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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N-linked glycosylation affects the potency, safety, immunogenicity, and pharmacokinetic clearance of several therapeutic proteins including monoclonal antibodies. A robust control strategy is needed to dial in appropriate glycosylation profile during the course of cell culture processes accurately. However, N-glycosylation dynamics remains insufficiently understood owing to the lack of integrative analyses of factors that influence the dynamics, including sugar nucleotide donors, glycosyltransferases, and glycosidases. Here, an integrative approach involving multi-dimensional omics analyses was employed to dissect the temporal dynamics of glycoforms produced during fed-batch cultures of CHO cells.

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As compared to other biotech products, viral vaccine manufacturing processes present some specific constraints linked to the cell substrates used. To remain competitively priced as well as profitable, bioprocess scientists are under pressure to develop methods for faster and more cost-efficient cell culture production. This has led to a shift from the use of expensive, two-dimensional T-flask and roller bottles to single-use, stirred tank bioreactors with microcarriers, or the adaptation of attachment-dependent cell lines such as BHK-21 for suspension culture. In this article, single-use, mini bioreactors are evaluated to determine if they are geometrically comparable to benchtop bioreactors (both glass and single-use vessels) and pilot-scale, single-use bioreactors for effectively modelling mammalian cell culture at 2 L and 50 L scale...
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It goes without saying that upgrades during a manufacturing shutdown pose less risk as there are no products being produced, but there are instances when it is critical to expand capacity or modify functionality while maintaining some processing. For example:

  • Manufacturers upgrade certain areas while maintaining process operations on adjacent lines
  •  Companies reconfigure equipment or add modular, flexible capabilities for the future
  • New or established manufacturers increase capacity incrementally while keeping supply on the market (and revenue flowing)

As George Wiker, Executive Director at AES Clean Technology, Inc.® , explains, these upgrades are a challenge for a variety of reasons. It is critical to maintain control of production and changes—and demonstrate proof of control—while keeping the involved workers and the products safe.

It is also important to note that a company will need to revalidate any system modified during a facility change, and the extent of the change may also require a re-inspection by the FDA.

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Original Publication Date: 12/2016

Just five or six years ago, bioprocessors viewed continuous bioprocessing skeptically. Now, seemingly, almost everyone is interested. Send to printer » Continuity Promotes Bioprocessing Intensity Moving From Batch Mode to Continuous Mode Moving From Batch Mode to Continuous Mode Concentrates Bioprocessing Wonderfully Concentrates Bioprocessing Wonderfully For example, fully continuous bioprocessing has become a top priority at Pall, which maintains a continuous processing laboratory in Westborough, MA. The company has also demonstrated its commitment to continuous bioprocessing through significant acquisitions of critical technology.

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Original Publication Date: 04/2016

Expansions and renovations to existing biological facilities, and construction of new facilities, provide a unique opportunity to rethink basic design strategies and use new technologies to build a better facility that will improve compliance. This article is the sixth in a six-part series on how single-use systems (SUS) are changing the modern biotechnology facility design and construction paradigm.

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Original Publication Date: 11/2016

Increasing expression rates in mammalian cell culture processes and the rapid implementation of single-use process technologies, encourage the biopharmaceutical industry to evaluate smaller footprint cleanroom infrastructures, respectively facility designs. The need for these smaller, but more flexible, manufacturing facility designs were accelerated by the requirement to establish in-country/for country manufacturing and by biosimilar developments, which require multi-product processing capabilities. Furthermore, therapies are changing and highly potent components necessitate robust containment and cell therapies the protection of the purely aseptic process. The call for higher flexibility and agility, besides rapid deployment and shorter time to run, generated a favorable view and adoption of innovations within the manufacturing area designs and cleanroom infrastructures. Traditional systems are getting replaced by more flexible and robust design and material options. Modular biomanufacturing facilities are adopted to gain the required flexibility and shorter time-to-run. To the benefit of the industry though, modular designs and materials evolve further, creating an enhanced toolbox of facility design choices. The evolution is not stopping at the materials and construction of the modular cleanroom infrastructures, but enhance further to standardized or platform systems, which can also reduce design timelines in future. Ultimately, at least common manufacturing processes should become a catalogue item to pick and chose from, to redline it and abbreviated the hours and hours spent to create the same anew.

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Original Publication Date: 01/2017

Robotic arms. Gloveless isolators. Manufacturing pods. Process modeling. Big data. Automation. Welcome to the future—or “next generation”—of pharmaceutical manufacturing, “Industry 4.0.” Pharmaceutical manufacturing is on the precipice of a paradigm change, particularly when it comes to biologic products. As biologic lots become more and more specic, some even personalized for individual patients, the need for exible, high-tech manufacturing equipment and solutions becomes critical.

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Original Publication Date: 03/2017

Regenerative medicine includes both cell and gene therapies. Currently 672 regenerative medicine companies operate around the world and 20 products have been approved by the US Food and Drug Administration (FDA). Of 631 ongoing clinical trials by the end of 2015 (1), over 40% are in oncology, followed in prominance by cardiovascular and infectious diseases. Here I focus on gene and cell therapy bioprocessing in which the final product delivered to patients are cells.

Cell therapies are either autologous (derived from a single patient, for that patient) or allogeneic (coming from a banked donor source, for many patients). Autologous therapies do not face risks associated with cell rejection; however, they are much more expensive to manufacture and distribute. Although Dendreon’s Provenge autologous cell therapy was approved by the FDA, it ultimately failed commercially because of its high cost to patients due to a manufacturing and distribution model that was inefficient. Cost of goods (CoG), manufacturing processes, and logistics are all critical to successful cell therapy commercialization, so they need to be considered along with clinical science from the inception of a cell therapy company. Three key enablers for success are manufacturing automation and single-use technologies; a diverse pipeline in modularized facilities; and sophisticated data acquisition and logistics.

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Original Publication Date: 04/2019

Biopharmaceutical products, and cell and gene therapies, are currently produced in fixed facilities that require a significant upfront, at-risk capital investment. Often, these traditional facilities are also product-dedicated, meaning that the facility lifecycle correlates to the product lifecycle and can require significant investment to retrofit for new applications.

Modular and mobile concepts offer an opportunity to shift from these large, fixed assets to networks of smaller, standardized manufacturing facilities. These can be built in less than half the time and in a way that defers costs until there is greater certainty about market demand and the probability of clinical and market success.

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Original Publication Date: 04/2019

Apoptosis is a form of programmed and controlled cell death that accounts for the majority of cellular death in bioprocesses. Cell death affects culture longevity and product quality; it is instigated by several stresses experienced by the cells within a bioreactor. Understanding the factors that cause apoptosis as well as developing strategies that can protect cells is crucial for robust bioprocess development. This review aims to a) address apoptosis from a bioprocess perspective; b) describe the significant apoptotic mechanisms linking them to the most relevant stresses encountered in bioreactors; c) discuss the design of operating conditions in order to avoid cell death...

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Continuous manufacturing (CM) is a process technology that has been used in the chemical industry for large‐scale mass production of chemicals in single‐purpose plants with benefit for many years. Recent interest has been raised to expand CM into the low‐volume, high‐value pharmaceutical business with its unique requirements regarding readiness for human use and the required quality, supply chain, and liability constraints in this business context. Using a fairly abstract set of definitions, this paper derives technical consequences of CM in different scenarios along the development–launch–supply axis in different business models and how they compare to batch processes. Impact of CM on functions in development is discussed and several operational models suitable for originators and other business models are discussed and specific aspects of CM are deduced from CM's technical characteristics. Organizational structures of current operations typically can support CM implementations with just minor refinements if the CM technology is limited to single steps or small sequences (bin‐to‐bin approach) and if the appropriate technical skill set is available. In such cases, a small, dedicated group focused on CM is recommended. The manufacturing strategy, as centralized versus decentralized in light of CM processes, is discussed and the potential impact of significantly shortened supply lead times on the organization that runs these processes. The ultimate CM implementation may be seen by some as a totally integrated monolithic plant, one that unifies chemistry and pharmaceutical operations into one plant. The organization supporting this approach will have to reflect this change in scope and responsibility. The other extreme, admittedly futuristic at this point, would be a highly decentralized approach with multiple smaller hubs; this would require a new and different organizational structure. This processing approach would open up new opportunities for products that, because of stability constraints or individualization to patients, do not allow centralized manufacturing approaches at all. Again, the entire enterprise needs to be restructured accordingly. The situation of CM in an outsourced operation business model is discussed. Next steps for the industry are recommended. In summary, opportunistic implementation of isolated steps in existing portfolios can be implemented with minimal organizational changes; the availability of the appropriate skills is the determining factor. The implementation of more substantial sequences requires business processes that consider the portfolio, not just single products. Exploration and implementation of complete process chains with consequences for quality decisions do require appropriate organizational support.

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Original Publication Date: 01/28/2015

This white paper provides a perspective of the challenges, research needs, and future directions for control systems engineering in continuous pharmaceutical processing. The main motivation for writing this paper is to facilitate the development and deployment of control systems technologies so as to ensure quality of the drug product. Although the main focus is on small‐molecule pharmaceutical products, most of the same statements apply to biological drug products. An introduction to continuous manufacturing and control systems is followed by a discussion of the current status and technical needs in process monitoring and control, systems integration, and risk analysis. Some key points are that: (1) the desired objective in continuous manufacturing should be the satisfaction of all critical quality attributes (CQAs), not for all variables to operate at steady‐state values; (2) the design of start‐up and shutdown procedures can significantly affect the economic operation of a continuous manufacturing process; (3) the traceability of material as it moves through the manufacturing facility is an important consideration that can at least in part be addressed using residence time distributions; and (4) the control systems technologies must assure quality in the presence of disturbances, dynamics, uncertainties, nonlinearities, and constraints. Direct measurement, first‐principles and empirical model‐based predictions, and design space approaches are described for ensuring that CQA specifications are met. Ways are discussed for universities, regulatory bodies, and industry to facilitate working around or through barriers to the development of control systems engineering technologies for continuous drug manufacturing. Industry and regulatory bodies should work with federal agencies to create federal funding mechanisms to attract faculty to this area. Universities should hire faculty interested in developing first‐principles models and control systems technologies for drug manufacturing that are easily transportable to industry. Industry can facilitate the move to continuous manufacturing by working with universities on the conception of new continuous pharmaceutical manufacturing process unit operations that have the potential to make major improvements in product quality, controllability, or reduced capital and/or operating costs. Regulatory bodies should ensure that: (1) regulations and regulatory practices promote, and do not derail, the development and implementation of continuous manufacturing and control systems engineering approaches; (2) the individuals who approve specific regulatory filings are sufficiently trained to make good decisions regarding control systems approaches; (3) provide regulatory clarity and eliminate/reduce regulatory risks; (4) financially support the development of high‐quality training materials for use of undergraduate students, graduate students, industrial employees, and regulatory staff; (5) enhance the training of their own technical staff by financially supporting joint research projects with universities in the development of continuous pharmaceutical manufacturing processes and the associated control systems engineering theory, numerical algorithms, and software; and (6) strongly encourage the federal agencies that support research to fund these research areas.

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Original Publication Date: 12/26/2014

There is a growing interest in realizing the benefits of continuous processing in biologics manufacturing, which is reflected by the significant number of industrial and academic researchers who are actively involved in the development of continuous bioprocessing systems. These efforts are further encouraged by guidance expressed in recent US FDA conference presentations. The advantages of continuous manufacturing include sustained operation with consistent product quality, reduced equipment size, high‐volumetric productivity, streamlined process flow, low‐process cycle times, and reduced capital and operating cost. This technology, however, poses challenges, which need to be addressed before routine implementation is considered. This paper, which is based on the available literature and input from a large number of reviewers, is intended to provide a consensus of the opportunities, technical needs, and strategic directions for continuous bioprocessing. The discussion is supported by several examples illustrating various architectures of continuous bioprocessing systems.

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Original Publication Date: 11/21/2014

We describe the key issues and possibilities for continuous final dosage formation, otherwise known as downstream processing or drug product manufacturing. A distinction is made between heterogeneous processing and homogeneous processing, the latter of which is expected to add more value to continuous manufacturing. We also give the key motivations for moving to continuous manufacturing, some of the exciting new technologies, and the barriers to implementation of continuous manufacturing. Continuous processing of heterogeneous blends is the natural first step in converting existing batch processes to continuous. In heterogeneous processing, there are discrete particles that can segregate, versus in homogeneous processing, components are blended and homogenized such that they do not segregate. Heterogeneous processing can incorporate technologies that are closer to existing technologies, where homogeneous processing necessitates the development and incorporation of new technologies. Homogeneous processing has the greatest potential for reaping the full rewards of continuous manufacturing, but it takes long‐term vision and a more significant change in process development than heterogeneous processing. Heterogeneous processing has the detriment that, as the technologies are adopted rather than developed, there is a strong tendency to incorporate correction steps, what we call below “The Rube Goldberg Problem.” Thus, although heterogeneous processing will likely play a major role in the near‐term transformation of heterogeneous to continuous processing, it is expected that homogeneous processing is the next step that will follow.

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Original Publication Date: 12/12/2014

Great hopes and expectations are linked to stem cells as a tool for drug discovery and to stem cell-derived products in therapeutic applications. Though several products have made it to commercial stage, most of the research is still performed in small scales using simple cultivation systems such as spinner flasks or T-flasks. Drug approval procedures however require a detailed knowledge of the process, reproducible results and a comprehensive documentation. At this point, the mentioned cultivation systems quickly reach their limits. To achieve production level yields these simple systems have limited economies of scale. Controlled bioreactors, widely established in traditional cell culture applications, can be the key to establish and optimize reproducible cultivation processes. Adapted to the special requirements of sensitive stem cells they provide a powerful tool for scale-up.

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Original Publication Date: 10/01/2014

Molded filling assemblies continue the shift to Single-Use processes that save time, reduce the risk of cross contamination and increase productivity between batches. Sterilized and ready to use, the assemblies feature molded junctions to reduce leak and entrapment risks and a multiport Tri-Clamp® design to minimize holdup volume and provide seamless flow.

  • Custom designed for filling vials or syringes
  • Made from AdvantaSil™ silicone tubing or weldable and sealable AdvantaFlex® TPE tubing
  • Tubing validation and extractables portfolios available upon request
  • Consult AdvantaPure’s experienced team of engineers to discuss your process requirements and design a customized solution.

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Original Publication Date: 03/01/2018

Information technology helps to integrate Quality by Design (QbD) and Process Analytical Technology (PAT) into standard laboratory procedures and increase efficiency in process and product development. The presented case study demonstrates how novel information technologies of an advanced Eppendorf DASGIP® Parallel Bioreactor System improved process development. Seamless integration of analytical data allowed for implementation of a predictive model control and comprehensive process automation.

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Original Publication Date: 06/01/2011

Increasing process complexity coupled with rising cost pressures and rapidly evolving regulatory requirements makes today’s process development eˆorts a special challenge. The pressure of achieving faster time-to-market for new and innovative biotechnological products has led to the need to optimize every element of the total development workflow. The following application note illustrates how the DASbox® Mini Bioreactor System combined with the BioBLU® 0.3c single-use vessels supports bioprocess development in human cell culture. Scale-down capabilities were investigated by comparison of 500 mL cultures in a DASGIP® Parallel Bioreactor System with 170 mL cultures in the DASbox using the BioBLU 0.3c single-use vessel.

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Original Publication Date: 06/1/2012

Single-use bioreactor solutions have been successfully established in animal and human cell culture in the last years. Now this technology is going to make its way for microbial applications. In the following case study reproducible process control was achieved with single-use mini bioreactors and 1 L single-use vessels running in parallel. Fermentation of E. coli K12 led to highly reproduclible results thus proving the tested rigid wall single-use stirred-tank vessels to be an appropriate tool to accelerate microbial process development and shorten time-to-market in biopharmaceutical industry.

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Original Publication Date: 10/1/2013

There is a growing awareness regarding the potential leaching of toxic or inhibitory chemicals from the plastic material of single-use bioreactors into cell culture medium. Based on a standardized cell culture test recommended by the German society for chemical engineering and biotechnology, DECHEMA®, we determined if there were no leachable chemicals from the Eppendorf BioBLU Single-Use Vessel material that affect cell culture performance. We did not observe any effect of leachables on CHO and Vero cell growth and viability, and the metabolic profile. The results suggest that the BioBLU Single-Use Vessels are safe for mammalian cell culture.

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Original Publication Date: 11/01/2016

We used Eppendorf packed-bed bioreactors filled with Fibra-Cel disks for the cultivation of Chinese Hamster Ovary (CHO) cells. The objective of this study was to compare process performance in BioBLU single-use and traditional glass packed-bed bioreactors. Alkaline phosphatase (ALKP)-secreting CHO cells were used to measure protein production in each bioreactor. Overall, the results from these comparisons suggest that there is no significant difference between the reusable and singleuse FibraCel basket systems for bench-scale production of recombinant proteins. Productivity of cells and collection of secreted proteins will not be hindered by the implementation of single-use bioreactor systems.

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Original Publication Date: 07/1/2012

Vero cells are anchorage-dependent cells that are widely used as a platform for viral vaccine production. In stirred-tank bioreactors, they are ordinarily grown on microcarriers. Fibra-Cel® disks are a promising alternative attachment matrix with a high surfaceto-volume ratio. They provide a three-dimensional environment that protects cells from damaging shear forces, helping to achieve high cell densities. In this study, we cultivated Vero cells in Eppendorf BioBLU 5p Single-Use Vessels pre-packed with Fibra-Cel. The process was controlled with a BioFlo® 320 bioprocess control station. We cultivated the cells in perfusion mode, which ensures a consistent supply of nutrients and the removal of toxic byproducts. We achieved the very high Vero cell density of approximately 43 million cells per mL, demonstrating great potential for Vero-cell-based vaccine production using Fibra-Cel packed-bed vessels.

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Original Publication Date: 10/1/2017

Single-gene disorders originate in the absence or loss of function of a protein due to a genetic mutation. Gene therapy is a promising therapeutic approach that delivers a normal version of the gene to affected cells to compensate for its missing or defective counterpart. It often employs viral vectors, such as recombinant Adeno Associated Viruses (rAAVs), to insert the genes. The insect cell line Sf9 provides a suitable host for virus production. Sf9 cells are cultured in suspension, and hence working volumes can be adapted to changing needs during process development and manufacturing much more easily than for adherent cell cultures. In this study, researchers at Généthon® developed a scale-down model for rAAV viral vector production in Sf9 cells using an Eppendorf DASbox® Mini Bioreactor System. Parallel experimentation in small working volumes allowed timeand cost-efficient evaluation of process performance.

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Original Publication Date: 03/01/2016

Dielectric spectroscopy (biocapacitance) is an up-and-coming technology for real time monitoring of biomass in cell culture processes and has opened the door for next-generation cell culture process control techniques such as automated on-demand nutrient feeding. In this case study we empirically demonstrate the lower limit of quantitation (LOQ), probe-to-probe consistency, and scalability of in situ biocapacitance probes using data generated from small- and large-scale Chinese hamster ovary (CHO) bioreactor cultures. The process understanding experiments culminated in the use of biocapacitance for process control in cGMP manufacturing environment... Learn More

The global biopharmaceutical market continues to grow and has been dominated by high volume products such as monoclonal antibodies. Within the next decade, however, biomanufacturers anticipate that their pipelines will become more diverse. Low volume-, highly potent products will enter the market and may require novel processes and smaller manufacturing scales. There has been an industry-wide increase in the adoption of single-use technologies for the commercial production of these biopharmaceuticals needed in low volumes. One of our multinational customers has adopted a single-use platform concept that will allow it to use the same process configurations for development and GMP manufacturing across its global locations.

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Original Publication Date: 01/01/2018

Process analytical technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community due to the potential for continuous real time quality assurance resulting in improved operational control and compliance. This paper presents a PAT application for one of the most commonly used unit operation in bioprocessing, namely liquid chromatography. Feasibility of using a commercially available online-high performance liquid chromatography (HPLC) system for real-time pooling of process chromatography column is examined. Further, experimental data from the feasibility studies are modeled and predictions of the model are compared to actual experimental data. It is found that indeed for the application under consideration, the online-HPLC offers a feasible approach for analysis that can facilitate real-time decisions for column pooling based on product quality attributes. It is shown that implementing this analytical scheme allows us to meet two of the key goals that have been outlined for PAT, that is, "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions." Finally, the implications of implementing such a PAT application in a manufacturing environment are discussed. The application presented here can be extended to other modes of process chromatography and/or HPLC analysis.
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Original Publication Date: 5/1/2008

Developing a bioprocess model can not only reduce cost and time in process development, but now also assist the routine manufacturing and guarantee the quality of the final products through Quality by Design (QbD) and Process Analytical Technology (PAT). However, these activities require a model based process design to efficiently direct, identify and execute optimal experiments for the best bioprocess understanding and optimisation. Thus an integrated model based process design methodology is desirable to significantly accelerate bioprocess development. This will help meet current urgent clinical demands and also lower the cost and time required. This thesis examines the feasibility of a model based process design for bioprocess optimisation. A new process design approach has been proposed to achieve such optimal design solutions quickly, and provide an accurate process model to speed up process understanding. The model based process design approach includes bioprocess modelling, model based experimental design and high throughput microwell experimentation. The bioprocess design is based on experimental data and a computational framework with optimisation algorithm. Innovative model based experimental design is a core part in this approach. Directed by the design objectives, the method uses D-optimal design to identify the most information rich experiments. It also employs Random design and Simplex to identify extra experiments to increase the accuracy, and will iteratively improve the process design solutions. The modelling and implementation method by high throughput experimentation was first achieved and applied to an antibody fragment (Fab’) precipitation case study. A new precipitation model based on phase equilibrium has been developed using the data from microwell experimentation, which was further validated by statistical tests to provide high confidence. The precipitation model based on good data accurately 6 describes not only the Fab’ solubility but also the solubility of impurities treated as a pseudo-single protein, whilst changing two critical process conditions: salt concentration and pH. The comparison study has shown the model was superior to other published models. The new precipitation model and the Fab’ microwell data provided the basis to test the efficiency and robustness of the algorithms in model based process design approach. The optimal design solution with the maximum objective value was found by only 5 iterations (24 designed experimental points). Two parameterised models were obtained in the end of the optimisation, which gave a quantitative understanding of the processes involved. The benefit of this approach was well demonstrated by comparing it with the traditional design of experiments (DoE). The whole model based process design methodology was then applied to the second case study: a monoclonal antibody (mAb) precipitation process. The precipitation model was modified according to experimental results following modelling procedures. The optimal precipitation conditions were successfully found through only 4 iterations, which led to an alternative process design to protein A chromatography in the general mAb purification platform. The optimal precipitation conditions were then investigated at lab scale by incorporating a depth filtration process. The final precipitation based separation process achieved 93.6% (w/w) mAb yield and 98.2 % (w/w) purity, which was comparable to protein A chromatography. Polishing steps after precipitation were investigated in microwell chromatographic experimentation to rapidly select the following chromatography steps and facilitate the whole mAb purification process design. The data generated were also used to evaluate the process cost through process simulations. Both precipitation based and protein A chromatography based processes were analysed by the process model in the commercial software BioSolve under several relevant titre and scale assumptions. The results showed the designed precipitation based processes was superior in terms of process time and cost when facing future process challenges.

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Original Publication Date: 06/1/2012

This paper examines the opportunities and challenges facing the pharmaceutical industry in moving to a primarily “continuous processing”‐based supply chain. The current predominantly “large batch” and centralized manufacturing system designed for the “blockbuster” drug has driven a slow‐paced, inventory heavy operating model that is increasingly regarded as inflexible and unsustainable. Indeed, new markets and the rapidly evolving technology landscape will drive more product variety, shorter product life‐cycles, and smaller drug volumes, which will exacerbate an already unsustainable economic model. Future supply chains will be required to enhance affordability and availability for patients and healthcare providers alike despite the increased product complexity. In this more challenging supply scenario, we examine the potential for a more pull driven, near real‐time demand‐based supply chain, utilizing continuous processing where appropriate as a key element of a more “flow‐through” operating model. In this discussion paper on future supply chain models underpinned by developments in the continuous manufacture of pharmaceuticals, we have set out;

  • The significant opportunities to moving to a supply chain flow‐through operating model, with substantial opportunities in inventory reduction, lead‐time to patient, and radically different product assurance/stability regimes.
  • Scenarios for decentralized production models producing a greater variety of products with enhanced volume flexibility.
  • Production, supply, and value chain footprints that are radically different from today's monolithic and centralized batch manufacturing operations.
  • Clinical trial and drug product development cost savings that support more rapid scale‐up and market entry models with early involvement of SC designers within New Product Development.
  • The major supply chain and industrial transformational challenges that need to be addressed.

The paper recognizes that although current batch operational performance in pharma is far from optimal and not necessarily an appropriate end‐state benchmark for batch technology, the adoption of continuous supply chain operating models underpinned by continuous production processing, as full or hybrid solutions in selected product supply chains, can support industry transformations to deliver right‐first‐time quality at substantially lower inventory profiles.

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Original Publication Date: 01/28/2015

This white paper focuses on equipment, and analytical manufacturers’ perspectives, regarding the challenges of continuous pharmaceutical manufacturing across five prompt questions. In addition to valued input from several vendors, commentary was provided from experienced pharmaceutical representatives, who have installed various continuous platforms. Additionally, a small medium enterprise (SME) perspective was obtained through interviews. A range of technical challenges is outlined, including: the presence of particles, equipment scalability, fouling (and cleaning), technology derisking, specific analytical challenges, and the general requirement of improved technical training. Equipment and analytical companies can make a significant contribution to help the introduction of continuous technology. A key point is that many of these challenges exist in batch processing and are not specific to continuous processing. Backward compatibility of software is not a continuous issue per se. In many cases, there is available learning from other industries. Business models and opportunities through outsourced development partners are also highlighted. Agile smaller companies and academic groups have a key role to play in developing skills, working collaboratively in partnerships, and focusing on solving relevant industry challenges. The precompetitive space differs for vendor companies compared with large pharmaceuticals. Currently, there is no strong consensus around a dominant continuous design, partly because of business dynamics and commercial interests. A more structured common approach to process design and hardware and software standardization would be beneficial, with initial practical steps in modeling. Conclusions include a digestible systems approach, accessible and published business cases, and increased user, academic, and supplier collaboration. This mirrors US FDA direction. The concept of silos in pharmaceutical companies is a common theme throughout the white papers. In the equipment domain, this is equally prevalent among a broad range of companies, mainly focusing on discrete areas. As an example, the flow chemistry and secondary drug product communities are almost entirely disconnected. Control and Process Analytical Technologies (PAT) companies are active in both domains. The equipment actors are a very diverse group with a few major Original Equipment Manufacturers (OEM) players and a variety of SME, project providers, integrators, upstream downstream providers, and specialist PAT. In some cases, partnerships or alliances are formed to increase critical mass. This white paper has focused on small molecules; equipment associated with biopharmaceuticals is covered in a separate white paper. More specifics on equipment detail are provided in final dosage form and drug substance white papers. The equipment and analytical development from laboratory to pilot to production is important, with a variety of sensors and complexity reducing with scale. The importance of robust processing rather than overcomplex control strategy mitigation is important. A search of nonacademic literature highlights, with a few notable exceptions, a relative paucity of material. Much focuses on the economics and benefits of continuous, rather than specifics of equipment issues. The disruptive nature of continuous manufacturing represents either an opportunity or a threat for many companies, so the incentive to change equipment varies. Also, for many companies, the pharmaceutical sector is not actually the dominant sector in terms of sales. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:821–831, 2015
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Original Publication Date: 12/1/2014

This paper assesses the current regulatory environment, relevant regulations and guidelines, and their impact on continuous manufacturing. It summarizes current regulatory experience and learning from both review and inspection perspectives. It outlines key regulatory aspects, including continuous manufacturing process description and control strategy in regulatory files, process validation, and key Good Manufacturing Practice (GMP) requirements. In addition, the paper identifies regulatory gaps and challenges and proposes a way forward to facilitate implementation.

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Original Publication Date: 01/14/2015

The availability of material for experimental studies is a key constraint in the development of full-scale bioprocesses. This is especially true for the later stages in a bioprocess sequence such as purification and formulation, where the product is at a relatively high concentration and traditional scale-down models can require significant volumes. Using a combination of critical flow regime analysis, bioprocess modeling, and experimentation, ultra scale-down (USD) methods can yield bioprocess information using only milliliter quantities before embarking on highly demanding full-scale studies. Learn More

Mammalian cell perfusion cultures represent a promising alternative to the current fed-batch technology for the production of various biopharmaceuticals. Long-term operation at a fixed viable cell density (VCD) requires a viable culture and a constant removal of excessive cells. Product loss in the cell removing bleed stream deteriorates the process yield. In this study, the authors investigate the use of chemical and environmental growth inhibition on culture performance by either adding valeric acid (VA) to the production media or by reducing the culture temperature with respect to control conditions. Learn More