Articles

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

In this study a model for an unconventional bioreactor, a U-loop fermentor is presented and compared to experimental data from a series of pH tracer pulse experiments. A good agreement between model and data, with very little error and computation time could be achieved when numerical diffusion combined with a carefully determined variable finite volume discretization method is employed to solve the system of partial differential model equations. The approach also overcame the obstacle posed by a constraint related to a complex pressure profile. An analysis of stability, controllability and observability if the reactor model is extended... Learn More

The main goal in biosimilar development is to increase Chinese Hamster Ovary (CHO) viability and productivity while maintaining product quality. Despite media and feed optimization during process development, depletion of amino acids still occurs. The aim of the work was to optimize an existing industrial fed batch process by preventing shortage of amino acids and to gather knowledge about CHO metabolism. Several process outputs were evaluated such as cell metabolism, cell viability, monoclonal antibodies (mAbs) production, and product quality. First step was to develop and supplement an enriched feed containing depleted amino acids. Learn More

Interferon-alpha receptor 1 (IFNAR1) is a target of interest for recombinant biotherapeutics that block the JAK/STAT pathway. In this work, we evaluate three different expression systems (baculovirus, human embryonic kidney 293 (HEK293×), and Chinese hamster ovary (CHO)) to improve expression of IFNAR1 ECD. We demonstrate the benefits of utilizing mammalian CHO cell transient transfection to increase expression titer, as well as an improved two-step purification process performed using immobilized metal affinity chromatography (IMAC) as the capture step and Ceramic Hydroxyapatite (CHT) Type II for HMW impurity removal in flow through mode. This process showed an 20-fold increase in productivity compared to the baseline process as measured by grams purified per liter of cell culture fluid. Learn More

The goal of cell culture process intensification is to increase volumetric productivity, generally by increasing viable cell density (VCD), cell specific productivity or production bioreactor utilization in manufacturing. In our previous study, process intensification in fed-batch production with higher titer or shorter duration was demonstrated by increasing the inoculation seeding density in combination with media enrichment. In this study, we further increased seeding density using perfusion N-1 seed cultures, which increased titers already at industrially relevant levels by 100% in 10-14 day bioreactor durations for four different mAb-expressing CHO cell lines. Learn More

The aim of this study was to obtain high-yielding cell cultures of Rubia cordifolia by applying gamma irradiation and subsequently scaling up for anthraquinone production in a bioreactor. Calli cultured on MS medium was irradiated at variable doses between 2 and 30 Gy. The callus cultures that were irradiated at 8 Gy accumulated a maximum alizarin level and a purpurin level during the M1T4 (fourth sub-cultures after gamma irradiation treatment) subculture, which was 6 fold and 11 fold higher than those of the nonirradiated callus cultures, respectively. Suspension cultures that originated from high-yielding callus cultures... Learn More

Single-use plastic culture devices are commonly used for two-dimensional (2-D) cell cultures. However, as oxygen is generally supplied by uni-directional diffusion from the top gas-liquid interface, 2-D cell cultures in the absence of medium flow may experience rapid exhaustion of dissolved oxygen at the bottom of culture wares with high cell densities. To overcome this issue, special cell culture devices utilizing the gas-permeable membrane at the bottom have been developed. In this study, cell growth, metabolic activity, and productivity were evaluated in gas-permeable culture devices compared to those from conventional gas-impermeable culture devices. Learn More

Oxygen transfer is a key element in aerobic fermentations, especially if the culture broth's rheology is non-Newtonian, as in the case of cultures of filamentous fungi. Viscosity negatively affects the volumetric mass transfer coefficient, but mechanisms involved in terms of change of interfacial area (a) and liquid-side mass transfer coefficient have still not been clearly identified. This lack of knowledge is in part due to the difficulty in measuring bubble size in viscous fluids. In this study, an innovative technique was used to measure bubble Sauter diameter in water and in xanthan gum solutions. Additional experiments were carried out to obtain the volumetric mass transfer coefficient and the global gas holdup... Learn More

The ability to predict an antibody's propensity for aggregation is particularly important during product development to ensure the quality and safety of therapeutic antibodies. We demonstrate the role of container surfaces on the aggregation process of three mAbs under elevated temperature and long-term storage conditions in the absence of mechanical stress. Our conclusion is that special care should be taken when interpreting size exclusion and particle count data from stability studies if different temperatures and vial types are involved. We propose a novel combination of methods to characterize vial surfaces and bulk solution for a full understanding of protein aggregation processes in a sample. Learn More

CHO cells are most prevalently used for producing recombinant therapeutics in biomanufacturing. Recently, more rational and systems approaches have been increasingly exploited to identify key metabolic bottlenecks and engineering targets for cell line engineering and process development based on the CHO genome-scale metabolic model which mechanistically characterizes cell culture behaviors. However, it is still challenging to quantify plausible intracellular fluxes and discern metabolic pathway usages considering various clonal traits and bioprocessing conditions. Thus, we newly incorporated enzyme kinetic information into the updated CHO genome-scale model (iCHO2291) and added enzyme capacity constraints within the flux balance analysis framework. Learn More

During the scale-up of a bioprocess, not all characteristics of the process can be kept constant throughout the different scales. This typically results in increased mixing times with increasing reactor volumes. The poor mixing leads in turn to the formation of concentration gradients throughout the reactor and exposes cells to varying external conditions based on their location in the bioreactor. This can affect process performance and complicate process scale-up. Scale-down simulators, which aim at replicating the large-scale environment, expose the cells to changing environmental conditions. This has the potential to reveal adaptation... Learn More

The ambr 15 has become the industry's standard automated microbioreactor system for mammalian cell culture. It has applications throughout the industry, most commonly for cell line screening and media/feed development. On each ambr 15 workstation, conditions in up to 48 × 15 mL bioreactors can be individually controlled while a liquid handler enables automated addition and removal of liquids during the process. Integrated cell counting, metabolite analysis and pH offset correction are also possible thereby reducing the operator interactions that are required. Extensive user and software manuals are supplied by the manufacturer, but in this chapter we describe additional ways of working that we have implemented in routine cell line screening using the ambr 15. Learn More

In 2004, the FDA published a guideline to implement process analytical technologies (PAT) in biopharmaceutical processes for process monitoring to gain process understanding and for the control of important process parameters. Viable cell concentration (VCC) is one of the most important key performance indicator (KPI) during mammalian cell cultivation processes. Commonly, this is measured offline. In this work, we demonstrated the comparability and scalability of linear regression models derived from online capacitance measurements. Learn More

There is a great need for high-throughput protein purification to produce protein molecules for research and therapeutics. Although there have been significant advancements made in automated multi-step chromatography and preparative in-process design-of-experiment (DOE) capabilities in commercial fast performance liquid chromatography (FPLC) instruments, almost all commercial FPLCs rely on a binary buffer mixing system, which hinders automated buffer preparation. Nevertheless, current-generation FPLCs are equipped with a quaternary mixer designed for limited in-line buffer preparation and preparative pH scouting DOE experiments. We decided to leverage the quaternary mixing capability by extending and re-programming AkTA Avant's quaternary valve into an automated in-process buffer preparation system to simplify automated purification requiring complex washing steps. Learn More

Perfusion is considered as the preferable unit operation mode for fully integrated continuous bioprocessing. However, the inherent complex process control, long process development times, and lack of suitable scale-down models for high-throughput screening are reasons why perfusion processes are still not routinely applied in cell culture technology. Advantages of perfusion are maintenance of a consistent cellular environment, a constant high-quality product flow, enhanced volumetric bioreactor productivity, and small lab footprint. Here, we provide guidelines for screening different proprietary but commercially available HyClone™ Cell Boost™ supplements in a Design of Experiment (DoE) approach... 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

Micro-bioreactors appear frequently in today's biotechnology 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. The micro-Matrix system consists of shaken disposable 24 deep square well plates in which each well is controlled individually for pH, dissolved oxygen (DO), and temperature. Learn More

Dissolved carbon dioxide (dCO2) is a well-known critical parameter in bioprocesses due to its significant impact on cell metabolism and on product quality attributes. Processes run at small-scale face many challenges due to limited options for modular sensors for online monitoring and control. Traditional sensors are bulky, costly, and invasive in nature and do not fit in small-scale systems. In this study, we present the implementation of a novel, rate-based technique for real-time monitoring of dCO2 in bioprocesses. A silicone sampling probe that allows the diffusion of CO2 through its wall was inserted inside a shake flask/bioreactor and then flushed with air to remove the CO2 that had diffused into the probe... Learn More

Mass transfer is known to play a critical role in bioprocess performance and henceforth monitoring dissolved O2 (DO) and dissolved CO2 (dCO2) is of paramount importance. At bioreactor level these parameters can be monitored online and can be controlled by sparging air/oxygen or stirrer speed. However, traditional small-scale systems such as shake flasks lack real time monitoring and also employ only surface aeration with additional diffusion limitations imposed by the culture plug. Here we present implementation of intensifying surface aeration by sparging air in the headspace of the reaction vessel and real-time monitoring of DO and dCO2 in the bioprocesses to evaluate the impact of intensified surface aeration. Learn More

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

Single-use bioreactors have increasingly been used in recent years, for both research and development as well as industrial production, especially in mammalian cell-based processes. Among the numerous single-use bioreactors available today, wave-mixed bags and stirred systems dominate. Wave-mixed single-use bioreactors are the system of choice for inoculum production, while stirred single-use bioreactors are most often preferred for antibody expression. For this reason, the present chapter describes protocols instructing the reader to use the wave-mixed BIOSTAT® RM 50 for cell expansion and to produce a monoclonal antibody (mAb) in Pall's Allegro™ STR 200 at pilot scale for the first time. All methods described are based on a Chinese hamster ovary (CHO) suspension cell line expressing a recombinant immunoglobulin G (IgG). Learn More

Increasing the cultivation volume from small to large scale can be a rather complex and challenging process when he method of aeration and mixing is different between scales. Orbitally shaken bioreactors (OSBs) utilize the same hydrodynamic principles that define the success of smaller-scale cultures, which are developed on an orbitally shaken platform, and can simplify scale-up. Here we describe the basic working principles of scale-up in terms of the volumetric oxygen transfer coefficient (kLa) and mixing time and how to define these parameters experimentally. The scale-up process from an Erlenmeyer flask shaken on an orbital platform to an orbitally shaken single-use bioreactor (SB10-X, 12 L) is described in terms of both fed-batch and perfusion-based processes. Learn More

The single-use stirred bioreactors are increasingly recognized as a viable alternative in animal cell culture due to the higher production capacity, increased flexibility, prevention of cross contamination, reduction of the cleaning cost, and shortened downtime. In this paper, the effects of the impeller speed, the volumetric gas flow rate, and the impeller type on the volumetric gas-liquid mass transfer coefficient in a single-use unbaffled angled-shaft bioreactor applicable for the animal cell culture were analyzed and compared with those attained for the baffled vertical-shaft bioreactors. The volumetric gas-liquid mass transfer coefficient (KLa) was experimentally determined by the simplified dynamic pressure method... Learn More

Mammalian cell culture processes are very important for the production of various recombinant proteins for clinical applications such as vaccines, antibodies and pharmaceuticals. The physiology of these cells is very sensitive, leading to the need of sophisticated process technology. A reliable sensor set-up is required for the control of major process parameters, such as pH, dissolved oxygen, carbon dioxide, cell density and feed rates. If these set-points are not controlled tightly, this has a potential impact on the product quality and quantity. Besides that, a steady cell concentration measurement is necessary to plan feeding strategies. Learn More

During the scale-up of a bioprocess, not all characteristics of the process can be kept constant throughout the different scales. This typically results in increased mixing times with increasing reactor volumes. The poor mixing leads in turn to the formation of concentration gradients throughout the reactor and exposes cells to varying external conditions based on their location in the bioreactor. This can affect process performance and complicate process scale-up. Scale-down simulators, which aim at replicating the large-scale environment, expose the cells to changing environmental conditions. Learn More

Increasing pressures on biomanufacturing costs have led to renewed interest in the development of single-use technologies that can be readily adapted to continuous processing. The objective of this study was to use commercially available hollow fiber membranes, originally designed for high-flux hemodialysis, for single pass tangential flow filtration with high conversions. Experiments were performed with solutions of Immunoglobulin G (IgG) using polysulfone hollow fiber membrane cartridges. The hollow fiber modules were able to provide more than 10-fold concentration of IgG in a single-pass. Stable operation was achieved during a continuous run for over a 120-h period... 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. We modified both Bio Rad NGC and ÄKTA Pure systems to run a three-column process (ProA to buffer exchange to SEC) enabling automated tandem affinity to SEC purification while minimizing the risk of SEC column fouling and subsequent cross-contamination. Learn More

There is a great need for high-throughput protein purification to produce protein molecules for research and therapeutics. Although there have been significant advancements made in automated multi-step chromatography and preparative in-process design-of-experiment (DOE) capabilities in commercial fast performance liquid chromatography (FPLC) instruments, almost all commercial FPLCs rely on a binary buffer mixing system, which hinders automated buffer preparation. Nevertheless, current-generation FPLCs are equipped with a quaternary mixer designed for limited in-line buffer preparation and preparative pH scouting DOE experiments. Learn More

The critical process parameters cell density and viability during mammalian cell cultivation are assessed by UV/VIS spectroscopy in combination with multivariate data analytical methods. This direct optical detection technique uses a commercial optical probe to acquire spectra in a label-free way without signal enhancement. For the cultivation, an inverse cultivation protocol is applied, which simulates the exponential growth phase by exponentially replacing cells and metabolites of a growing Chinese hamster ovary cell batch with fresh medium. For the simulation of the death phase, a batch of growing cells is progressively replaced by a batch with completely starved cells. Learn More

Driven by epidemic events and by governmental vaccination programs, there is a rising demand for development of new vaccines and the industry is growing at a double-digit rate. The vaccine industry is facing the challenge of developing new products to serve so far unmet needs and fulfilling the demands on dose numbers, both in an economically viable way.

Upstream bioprocessing is an important piece of the puzzle. High titer, robustness of the process, constant product quality, fast turn-around times, and scalability are some of the success factors. With a comprehensive portfolio of scalable bioreactor and fermentor systems, software, single-use bioreactors, and worldwide service, Eppendorf strives to support bioprocess engineers in tackling these challenges.

With this ebook we would like to share expert views and case studies on some of the hot topics in vaccine bioprocess development. We hope the information on viral vector production, continuous bioprocessing, and process scale-up will prove useful for your own development projects.

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Continuous countercurrent tangential chromatography (CCTC) enables steady-state continuous bioprocessing with low-pressure operation and high productivity. CCTC has been applied to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest and postcapture polishing of mAb; however, these studies were performed with commercial chromatography resins designed for conventional column chromatography. In this study, a small particle size prototype agarose resin with lower cross-linking was co-developed with industrial partner Purolite and tested with CCTC. Due to increased binding capacity and faster kinetics... 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... Learn More

Biotechnology manufacturing has gradually evolved over the years from traditional batch mode operation to more continuous modes of operation with the implementation of continuous perfusion cell culture. Recently, with technical advancements in continuous chromatography and an increased focus on single-use systems, focus has shifted to an integrated upstream and downstream continuous process. However, due to technical constraints and lack of small-scale models, most theoretical continuous manufacturing designs focus on a hybrid continuous system with one or more dedicated virus removal / inactivation steps remaining in batch mode via traditional hold tanks (e.g., low pH inactivation) or as a dedicated offline step (e.g., virus filtration). Learn More

In situ measurement to determine mammalian cell number in a non-invasive, non-destructive and reagent-free manner is needed to enable continuous cell manufacturing. An analytical method is presented for non-invasive cell counting by conducting multiwavelength spectral analysis of mammalian cells achieving a minimal detectable cell count of 62,500 at 295 nm. Light absorbance was insensitive to culture volume, giving an absolute cell count rather than a concentration. The activation state of cells was also considered. The study was extended to quantification within polymeric microcapsules as an advanced substrate for mammalian cell growth... Learn More

Everything from your PC and smartphone to your clock, refrigerator and automobile have sensors, memory and communication capabilities, increasingly known as the internet of things (IoT). Incredible intelligence is being built into just about every device we touch, and, if it’s not built in, the information is available via communication with “the cloud.”

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Time-Gated Surface-Enhanced Raman spectroscopy (TG-SERS) was utilized to assess recombinant protein production in Escherichia coli. TG-SERS suppressed the fluorescence signal from the biomolecules in the bacteria and the culture media. Characteristic protein signatures at different time points of the cell cultivation were observed and compared to conventional continuous wave (CW)-Raman with SERS. TG-SERS can distinguish discrete features of proteins such as the secondary structures and is therefore indicative of folding or unfolding of the protein. A novel method utilizing nanofibrillar cellulose as a stabilizing agent for nanoparticles and bacterial cells was used for the first time... Learn More

Single-use (SU) technology has become an important part of biotechnology research and commercial biomanufacturing. Sustainability is a long-term holistic approach that evaluates how biological systems remain diverse and productive over time, and also considers concerns of a more immediate and nonbiological focus. Sustainability emphasizes the ability to use natural resources in a way that indefinitely protects the integrity (and limits the fouling or depletion) of existing biological and other environmental systems. Several initial environmental analyses performed for SU biologics production illustrated the need for rational and comprehensive analysis techniques such as life cycle assessment to understand the total, specific, and "cradle-to-grave" environmental strain imposed by both durable and SU approaches. "End-of-life" considerations are a big part of most discussions about SU and the environment because of the visibility of the pre and post-process material. 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. 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. This in turn enables selective and targeted process controls to respond in an appropriate manner to process disturbances. Learn More

This study provides a list of cold-inducible genes and endogenous cold-inducible promoters of CHO cells. Transcriptome data before and after a temperature shift from 37 to 33 °C are analyzed to identify 94 cold-inducible genes, which are highly expressed and have a high fold change in expression after the temperature shift. Cold-inducible promoters are identified from the top ten cold-inducible genes, showing up to 11-fold increased luciferase expression at lowered temperature in transient transfections. Additionally, several common transcription factor binding sites are identified in the top cold-inducible promoter sequences and expression... Learn More

Biomanufacturing relies on numerous pieces of equipment working in concert to produce life-altering therapeutics. The equipment relies on various subsystems to achieve the desired results. In a bioreactor, one of the most essential subsystems provides gas management for the gases necessary for cellular metabolism. At the heart of the gas management subsystem is the thermal mass flow controller (MFC), a component that precisely measures and controls the delivery of gases to the bioprocess.

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Original Publication Date: 11/2019

Iron-free HPLC systems, better known as biocompatible systems, are generally regarded to be chemically more inert compared to conventional HPLC systems. In this work, we studied the chromatographic behavior of some classes of compounds of pharmaceutical interest, analyzed with iron-free systems. Issues typically associated with metal contamination, i.e. strong peak tailing, were observed when using an amide polar-embedded column. Effects of the contamination were visible when anhydrous methanol-acetonitrile was used, indicating that this solvent, albeit generally considered safe for conventional HPLC systems, induce corrosion of iron-free systems. Learn More

Process intensification in mammalian cell culture-based recombinant protein production has been achieved by high cell density perfusion exceeding 108 cells/mL in the recent years. As the majority of therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cells, intensified perfusion processes have been mainly developed for this type of host cell line. However, the use of CHO cells can result in non-human posttranslational modifications of the protein of interest, which may be disadvantageous compared with human cell lines. In this study, we developed a high cell density perfusion process of Human Embryonic Kidney (HEK293) cells producing recombinant human Erythropoietin (rhEPO). The results from our study show that human cell lines, such as HEK293 can be used for intensified perfusion processes. 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. Learn More

Some antibodies exhibit elevated viscosity at high concentrations, making them poorly suited for therapeutic applications requiring administration by injection such as subcutaneous or ocular delivery. Here we studied an anti-IL-13/IL-17 bispecific IgG4 antibody, which has anomalously high viscosity compared to its parent monospecific antibodies. The viscosity of the bispecific IgG4 in solution was decreased by only ~30% in the presence of NaCl, suggesting electrostatic interactions are insufficient to fully explain the drivers of viscosity. Intriguingly, addition of arginine-HCl reduced the viscosity of the bispecific IgG4 by ~50%... Learn More

Just recently, chemical cross-linking combined with mass spectrometry (XL-MS) has emerged as valuable tool to study protein interaction networks on the system-wide level. The current challenges in XL-MS are to develop robust workflows enabling a comprehensive capture of dynamic biological assemblies in their native environment in a routine manner. In this review, we will highlight both the latest technological developments as well as selected applications of XL-MS for investigating protein networks in cells, organisms, and tissue. In addition, different bioinformatics tools for data analysis will be presented. In light of these exciting new developments... Learn More

The monoclonal antibody (mAb) market has changed rapidly in the past 5 years: it has doubled in size, becoming dominated by fully human molecules, launched bispecific molecules, and faced competition from biosimilars. We summarize the market in terms of therapeutic applications, type and structure of mAbs, dominant companies, manufacturing locations, and emerging markets. Learn More

Modular design is at the foundation of contemporary engineering, enabling rapid, efficient, and reproducible construction and maintenance of complex systems across applications. Remarkably, modularity has recently been discovered as a governing principle in natural biological systems from genes to proteins to complex networks within a cell and organism communities. The convergent knowledge of natural and engineered modular systems provides a key to drive modern biotechnology to address emergent challenges associated with health, food, energy, and the environment. Here, we first present the theory and application of modular design in traditional engineering fields. Learn More

Screening for novel producer strains and enhanced therapeutic production at reduced cost has been the focus of most of the biopharmaceutical industries. The obligation to carry out prolonged intensive pilot scale experiments gave birth to micro-scale bioreactor systems. Screening large number of microorganisms using shake flasks and benchtop bioreactors is tedious and consumes resources. Microbioreactors that mimic benchtop bioreactors are capable not only of high throughput screening of producer strains, but also aid in optimizing the growth kinetics and expression of proteins. Modern technology has enabled the collection of precise online data for variables such as optical density (OD), pH, temperature, dissolved oxygen (DO), and adjusting in mixing inside microreactors. 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

The increasingly competitive nature of the market for biopharmaceuticals is exacerbating the need for greater cost efficiency within the industry. This chapter describes how biomanufacturers can streamline early-stage development and rapidly configure a standard and well-qualified manufacturing single-use process by adopting the platform approach. Single-use platforms are available for a wide range of biological entities including mAbs, viral vectors, antibody-drug conjugates, and regenerative medicines. Single-use facilities require a lower capital investment than their multiuse stainless steel equivalents even if the costs of consumables are higher. Learn More

The Raw Material Risk Management workstream has launched a new, raw material risk assessment tool aimed at helping industry identify and prioritize around the challenging question of material fit. In our high-stakes, highly regulated environment, as suppliers and manufacturers strive to meet a perpetual tide of new "regulatory standards, the supplier-biomanufacturer relationship can become strained. This new tool standardizes and structures the risk assessment process, thereby improving communication between - and within - manufacturers and suppliers." Learn More

The continuous production of monoclonal antibodies (mAb) with the help of disposable equipment poses one of the future major changes in the pharmaceutical industry. Consequently, also continuous viral clearance needs to be developed. The coiled flow inverter (CFI) was successfully implemented in the continuous downstream as a residence time module for low pH viral inactivation. As the elution profile of the upstream continuously operated Protein A chromatography results in fluctuating pH values, the pH level distribution inside the CFI is highly relevant. This work presents a detailed investigation of pH level distribution inside the CFI at varying inlet conditions with the help of computational fluid dynamics (CFD) simulation... Learn More

In this study the Single-Pass-Tangential-Flow-Filtration (SPTFF) concept for continuous ultrafiltration in bioprocessing is investigated. Based on a previously validated physico-chemical model for a single ultrafiltration cassette, the transfer to a multistage SPTFF is predicted and validated experimentally by concentration steps for bovine serum albumin (BSA) and the monoclonal antibody immunoglobulin G (IgG) are compared. The model applied for the ultrafiltration membrane contains the Stagnant Film Model (SFM) for concentration polarization, as well as the Osmotic Pressure Model (OPM) and the Boundary Layer Model (BLM) for the mass transfer through the membrane. Learn More

The development of mammalian cell perfusion cultures is still laborious and complex to perform due to the limited availability of scale-down models and limited knowledge of time- and cost-effective procedures. The maximum achievable viable cell density, minimum cell-specific perfusion rate, cellular growth characteristics, and resulting bleed rate at steady-state operation are key variables for the effective development of perfusion cultures. In this study, we developed a stepwise procedure to use shake tubes in combination with benchtop bioreactors for the design of a mammalian cell perfusion culture at high productivity and low product loss in the bleed for a given expression system. Learn More

The objective of this mini-review is to provide an overview of: the history of bioprocess affinity chromatography, the current state of platform processes based on affinity capture steps, the maturing field of custom developed bioprocess affinity resins, the advantages of affinity capture based downstream processing in comparison to other forms of chromatography, and the future direction for bioprocess scale affinity chromatography. The use of affinity chromatography can result in economic advantages by enabling the standardization of process development and the manufacturing processes and the use of continuous operations in flexible multiproduct production suites. Learn More

In this study, a scale-down model representing commercial-scale cell culture process of adalimumab biosimilar HS016 was first developed based on constant power per volume principle and then qualified by multivariate data analysis and equivalence test method. The trajectories of the bench-scale process lie in the middle of the control range of large-scale process, built by multivariate evolution model based on nutrients, metabolites, and process performance datasets. This indicates that the small-scale process performance is comparable with that of the full-scale process. Learn More

We present a straightforward protocol for reverse genetics in cultured mammalian cells, using CRISPR/Cas9-mediated homology-dependent repair (HDR) based insertion of a protein trap cassette, resulting in a termination of the endogenous gene expression. Complete loss of function can be achieved with monoallelic trap cassette insertion, as the second allele is frequently disrupted by an error-prone non-homologous end joining (NHEJ) mechanism. The method should be applicable to any expressed gene in most cell lines, including those with low HDR efficiency, as the knockout alleles can be directly selected for. Learn More

Viral inactivation plays a critical role in assuring the safety of monoclonal antibody (mAb) therapeutics. Traditional viral inactivation involves large holding tanks in which product is maintained at a target low pH for a defined hold time. The drive toward continuous processing and improved facility utilization has provided motivation for development of a continuous viral inactivation process. To this end, a lab-scale prototype viral inactivation system was designed, built, and characterized. Multiple incubation chamber designs are evaluated to identify the optimal design that enables narrow residence time distributions in continuous flow systems. Learn More

In the area of biological drug development, high throughput (HT) technologies are key to identifying the most promising therapeutic candidate in a time-efficient and market-competitive manner. While efficient cloning and expression methods exist, HT downstream processing mainly relies on liquid handling workstations applying miniaturized chromatography columns or resin-based 96-well plates to shorten process development time. In this work, we devised a unique chromatography setup enabling an unattended two-step purification of IgGs on the milligram scale directly from 35 ml clarified cell supernatants. Learn More

Over the last decade, the growing use of single-use technology (SUT) in the biopharmaceutical industry has transformed how drugs are developed and manufactured. Traditional methods using large stainless-steel bioreactors with costly clean-in-place and sterilize-in-place systems have been replaced, in most cases, by more efficient SUT bioreactors. Not only do SUT bioreactors reduce the costs associated with drug manufacturing, but they also offer more flexibility, allowing companies to streamline operations and increase productivity. However, as many benefits as there are to SUT, there is one critical issue drug companies must address when transitioning to plastic equipment, and that is the presence of extractables and leachables (E&L). Learn More

Ingenza cooperated with Hamilton in order to plug the signal from VisiFerm sensors directly into their control towers, so that they are linked to bioreactor RPM and aeration on a cascade set-point. This enabled Ingenza to bring the benefit of optical DO sensors into their existing bioreactor. According to Alison Arnold, Ingenza's Head of Fermentation and Microbiology, beside the robustness of the digital signal, the main benefits of such solution, compared to an analog one, are the following: Time-savings, No downtime risk and Automated efficiency. This makes the company's fermentation processes more efficient. Learn More

An experimental feasibility study on continuous bioprocessing in pilot-scale of 1 L/day cell supernatant, that is, about 150 g/year product (monoclonal antibody) based on CHO (Chinese hamster ovary) cells for model validation is performed for about six weeks including preparation, start-up, batch, and continuous steady-state operation for at least two weeks stable operation as well as final analysis of purity and yield. A mean product concentration of around 0.4 g/L at cell densities of 25 × 106 cells/mL was achieved. After perfusion cultivation with alternating tangential flow filtration (ATF), an aqueous two-phase extraction (ATPE) followed by ultra-/diafiltration (UF/DF)...  Learn More

Despite the recent explosion in the use of monoclonal antibodies (mAbs) as drugs, it remains a significant challenge to generate antibodies with a combination of physicochemical properties that are optimal for therapeutic applications. We argue that one of the most important and underappreciated drug-like antibody properties is high specificity - defined here as low levels of antibody non-specific and self-interactions - which is linked to low off-target binding and slow antibody clearance in vivo and high solubility and low viscosity in vitro. Here, we review the latest advances in characterizing antibody specificity and elucidating its molecular determinants as well as using these findings to improve the selection and engineering of antibodies with extremely high, drug-like specificity.

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Cell culture is a ubiquitous and flexible research method. However, it heavily relies on plastic consumables generating millions of tons of plastic waste yearly. Plastic waste is a major and growing global concern. Here we describe a new cell culture dish that offers a culture area equivalent to three petri dishes but that is on average 61% lighter and occupies 67% less volume. Our dish is composed of a lid and three thin containers surrounded by a light outer shell. Cell culture can be performed in each of the containers sequentially. The outer shell provides the appropriate structure for the manipulation of the dish as a whole. The prototype was tested by sequentially growing cells in each of its containers.

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High-pressure liquid chromatography employing the multicolumn countercurrent solvent gradient purification (MCSGP) process principle has been developed as a novel purification technology for peptides produced by chemical synthesis. MCSGP offers a step change in efficiency compared to batch HPLC processing. Peptides can be purified at preparative/production scale with significantly higher yield without compromising target purity. The process also allows an up to 10-fold higher productivity with typically 80% lower solvent consumption, providing an overall attractive economical production scenario and allowing pushing of the boundary of economic synthesis of long peptides to realize savings of millions of US dollars.

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There is a trend across the pharmaceutical sector toward process intensification and continuous manufacturing to produce small-molecule drugs or biotechnology products. For biotechnology products, advancing the manufacturing technology behind upstream and downstream processes has the potential to reduce product shortages and variability, allow for production flexibility, simplify scale-up procedures, improve product quality, reduce facility footprints, increase productivity, and reduce production costs. This work examines the current scientific and regulatory landscape surrounding the implementation of integrated continuous biomanufacturing.

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Magnetic separation is a promising alternative to conventional methods in downstream processing. This can facilitate easier handling, fewer processing steps, and more sustainable processes. Target materials can be extracted directly from crude cell lysates in a single step by magnetic nanoadsorbents with high-gradient magnetic fishing (HGMF). Additionally, the use of hazardous consumables for reducing downstream processing steps can be avoided. Here, we present proof of principle of one-step magnetic fishing from crude Escherichia coli cell lysate of a green fluorescent protein (GFP) with an attached hexahistidine (His6)-tag, which is used as the model target molecule. The focus of this investigation is the upscale to a liter scale magnetic fishing process in which a purity of 91% GFP can be achieved in a single purification step from cleared cell lysate.

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Historically, manufacturers have used a 'waterfall' approach when designing and building their production facilities, sequentially resolving and specifying all aspects up front and in detail for each project, over and over again. These projects can take up to five years before reaching full operation and have an obsolescence risk because technology and solutions have often moved on by the time the project is completed. Even an agile project can take significant time to complete due to the number of inherent iterative design loops. These traditional projects can be expensive to build and modify, and may be inflexible if they have to change to accommodate new products. A new approach and mindset are needed to change the way that manufacturing facilities are designed and constructed.

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A large-scale production of clinical-grade MSCs and their derived products is essential due to their immense therapeutic potential. Even though 3D bioreactors are cost efficient in scale up, the plastic adherence of MSCs, makes expansion in suspension cultures challenging. A variety of microcarriers (MCs) allow plastic adherent cells to grow on their surface while maintaining cells in suspension within a bioreactor for expansion. However, this leads to loss of cells, particularly during separation of cells from the carriers. To overcome this, we identified "dissolvable microcarriers" such as Corning Synthemax II dissolvable MCs, which removes the filtration step.

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In the biopharmaceutical industry, mammalian cell culture systems, especially Chinese hamster ovary (CHO) cells, are predominantly used for the production of therapeutic glycoproteins. Glycosylation is a critical protein quality attribute that can modulate the efficacy of a commercial therapeutic glycoprotein. Obtaining a consistent glycoform profile in production is desired due to regulatory concerns because a molecule can be defined by its carbohydrate structures. An optimal profile may involve a spectrum of product glycans that confers a desired therapeutic efficacy, or a homogeneous glycoform profile that can be systemically screened for.

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Incorporating affinity chromatography in vaccine purification has long been attempted by researchers to improve unit yield and purity with the secondary goal of reducing the number of downstream process operations. Despite the success in laboratory-scale proof of concept, implementation of this technique in pilot or cGMP manufacturing has rarely been realized due to technical and economic challenges in design and manufacturing of ideal ligands as well as availability of high-productivity chromatography media. This paper reviews evolving technologies in engineered ligands and chromatography media that are encouraging companies to re-visit the possible use of affinity chromatography in larger scale vaccine purification.
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Ammonia has been reported to be toxic and inhibitory for mammalian cell cultures for many years. Reduction of growth rates and maximal cell densities in batch cultures, changes in metabolic rates, perturbation of protein processing and virus replication have been reported. However, cellular mechanisms of ammonia toxicity are still the subject of controversy and are presented here. The physical and chemical characteristics of ammonia and ammonium are important, with the former capable of readily diffusing across cellular membranes and the latter competing with other cations for active transport by means of carrier proteins.

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Monoclonal antibodies (mAbs) used as therapeutics often require formulation at high concentrations to minimize administration volume. High concentration poses an increased risk of instability, primarily via complicated aggregation pathways. Identification of consistently reliable tools to predict longer term stability based on initial data remains a challenge in the biotech industry, especially in the context of protein aggregation. Aggregation is influenced by both colloidal and conformational stability.  In this work, we evaluate the ability of these methods to predict the long-term aggregation for a series of mAbs based on their intrinsic molecular properties.

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A novel, alternative intensified cell culture process comprised of a linked bioreactor system is presented. An N-1 perfusion bioreactor maintained cells in a highly proliferative state and provided a continuous inoculum source to a second bioreactor operating as a continuous-flow stirred-tank reactor (CSTR). An initial study evaluated multiple system steady-states by varying N-1 steady-state viable cell densities, N-1 to CSTR working volume ratios, and CSTR dilution rates. After identifying near optimum system steady-state parameters yielding a relatively high volumetric productivity while efficiently consuming media, a subsequent lab scale experiment...

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In recent years, microbioreactor (MBR) systems have evolved towards versatile bioprocess engineering tools. They provide a unique solution to combine higher experimental throughput with extensive bioprocess monitoring and control, which is indispensable to develop economically and ecologically competitive bioproduction processes. MBR systems are based either on down-scaled stirred tank reactors or on advanced shaken microtiter plate cultivation devices. This review will discuss the current state of the art in the field of MBR systems and we can readily conclude that their importance for industrial biotechnology will further increase in the near future.

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Poor solubility is a common challenge encountered during the development of high concentration monoclonal antibody (mAb) formulations, but there are currently no methods that can provide predictive information on high-concentration behavior of mAbs in early discovery. We explored the utility of methodologies used for determining extrapolated solubility as a way to rank-order mAbs based on their relative solubility properties. We devised two approaches to accomplish this: 1) vapor diffusion technique utilized in traditional protein crystallization practice, and 2) polyethylene glycol (PEG)-induced precipitation and quantitation by turbidity.

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Thorough characterisation is essential for efficient and knowledge-led cell culture process development in biomanufacturing. Despite diverse applications of rocked bag bioreactors, there is currently little understanding of the fundamental determinants of fluid mixing and mass transfer, and the effects that these would have on cell culture kinetics, product quality and cell physiology. A rocked bag bioreactor has been fully evaluated at 10 to 50 L scale. Under typical operating conditions, single-use rocked bag bioreactor tm were found to vary from 7-71 s, kLa(O2) from 3.5-29 h -1 and kLa(CO2) from 0.6-2.7 h -1 , with the rocking rate found to cause gas entrainment above 20 min-1 . A GS-CHO cell line cultured under controlled fed-batch conditions at low rocking rate to produce surface aeration achieved significantly higher cell specific antibody productivities. However, these cells were significantly less robust at harvest than cells cultured in the presence of a dispersed gas phase in rocked bags or stirred tanks. A fabricated rocked bag mimic was fluid dynamically characterised using particle image velocimetry. It was found that increasing rocking rate from 25 to 42 min-1 produced an 8-fold increase in turbulence kinetic energy, giving the rocked bag similar fluid dynamic characteristics to a stirred tank. The gas entrainment noted at higher rocking rates was connected to the fluid transitioning out of phase at higher rocking rates. A detailed cell culture kinetic, physiological and transcriptomic evaluation demonstrated that cells cultured in the rocked bag operated to entrain gas matched very closely those cultured in a stirred tank. Cells cultured in a bubble free environment exhibited several indications of higher stress, despite identical cell culture kinetics to the stirred tank. In a second industrial GS-CHO cell line, the specific productivity of the cells cultured in entrained gas phase bags was again found to be lower than those cells cultured in surface aerated bags, however the product quality was not significantly impacted. Abstract 5 In summary, this work demonstrates the flexibility of rocked bags as alternative single-use bioreactor designs.

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