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

Membrane systems play an essential role in the production of all biopharmaceuticals. This includes initial clarification of cell culture fluid by both depth filtration and tangential flow filtration, biomolecule purification by membrane adsorbers, sterilizing grade filtration and bioburden reduction by normal flow filtration, and virus removal by specially designed virus filtration membranes. Recent developments in biomanufacturing are driving new innovations in membrane technology to address the needs associated with continuous bioprocessing, single-use flexible manufacturing, and the production of more complex biotherapeutics for gene and cell-based therapies. 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

Four Protein A resins from leading industry vendors, including AmsphereTM A3, were screened for use in a capture step for a Herceptin biosimilar. Multiple parameters were assessed for their performance against the platform resin. Initially, a dynamic binding capacity (DBC) determination was made using a standard 4-minute residence time (RT) using purified protein. Performance of the resins were then screened for recovery and purity using harvested cell culture fluid (HCCF) where the mAb load was 80% of DBC at 10% breakthrough (BT). It was observed that substituting Amsphere A3 in the platform capture process led to a 40% increase in DBC relative to two of the resins tested including the platform resin and a 25% DBC increase over the third comparator resin. 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

Virus filtration is a critical process in the production of biotherapeutics and drug products derived by plasma fractionation. The filterability of process solutions on virus removal filters is largely dependent on preceding downstream process steps, and column chromatography can have a particularly large impact on the throughput and flux of virus filtration. Filterability (throughput and flux) on Planova BioEX was greatly reduced for mAb and plasma IgG spiked with aggregate, and filterability improvement achieved by processing with chromatography resins (modified CEX, mixed-mode AEX and normal AEX column) was specific to protein solution. 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

The purification and manufacture of recombinant proteins, especially those lacking affinity handles, often requires multiple chromatography steps and complicated feedstream conditioning, resulting in high production costs and relatively low recovery. These issues may be exacerbated by low protein expression, pH sensitivity, and limited stability. Mixed-mode chromatography resins have become an important tool for the purification of such proteins. The objective of this study was to purify one such recombinant protein, EPA, from E. coli fermentate to >95% purity and recovery. 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

Cation exchange chromatography (CEX) is an essential part of most monoclonal antibody (mAb) purification platforms. Process characterization and root cause investigation of chromatographic unit operations are performed using scale down models (SDM). SDM chromatography columns typically have the identical bed height as the respective manufacturing-scale, but a significantly reduced inner diameter. While SDMs enable process development demanding less material and time, their comparability to manufacturing scale can be affected. Mechanistic models can help to close gaps between scales and reduce experimental efforts compared to experimental SDM applications. 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

Q Lock is a fully configurable single-use quick locking system specifically developed to replace silicone tube sets in bioprocessing applications with pressures up to 100 psi. These fittings are available in various port configurations like elbows, tees, reducers, crosses, etc., in sizes from 0.25" to 1.50 and are manufactured from certified USP Class IV materials (PVFD). 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

Tangential flow filtration (TFF) has many advantages for bioreactor harvesting, as the permeate could be introduced directly to the subsequent capture step. However, membrane fouling has limited its widespread use. This is particularly problematic given the high cell densities encountered today. Here, a reverse asymmetric membrane, where the more open surface faces the feed stream and the tighter barrier layer faces the permeate stream, has been investigated. Our results suggest that using a reverse asymmetric membrane could avoid severe flux decline associated with fouling of the barrier layer during bioreactor harvesting. 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

TFF Process Control System is an extremely valuable tool in biopharm and viral vector for process development & pilot scale production. Its dynamic concentration mode (Fed-batch) can perform very high concentration factors (>20x) in a completely automated fashion with no user intervention. 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

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. 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

We report a mid-IR transmission setup for the analysis of the protein amide I and amide II band in aqueous solutions that achieves a limit of detection as low as 0.0025 mg mL-1, outperforming our previous results and other state-of-the-art mid-IR-based techniques by almost an order of magnitude. This large improvement is made possible by combining the latest-generation external cavity-quantum cascade laser (EC-QCL) operated at room temperature with an optimized double-beam optical setup that adjusts the path length (26 μm) to ensure robust sample handling. 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

Efficient virus recovery and impurity removal arechallenges in the purification of viruses such as influenza A virus subtype H1N1. As a capture chromatography medium, high capacity anion exchange resin, resulted in high virus recovery and excellent impurity removal in a single purification step. Learn More

This chapter discusses the role of membrane processes, namely, microfiltration, ultrafiltration, nanofiltration, and reverse osmosis in the field of bioprocess engineering. Membrane processes are widely accepted and used techniques in separation and filtration applications because of their unique and beneficial properties, such as low cost, environment friendliness, ease to scale up, ease to integrate with other processes, and compactness. Therefore, their use and implementation in various fields of bioprocess engineering helps in saving a lot of energy and resources. 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

This white paper examines the material-handling challenges pertaining to flow rate and pressure in chromatography, virus filtration and TFF processes and illustrate why the design and operation of the quaternary diaphragm pump - rather than other technologies such as the lobe or peristaltic (hose) pump - makes it ideal for use in critical biopharmaceutical-manufacturing applications. Additionally, the paper will show how the quaternary diaphragm pump's ability to operate consistently whether in a fixed stainless-steel production regime or in the increasingly popular single-use applications gives it the versatility to optimize biopharmaceutical-manufacturing maintenance, downtime, changeover and operational costs. Learn More

The aim of this handbook is to guide and inspire beginners as well as experts toward successful Western blotting based on the latest Cytiva technology and know-how. It provides expertise and support, describing the complete Western blotting workflow, from sample preparation to detection and analysis to achieve optimal results. 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

Continuous downstream processing is increasingly evaluated and implemented in the biopharmaceutical industry. For the capture of monoclonal antibodies using protein A affinity chromatography, periodic countercurrent processes have been described. While general process principles have been fully understood, the focus has shifted to important manufacturing-related aspects such as scale-up, control and validation. 

As part of a process validation procedure, this ~30 minute seminar presents an outline of a risk-based model-assisted process characterization approach for the twin column capture process CaptureSMB. The basic procedure uses process description, risk analysis and ranking, parameter testing and statistical analysis as main elements. It is shown how modeling can be used to significantly lower the experimental burden of twin column capture process validation and find optimal process operating ranges. 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

New biopharmaceutical drug products that alter and even save lives can be very profitable for their developers. They are, however, costly to develop and represent significant investment, particularly at the end of the manufacturing process. For this reason, safe cost-effective frozen storage and transport are critical, especially as drugs transition from R&D through clinical trial and to higher volume production. The objectives for choosing a new storage/transport method include minimizing both capital and operating expense, reducing facility space requirements, and cutting time and energy for clean-in-place and steam- in-place processes. At the same time, valuable product must be protected from cross-batch or other sources of contamination. 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

Although several compelling benefits for bioprocess intensification have been reported, the need for a streamlined integration of perfusion cultures with capture chromatography still remains unmet. Here, a robust solution is established by conducting tangential flow filtration-based perfusion with a wide-surface pore microfiltration membrane. The resulting integrated continuous bioprocess demonstrated negligible retention of antibody, DNA, and host cell proteins in the bioreactor. Further discussion regarding the potential membrane fouling mechanisms is also provided by comparing two membranes with different surface pore structures and the same hollow fiber length, total membrane area, and chemistry. 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

Many viruses utilize cell-surface glycans as receptors for host cell entry. Viral surface glycoproteins specifically interact with glycan motifs, which strongly contributes to viral tropism. Recently, the interactions between host cell glycan receptors and the mumps virus (MuV) hemagglutinin-neuraminidase (MuV-HN) protein were characterized by determining the co-crystal structure of MuV-HN in complex with glycan receptors. Here, we describe protocols for large-scale expression, purification and crystallization of MuV-HN proteins for structural analyses and glycan-binding assays with the overarching goal of investigating glycan-protein interactions. 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

Biopharmaceutical protein production plays an increasingly important role in human health, including the treatment and prevention of cancer and immune diseases. With the rapid development of synthetic biology technology, cell-free protein synthesis (CFPS) has been regarded as a powerful tool to manufacture a variety of proteins because of its remarkable advantages over living-cell protein synthesis. Working with an efficient and easily scalable reactor, CFPS can meet the requirements of flexible on-demand protein synthesis. To achieve this goal, a new continuous-flow CFPS platform was developed using a tube-in-tube reactor in this study. 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

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 at ultrafiltration and diafiltration (UF/DF) unit operation of biologics manufacturing. 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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A novel version of bead -based assays with fluorescence detection enables the high-throughput analysis of antibodies and proteins. The protocols are carried out in special 384-well plates, require very few manual interventions, and are easy to automate. Here we describe how the technology can be used to determine antibody titers and screen for product glycosylation, a critical quality attribute, early in cell line and bioprocess development. Learn More

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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Counterflow centrifugal (CFC) systems have been applied to cell manufacturing for the processes of cell selection and buffer exchange. The recently developed Rotea™ (Scinogy) is a CFC device that enables closed automated buffer exchange for small-scale cell manufacturing. However, the fluid dynamics within the processing chamber is complex and are influenced by a number of factors, including flowrate, centrifugal force, and buffer density. The impact of individual or compounded effects on cell quality is unclear. This study aims to determine the critical factors for mesenchymal stem cell (MSC) processing using the Rotea™. Learn More

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

Affinity capture represents an important step in downstream processing of proteins, and it is conventionally performed through a chromatographic process. The performance of this step highly depends on the type of matrix employed. In particular, resin beads and convective materials, such as membranes and monoliths, are the commonly available supports. The present work deals with non-competitive binding of bovine serum albumin (BSA) on different chromatographic media functionalized with Cibacron Blue F3GA (CB). The aim is to set up the development of the purification process starting from the lab-scale characterization of a commercially available CB resin. 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

We describe an affinity purification-mass spectrometry (AP-MS) method for probing the interactome of a special targeting protein. The AP was implemented with monolithic micro immobilized metal ion affinity chromatography columns (m-IMAC) which were prepared by photoinitiated polymerization in the tip of a pipet (spin-tip columns). The recombinant His6-tagged protein (bait protein) was reversibly immobilized on the affinity column through the chelating group nitrilotriacetic acid (NTA)-Ni2+. The bait protein and its interacting partners can be easily eluted from the affinity matrix. The pulled-down cellular proteins were then analyzed with label-free quantitative proteomics. 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

Cleaning validation acceptance criteria in multiproduct facilities are established using maximum allowable carryover calculations. Carryover calculations incorporate the shared equipment surface area between two products to ensure that an acceptable limit for residue from the previously manufactured product to the subsequent product is determined. Until now, this assumption has been based on empirical knowledge without experimental data quantifying the clearance or removal of potential residues. We present a three-part study that determined the effects of cleaning conditions on selected monoclonal antibodies (mAbs) and the generation of degraded fragments... 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

Technology to increase productivity in the capture chromatography step during the purification of bio-molecules (e.g. mAb) by 2 to 3-fold while reducing Protein A and buffer usage by up to 50% is presented in this 30 minute presentation.  Animations to illustrate the technique will quickly provide the webinar attendee with insight of how the process mimics conventional batch LPLC purification - but allows continuous chromatography. Economics of the process as applied to mAbs illustrate the impact of incorporating the patented CaptureSMB process on plant productivity and cost-reduction. Information on scale-up and GMP scale equipment presently in use at multiple biopharmaceutical companies will be shown. 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

Bioprocess intensification can be achieved through high cell density perfusion cell culture with continuous protein capture integration. Protein passage and cell retention are commonly accomplished using tangential flow filtration systems consisting of microporous membranes. Significant challenges, including low efficiency and decaying product sieving over time, are commonly observed in these cell retention devices. Here, we demonstrate that a macroporous membrane overcomes the product sieving challenges when comparing to several other membrane chemistries and pore sizes within the microporous range. This way, variable chromatography column loading is avoided... 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

This whitepaper, authored by Kaiser Optical, discusses the role of Raman in extending process understanding to downstream bioprocesses to ensure high-quality biological products. Rapid and non-destructive spectroscopic methods offer monitoring and control options for bioprocessdownstream operations, such as in situ monitoring of purification cycles, buffer identification, protein aggregation, product identification, and quality control. 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

Experience of multiple end-users employing GMP scale pilot systems using twin-column continuous capture and sequential batch (polishing) chromatography on actual mAb feed-stock is presented in this data-based presentation. Economics as modeled by end-users is also shown. During this 30 minute webinar a number of case studies showing the successful operation of a twin-column capture process (CaptureSMB) for monoclonal antibodies in GMP pilot scale are reviewed. Compared to traditional batch chromatography, specific productivity and resin capacity utilization by up to 2-3 fold and 30-60%, respectively. 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

The bacterial resistance caused by the irrational usage of antibiotic has become a global public health crisis. Phage therapy is expected to be an alternative to antibiotics for the treatment of drug-resistant infections. However, industrial separation and purification of phage are cumbersome, time-consuming, high cost, or low efficiency. In this study, two-step salting-out extraction of Klebsiella phage was evaluated. This work provides a potential method for industrial phage purification and has a great significance for the future development of phage therapy. 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

In this case study the author, AdvantaPure, describes the design and implementation of a custom molded filling assembly used in final fill. The primary objective of the customer was to re-design the current filling process to minimize potential assembly leak points and maintain sterility assurance level (SAL 10-6). Additionally, the customer was looking to maximize throughput and repeatedly produce accurate fill volumes. 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

Affinity purification, such as Protein A (ProA) followed by size exclusion chromatography (SEC) remains a popular method to obtain research scale proteins. With the need for higher throughput protein production increasing for discovery research, there is substantial interest in the automation of complex protein purification processes, which often start with a ProA step followed by SEC. However, the harsh elution conditions from ProA based chromatography can destabilize some proteins resulting in particulates, which in turn can cause column fouling and potential cross-contamination of subsequent purifications. 

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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

Continuous downstream processing is increasingly evaluated and implemented in the biopharmaceutical industry. For the capture of monoclonal antibodies using protein A affinity chromatography, periodic counter-current processes have been described. While general process principles have been fully understood, the focus has shifted to important manufacturing-related aspects such as scale-up, control and validation.

As part of a process validation procedure, we present an outline of a risk-based model-assisted process characterization approach for the twin column capture process CaptureSMB. The basic procedure uses process description, risk analysis and ranking, parameter testing and statistical analysis as main elements. It is shown how modeling can be used to significantly lower the experimental burden of twin column capture process validation and find optimal process operating ranges.

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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

In this presentation, the fundamental principles of virus filtration as well as its practical execution will be discussed. In addition, participants will gain an in-depth understanding of filterability, validation and the local regulatory framework surrounding virus filtration processes. By the conclusion of the session, all attendees should be able to conduct a virus filterability trial and will be well equipped to have appropriate discussions with validation labs. 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

A well optimized and characterized chromatographic purification process is critical for robust and cost-effective manufacturing. Novasep has accumulated 30 years of experience for streamlining process development of preparative chromatographic processes, based on the successful execution of more than 30 R&D and production projects each year. In this webinar, we discuss large scale chromatographic purification processes for APIs, from development to scale-up, through some case studies based on real projects. 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

Drug formulation design is used to develop a drug delivery system (DDS) suitable for the administration of therapeutics while maintaining their concentration within the therapeutic range for the desired duration to achieve a therapeutic effect. It is important to thoroughly understand the required chemical, interfacial, mechanical, and biological properties of the formulation before selecting a suitable biomaterial for drug delivery purposes. This chapter illustrates the properties of DDSs required for various routes of drug administration with a specific focus on targeting drugs to the brain, colon, and cancer tissues. 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

Biological centrifugation is a process that uses centrifugal force to separate and purify mixtures of biological particles in a liquid medium. It is a key technique for isolating and analyzing cells, subcellular fractions, supramolecular complexes, and isolated macromolecules such as proteins or nucleic acids. The development of the first analytical ultracentrifuge by Svedberg in the late 1920s and the technical refinement of the preparative centrifugation technique by Claude and colleagues in the 1940s positioned centrifugation technology at the center of biological and biomedical research for many decades. Today, centrifugation techniques represent a critical tool for modern biochemistry and are employed in almost all invasive subcellular studies. 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 Car9 affinity tag is a dodecameric silica-binding peptide that can be fused to the N- and C-termini of proteins of interest to enable their rapid and inexpensive purification on underivatized silica in a process that typically relies on l-lysine as an eluent. Here, we show that silica paper spin columns and borosilicate multi-well plates used for plasmid DNA purification are suitable for recovering Car9-tagged proteins with high purity in a workflow compatible with high-throughput experiments. Spin columns typically yield 100 μg of biologically active material that can be recovered in minutes with low concentrations of lysine. 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

Chaperones are a diverse class of molecules known for increasing thermo-stability of proteins, preventing protein aggregation, favoring disaggregation, increasing solubility and in some cases imparting resistance to proteolysis. These functions can be employed for various biotechnological applications including point of care testing, nano-biotechnology, bio-process engineering, purification technologies and formulation development. Here we report that the N-terminal domain of Pyrococcus furiosus l-asparaginase, (NPfA, a protein chaperone lacking α-crystallin domain) can serve as an efficient, industrially relevant, protein additive. 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

Protein biopharmaceuticals, among which interferon alpha-2b (IFNα-2b) that can be used in the treatment of chronic hepatitis C and hairy cell leukemia, have become an indispensable product of current medicine. However, their current high costs derived from the lack of cost-effective downstream strategies still limits their widespread use. Polymer-based aqueous two-phase systems (ATPS) or aqueous biphasic systems (ABS) can be used in biopharmaceuticals purification. This work investigates the application of ionic liquids (ILs) as adjuvants (at 5 wt%) in ATPS constituted by polyethylene glycol with a molecular weight of 600 g mol−1 (PEG 600) and polypropylene glycol with a molecular weight... Learn More

Reliable scale-up of biopharmaceutical production processes is key in Quality by Design. In this study, a model-based workflow is described to evaluate the bioprocess dynamics during process transfer and scale-up computationally. First, a mathematical model describes the bioprocess dynamics of different state variables (e.g., cell density, titer). Second, the model parameter probability distributions are determined at different scales due to measurement uncertainty. Third, the quantified parameter distributions are statistically compared to evaluate if the process dynamics have been changed. This workflow was tested for the scale-up of an antibody-producing CHO fed-batch process. Significant differences were identified... Learn More

Protein purification processes in basic research using ÄKTA™ liquid chromatography systems are often limited to single sample injections and simple one-column purifications. Because many target proteins in structural biology require complex purification protocols the work easily becomes laborious. To streamline and accelerate downstream protein production, an ALIAS™ autosampler and a modular sample in-line dilution process coupled to ion-exchange chromatography were incorporated into the workflow to automate two of the most commonly performed purification strategies - ion-exchange to size exclusion and nickel-ion metal affinity to size exclusion. Learn More

The development of a process to manufacture drugs for clinical studies requires the right analytical data to make the final process decisions. Some of the critical data used in making the decisions in microbial fermentations are the sugars (glucose/glycerol), acetate, and phosphate levels. The sugar levels help to determine the amount of feed needed at the growth rate of the cells. Excess feed results in the accumulation of acetic acid in the fermentation broth which is toxic to the cells. For processes where the induction mechanism involves the depletion of phosphate (PhoA process), the level of Phosphate in the media needs to be below a threshold for expression. This session discusses the analytical data that was generated to determine the final process to be used in cGMP manufacturing to produce clinical materials. Learn More

Specifically tailored amino acid-based formulations were previously shown to have a high potential to avoid stress-mediated degradation of complex molecules such as monoclonal antibodies and viral vectors. By using adenovirus 5 (Ad5) as a model, we studied whether such formulations may also efficiently protect viral vectors in thermal stress experiments and during long-term liquid storage. Algorithm-based amino acid preselection using an excipient database and subsequent application of design of experiments (DoE) in combination with a 37°C challenging model enabled the prediction of long-term storage stability of Ad5. Learn More

Recombinant protein production in the baculovirus expression vector system (BEVS) has emerged as a system of choice for the production of recombinant human proteins for R&D purposes. Scale-up protein production in insect cells past the one or two liter volume generally utilizes disposable cellbag bioreactors that provide a means to scale to the 5-25L range in a single vessel. However, cellbags can be expensive and their use requires capital investment in dedicated rocker platforms and their associated air pumps and exhaust heaters. Additional equipment, such as tube welders and liquid pumps are often also deployed for the sterile transfer of media outside of a biosafety cabinet. Learn More

CGMP regulations stipulate that sampling and testing of in-process materials and drug products requires control procedures to "be established to monitor the output and to validate the performance of those manufacturing processes that may be responsible for causing variability in the characteristics of in-process material and the drug product". Learn More

In this presentation,Kristin O'Neill at Merck discusses the addition of Flownamics' fully automated system for bioreactor sampling, analysis, and feed control and how it provided an opportunity to integrate a substantial amount of discrete sample data into the PI system. This innovative use case enabled remote monitoring of all aspects of the cell culture process as well as provided a tool to monitor the system health of the auotsampler itself. This novel application of bringing discrete data from the autosampler to the PI system uses both PI Vision and PI AF. Learn More