts in the biopharmaceutical industry to enhance productivity and reduce manufacturing costs include development of intensified, linked, and/or continuous processes. One approach to improve productivity and process economics of the polishing step (i.e. anion exchange chromatography) is to pre‐concentrate the product intermediate using a single‐pass tangential flow filtration step before loading on the resin. This intensification of the polishing step consequently leads to changes in product intermediate concentration for subsequent virus filtration operations, potentially impacting filter performance and methods for evaluating viral clearance.
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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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Continuous processing is already well accepted on the upstream processing side, since perfusion mode culture has been in use for decades at an industrial scale. Justifications for switching from batch to continuous chromatography are numerous. Besides, the rising demand of biologics suggests reduced processing and labor costs, footprint reduction, flexibility increase, more stringent requirements for controlled, more consistent and improved quality; and most importantly, the requirement for higher productivity and therefore overall manufacturing cost reduction. This white paper highlights why and how to intensify your bioprocess.
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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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The primary goal of downstream processing is the isolation and purification of the desired protein or nucleotide from the bacterial or animal cells that produce it in a fermentation process. The fermentation processes that are used by biopharmaceutical manufacturers have shown to lead to increasing quantities of therapeutic proteins. Manufacturers are increasingly recognizing the present need for improvement and have shifted their focus from improving the upstream process to improving the downstream process. Umang Trivedi said, ‘’The problem that we are facing right now is some of the inefficiencies in the process which are being brought to the forefront because of the increased titres, increased cell densities that are coming out of the upstream processes.’’ Challenges in downstream bioprocessing are driven by the pipeline growth of variable and complex emerging molecules such as bispecific antibodies, monoclonal antibodies (mAbs), antibodydrug conjugates (ADCs), and viral vectors for gene and cell therapies. Learn More

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

In this presentation we will review the new Revision Annex 1 with respect to Single Use Systems and consider initial designs for Single Use Systems, including; design concepts/ location of bacterial retention filters inside or outside the Isolator or RABS/mock-ups. We will also Look at the handling and set-up of Single Use Systems, as well as the sterilization process and product filter preparation steps. Finally, we will uncover the approach to qualification for Single Use Systems. 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

AdvantaPass® Cleanroom Portal for Aseptic Fluid Transfer - The aseptic transfer of liquids between two areas of different grade classification poses a challenge in Single-Use facility design and operation. The main objective of the customer was to quickly transfer fluid from one area to another while minimizing the risk of contamination. In addition, the customer was looking to maximize efficiency and reduce operator error. 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

We designed small‐scale virus filtration models to investigate the impact of the extended process times and dynamic product streams present in continuous manufacturing. Our data show that the Planova 20N and BioEX virus filters are capable of effectively removing bacteriophage PP7 (>4 log) when run continuously for up to 4 days. Additionally, both Planova 20N and BioEX filters were able to successfully process a mock elution peak of increased protein, salt, and bacteriophage concentrations with only an increase in filtration pressure observed during the higher protein concentration peak. These experiments demonstrated that small‐scale viral clearance studies can be designed to model a continuous virus filtration step with specific process parameters. 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

Frost & Sullivan recently invited academic and industry leaders in biopharmaceutical manufacturing to participate in a new and unique thought leadership forum, our Virtual Think Tank series. This forum brought together leading minds in manufacturing to discuss challenges, strategies, techniques, and barriers to new technology implementation in monoclonal antibody harvesting. 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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New industry trends often lead to new demands. As you work on an increasing range of products, which all still require a high-quality output, your process can become more complex. It can be a challenge to meet your yield and cost goals. These challenges bring the need for newer technologies. Review key process challenges and some options to overcome them. Newer technology, such as upstream chromatographic filters, may enable cleaner Proten A eluent and reduce the number of downstream unit operations required. Learn More

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

This study introduces a next-generation flow cytometry method multiplexing the CellROX® Green and Propidium Iodide probes for the simultaneous measurement of free total reactive oxygen species (ROS) and membrane integrity, respectively. The multiparameter method was compared to the single-parameter assays, measuring either ROS or membrane integrity, for the ability to evaluate the fitness of Lactobacillus rhamnosus GG (LGG) after freeze drying, spray drying and H2O2-mediated oxidative stress. 
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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

Many laboratory studies in cryptosporidial research require a source of purified oocysts. Sources can include experimentally infected laboratory animals or from samples collected from naturally infected animals and from clinical cases of human cryptosporidiosis. Purification of oocysts can be accomplished with readily available laboratory equipment including tabletop centrifuges and microcentrifuges. Following purification, oocysts can be stored in antibiotic-supplemented buffers or in 2.5% aqueous potassium dichromate for over 6 months. 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 effects of activation of the unfolded protein response (UPR) via co-expression of the HAC1i gene on the production of the recombinant Pseudomonas aeruginosa elastase (rPAE) in Pichia pastoris GS115 were evaluated in this study. The results showed that expression of the HAC1i gene significantly increased the level of Kar2p (a hallmark of UPR activation) in P. pastoris GS115, demonstrating activation of the UPR. This gene did not affect the growth of yeast in the buffered glycerol-complex medium but stimulated its growth in the buffered methanolcomplex medium. 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

This video demonstrates the flexibility and crush resistance of AdvantaSil Ultra Low Temperature Silicone Tubing. This unique fluid flow product is designed for applications where the tubing material is exposed to extremely low temperatures. It's the first tubing of its kind and the optimal choice for applications related to bulk drug storage and transport. 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

This ebook highlights the use of chromatography resins and their unique functionalities as powerful tools for virus purification and includes approaches that accelerate method development, thus allowing scientists to fine-tune selectivity and optimize downstream purification platforms. 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

Analytical centrifugation is a fractionation technique that relies on the observation of colloidal movement during centrifugation. Centrifuges with various optics (turbidity, absorption, interference, and x-ray attenuation) are commercially available and offer maximum acceleration up to 250,000 g. The best established and ISO standardized application is the determination of a highly resolved size distribution of polydisperse materials in the size range from < 1 nm to 10 μm. The conversion from the original metric of the detector to volume metrics is highly established and was recently extended to reliable conversion to number metrics. 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

In this brief presentation we focus on one unique functionality of the multi-functional Contichrom TWIN Capture LPLC: the sequential batch ("pool-less") polishing operation. Our twin-column system allows the sequential purification of two orthogonal chromatographic modes where the purified product of the first column is directly loaded onto the second column without any storage tanks in between. This sequential operation can be applied when connecting a capture step with a sequential polishing step or two polishing steps. Case studies will be presented that highlight the advances of the sequential operation, such as increased productivity, buffer savings, and reductions in OpEX and CapEx. 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

Recent reports show that ion-exchange chromatography (IEC) is an effective tool for the isolation and purification of adenovirus. However, during the separation and purification, host cell protein and DNA, as well as serum from the culture medium, can non-specifically occupy numerous binding sites of the chromatography packings, thereby reducing the binding between the adenovirus and packing media. We here report a novel method for highly efficient purification of adenoviruses by increasing the salt concentrations of the samples to be ultrafiltrated by tangential flow filtration, the diafiltration buffer, and the samples for IEC purification. 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