Articles

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

In recent years significant progress has been made in the miniaturization of protein expression and purification. As a consequence of miniaturization, the protein purification is restricted to a one-step process. In addition, the amount of purified protein is usually in the μg-range. This might be suitable if a sensitive initial screening assay is available. However, when larger amounts of proteins are required robotic platforms are no longer appropriate. To address this topic we have used the NGC chromatography system for automated purification of up to five samples using a three-step purification procedure. Learn More

The use of Raman in Bioprocess development has shown great potential for process understanding and monitoring although there are still some challenges and limitations in performance when conditions such as clone, media or scale are changed during bioprocess development. This study proposes different strategies to balance the different information content of multiple mammalian cell cultivations produced during a bioprocess development program when several conditions are investigated. The aim is to serve as a blueprint to how can PAT approaches be best developed in parallel to bioprocess development. Learn More

Controlling acidic charge variants is critical for an industrial bioprocess due to the potential impact on therapeutic efficacy and safety. Achieving a consistent charge variant profile at manufacturing scale remains challenging and may require substantial resources to investigate effective control strategies. This is partially due to incomplete understanding of the underlying causes for charge variant formation during the cell culture process. To address this gap, we examined the effects of four process input factors (temperature, iron concentration, feed media age, and antioxidant (rosmarinic acid) concentration) on charge variant profile. These factors were found to affect the charge profile by modulating the cell culture oxidative state. Learn More

The Bioprocess Systems Alliance published its first component quality test matrices in 2007. The state purpose of this work was to “help guide users when making their selections and (to) facilitate qualification, validation, and use of single-use products.”
This 2015 document is an update to the original publication necessitated by the increase in the number of components the are currently incorporated into single-use assemblies as well as the introduction of new single-use products to the market. For example, single use sensors and chromatography column products were not a part of the 2007 matrices publication. This update also accounts for changes to common practices followed by single-use component and assembly providers.
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Single-use technology is fast growing in the biopharmaceutical industry.  Often, designing new single-use systems involves a long, iterative process between end-user and supplier to ensure quality, regulatory, and technical requirements are met.  Wouldn’t it be convenient if all these requirements were captured in a toolkit? BioPhorum and BPSA have created new templates aligned with industry standards (i.e. ASTM E3051) which will simplify the single-use design process.
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This white paper, The Role of Single-Use Polymeric Solutions in Enabling Cell and Gene Therapy Production:

  • Defines and maps commonalities between bioprocessing and CGT
  • Provides high level documentation to identify information gaps
  • Shares best practices to scale up and scale out
  • Identifies regulatory and technical requirements of each stage
  • Provides education around single-use technologies and what is available
  • Defines the language

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Miniaturized stirred bioreactors (MSBRs) are gaining popularity as a cost-effective approach to scale-down experimentation. However, realizing conditions that reflect the large-scale process accurately can be challenging. This article highlights common challenges of using MSBRs for scale-down. The fundamental difference between oxygen mass transfer coefficient (kLa) and oxygen transfer rate scaling is addressed and the difficulty of achieving turbulent flow and industrially relevant tip speeds is described. By highlighting these challenges, the article aims to create more awareness of these difficulties and to contribute to improved design of scale-down experiments. Learn More

This extended abstract book captures some of the presentations and posters from this very exciting conference. We hope that this book will serve as a resource and summary of the first-rate talks and discussions, as well as encourage you to participate in future events in this HTPD conference series.
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Original Publication Date: 2017

The advent of single-use bioprocess systems used for the delivery, storage or manufacture of biopharmaceuticals has introduced a new potential source for extractables and leachables (E&L) as these systems are comprised of polymeric materials. Several industry working groups, the FDA and USP have issued guidance and draft guidance on E&L analyses for a variety of applications. These documents typically indicate that mass spectrometry should be applied for discovery of E&L's but provide little guidance as to the exact analytical methodology which should be used. We investigated the extractable profiles of a model single-use bioprocessing system consisting of a single-use bioprocess bag, connector tubing, and a hydrophilic disk filter including filter housing. Extractions were performed in water, ethanol, ethanol/water (50:50) and saline solutions. Extracts were analyzed using a stepwise analytical methodology including a variety of screening and mass spectrometry methods We then used this model system to demonstrate the use of recursive feature finding to automatically detect unique extractables followed by statistical filtering to focus on differentially present extractables which were above the analytical evaluation threshold (AET). We further show the significant affects of standard selection on the number of compounds determined to be above AET when reducing liquid chromatography-mass spectrometry (LC/MS) data. A relative response factor database consisting of 14 structurally diverse commercially available polymer additives was used to arrive at an LC/MS identification threshold. The results of this study demonstrate that significant care should be taken when selecting standards for LC/MS analysis to avoid under reporting of extractables and leachables.
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Original Publication Date: 2/20/18

This review provides an overview and a critical discussion of novel possibilities of applying soft sensors for on-line monitoring and control of industrial bioprocesses. Focus is on bio-product formation in the upstream process but also the integration with other parts of the process is addressed. The term soft sensor is used for the combination of analytical hardware data (from sensors, analytical devices, instruments and actuators) with mathematical models that create new real-time information about the process. In particular, the review assesses these possibilities from an industrial perspective, including sensor performance, information value and production economy. The capabilities of existing analytical on-line techniques are scrutinized in view of their usefulness in soft sensor setups and in relation to typical needs in bioprocessing in general. The review concludes with specific recommendations for further development of soft sensors for the monitoring and control of upstream bioprocessing.
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Original Publication Date: 4/20/17

The application of PAT for in-line monitoring of biopharmaceutical manufacturing operations has a central role in developing more robust and consistent processes. Various spectroscopic techniques have been applied for collecting real-time data from cell culture processes. Among these, Raman spectroscopy has been shown to have advantages over other spectroscopic techniques, especially in aqueous culture solutions. Measurements of several process parameters such as glucose, lactate, glutamine, glutamate, ammonium, osmolality and VCD using Raman-based chemometrics models have been reported in literature. The application of Raman spectroscopy, coupled with calibration models for amino acid measurement in cell cultures, has been assessed. The developed models cover four amino acids important for cell growth and production: tyrosine, tryptophan, phenylalanine and methionine. The chemometrics models based on Raman spectroscopy data demonstrate the significant potential for the quantification of tyrosine, tryptophan and phenylalanine. The model for methionine would have to be further refined to improve quantification.
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Original Publication Date: 10/3/17

In this study, we prepared protein A grafted magnetic nanoparticles for the industrial large-scale purification of antibodies with enhancement of binding capacity and immobilization by controlled orientation with chlorophenylsilane (CPTMS) on the surface. For site-specific immobilization of protein A, genetically modified protein A with a cysteine residue was expressed in E. coli and purified by affinity chromatography. To improve the surface area to volume ratio and increase the immobilization amount of protein A, chlorophenylsilane functionalized magnetic nanoparticles (CPTMS@MNPs) were prepared, which are smaller nanoparticles with an average diameter of 20 nm compared to commercial magnetic microparticles (Dynabeads) with an average size of 2.8 μm. The CPTMS@MNPs showed the enhancement of protein A immobilization and binding capacity to antibodies, being 11.5-fold and 7-fold higher than those of commercial Dynabeads, respectively. In addition, the CPTMS@MNPs retained about 80% of the initial protein binding capacity until the third stage of recycling. Therefore, protein A grafted CPTMS@MNPs may be useful for the industrial large-scale purification of antibodies.
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Original Publication Date: 11/2/17

Stirred tank single-use bioreactors have proven their capability to successfully replace their stainless-steel counterparts in the biopharmaceutical industry. To date, however, only a five-fold volume expansion is achievable in a single-use stirred tank bioreactor, which in turn, necessitates intermediate equipment to scale up the culture to production volume. This study introduces a two-chamber single-use bioreactor that is capable of cell culture expansion from 1 to 50 L. The prototype is a proof of concept that can help users save costs of purchase and qualification of equipment, reduce factory footprint, and reduce the risk of contamination during culture transfer from one intermediate bag to another. The prototype is made of two chambers of different volumes, interconnected as a single, closed system. The design and construction of the prototype is described in detail and results from the engineering characterization (e.g., mixing time, power input per unit volume, and oxygen mass transfer coefficient) are reported for both chambers. The results are in good agreement with general criteria proposed in literature for bioreactor design and with published data for commercially available bioreactors. Further, the concept is not limited to the two-chamber design presented in this study; additional chambers can be integrated along with different volumes, geometries, mixing, sparging, and heating approaches.
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Original Publication Date: 2/15/18

In a conventional protein downstream processing (DSP) scheme, chromatography is the single most expensive step. Despite being highly effective, it often has a low process throughput due to its semibatch nature, sometimes with nonreproducible results and relatively complex process development. Hence, more work is required to develop alternative purification methods that are more cost-effective, but exhibiting nearly comparable performance. In recent years, surfactant precipitation has been heralded as a promising new method for primary protein recovery that meets these criteria and is a simple and cost-effective method that purifies and concentrates. The method requires the direct addition of a surfactant to a complex solution (e.g. a fermentation broth) containing the protein of interest, where the final surfactant concentration is maintained below its critical micelle concentration (CMC) in order to allow for electrostatic and hydrophobic interactions between the surfactant and the target protein. An insoluble (hydrophobic) protein-surfactant complex is formed and backextraction of the target protein from the precipitate into a new aqueous phase is then carried out using either solvent extraction, or addition of a counter-ionic surfactant. Importantly, as highlighted by past researchers, the recovered proteins maintain their activity and structural integrity, as determined by circular dichroism (CD). In this review, various aspects of surfactant precipitation with respect to its general methodology and process mechanism, system parameters influencing performance, protein recovery, process selectivity and process advantages will be highlighted.
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Original Publication Date: 04/21/17

Glycosylation is recognized as a Critical Quality Attribute for therapeutic glycoproteins such as monoclonal antibodies, fusion proteins and therapeutic replacement enzymes. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for their discovery, development and quality control. The aim of this review is to highlight relevant and recent advances in analytical technologies for characterization of biotherapeutic glycoproteins. The review gives an overview of the glycosylation trends of biotherapeutics approved in 2016 and 2017 by FDA. It describes current and novel analytical technologies for characterization of therapeutic glycoproteins and is explored in the context of released glycan, glycopeptide or intact glycoprotein analysis. Ultra performance liquid chromatography, mass spectrometry and capillary electrophoresis technologies are explored in this context. There is a need for the biopharmaceutical industry to incorporate novel state of the art analytical technologies into existing and new therapeutic glycoprotein workflows for safer and more efficient biotherapeutics and for the improvement of future biotherapeutic design. Additionally, at present, there is no 'gold-standard' approach to address all the regulatory requirements and as such this will involve the use of orthogonal glycoanalytical technologies with a view to gain diagnostic information about the therapeutic glycoprotein.
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Original Publication Date: 11/22/17

mAbs are successful biopharmaceuticals playing an important role in the treatment of cancer, autoimmunity and inflammatory diseases. Mammalian cells are the dominant system for the recombinant production of the biopharmaceuticals due to complex protein processing machinery for proper protein folding and assembly. Like other proteins, mAbs tend to form higher molecular weight aggregates during manufacturing, which negatively influence quality, safety and efficacy of the biotherapeutics. The protein aggregates can be removed during downstream processing, but this cost-intensive step leads to a reduction of process yields. An alternative to this expensive removal of HMW species during DSP represents the reduction of protein aggregates at its origin during upstream processing using bioprocess optimization. Such bioprocess optimization has been performed mainly to improve cell growth, product yield and glycosylation instead of decreasing protein aggregation of mAbs. One reason for this is the lack of analytical methods for the upstream characterization of mAb aggregation. In order to study protein aggregation upstream several methods were established for the characterization of soluble protein aggregates and large aggregate particles in cell culture samples. Using these novel methods cell culture conditions were screened for their influence on protein aggregation in cell cultures of Chinese hamster ovary (CHO) production cell lines. After identification of the most critical factors influencing protein aggregation, conditions were found for the reduction of aggregates in the cultures of the tested cell lines. Strikingly, the aggregate reducing conditions were validated in another cell culture medium and another CHO production cell line. Furthermore, it was revealed that leachables from cell culture vessels like Bisphenol A do not influence protein aggregation during bioprocessing, but can influence cellular performance of production cell lines. Moreover, this study revealed that the production enhancer VPA increased the specific productivity of mAbs produced in CHO, but also induced protein aggregation in a concentration dependent manner and negatively influenced the glycosylation pattern.
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Original Publication Date: 02/23/17

A method for the identification of leachables in chemically defined media for CHO cell culture using dispersive liquid-liquid microextraction (DLLME) and UHPLC-MS is described. A Box-Behnken design of experiments (DoE) approach was applied to obtain the optimum extraction conditions of the target analytes. Performance of DLLME as extraction technique was studied by comparison of two commercial chemically defined media for CHO cell culture. General extraction conditions for any group of leachables, regardless of their specific chemical functionalities can be applied and similar optimum conditions were obtained with the two media. Extraction efficiency and matrix effects were determined. The method was validated using matrix-matched standard calibration followed by recovery assays with spiked samples. Finally, cell culture media was incubated in 7 single use bioreactors (SUBs) from different vendors and analysed. TBPP was not detected in any of the samples, whereas DtBP and TBPP-ox were found in all samples, with bDtBPP detected in six SUBs. This method can be used for early identification of non-satisfactory SUB films for cultivation of CHO cell lines for biopharmaceutical production.
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Original Publication Date: 08/25/17

Microbial bioprocesses need to be designed to be transferable from lab scale to production scale as well as between setups. Although substantial effort is invested to control technological parameters, usually the only true constant parameter is the actual producer of the product: the cell. Hence, instead of solely controlling technological process parameters, the focus should be increasingly laid on physiological parameters. This contribution aims at illustrating a workflow of data life cycle management with special focus on physiology. Information processing condenses the data into physiological variables, while information mining condenses the variables further into physiological descriptors. This basis facilitates data analysis for a physiological explanation for observed phenomena in productivity. Targeting transferability, we demonstrate this workflow using an industrially relevant Escherichia coli process for recombinant protein production and substantiate the following three points: (1) The postinduction phase is independent in terms of productivity and physiology from the preinduction variables specific growth rate and biomass at induction. (2) The specific substrate uptake rate during induction phase was found to significantly impact the maximum specific product titer. (3) The time point of maximum specific titer can be predicted by an easy accessible physiological variable: while the maximum specific titers were reached at different time points (19.8 ± 7.6 h), those maxima were reached all within a very narrow window of cumulatively consumed substrate dSn (3.1 ± 0.3 g/g). Concluding, this contribution provides a workflow on how to gain a physiological view on the process and illustrates potential benefits.
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Original Publication Date: 09/20/16

Adoption of Quality by Design (QbD) principles, regulatory support of QbD, process analytical technology (PAT), and continuous manufacturing are major factors effecting new approaches to pharmaceutical manufacturing and bioprocessing. In this review, we highlight new technology developments, data analysis models, and applications of Raman spectroscopy, which have expanded the scope of Raman spectroscopy as a process analytical technology. Emerging technologies such as transmission and enhanced reflection Raman, and new approaches to using available technologies, expand the scope of Raman spectroscopy in pharmaceutical manufacturing, and now Raman spectroscopy is successfully integrated into real-time release testing, continuous manufacturing, and statistical process control. Since the last major review of Raman as a pharmaceutical PAT in 2010, many new Raman applications in bioprocessing have emerged. Exciting reports of in situ Raman spectroscopy in bioprocesses complement a growing scientific field of biological and biomedical Raman spectroscopy. Raman spectroscopy has made a positive impact as a process analytical and control tool for pharmaceutical manufacturing and bioprocessing, with demonstrated scientific and financial benefits throughout a product's lifecycle
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Original Publication Date: 01/01/17

The BPSA has debuted its new technical guide to Design, Control and Monitoring of Single-Use Systems for Integrity Assurance. The uptake of single-use technologies (SUT) in more critical good manufacturing practices (cGMP) processes and applications has made assurance of integrity a critical quality attribute for both suppliers and end-users. The document provides recommendations to both suppliers and end-users in the single-use technology industry regarding strategies, tools and procedures that can assist in providing enhanced assurance of integrity of single-use systems. It can help end-users convey their specific requirements to the supplier. In turn, suppliers can use the document to demonstrate what they can provide to the end-user.

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Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented—one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research.
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This article discusses the status quo of the Cabilly patents, their scope of protection and the role these patents play for the therapeutic antibody industry in Europe and the US.

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A two part guide covering definitions, regulatory issues, risk assessment, program evaluation and program execution as part of process validation for implementation of single-use systems. Keywords: extractables, leachables, regulatory, compliance, CFR, FDA, 211.65, Title 21, CDER, DMF, BMF, QA,RA, environmental, control.

Author: Extractables and Leachables Subcommittee of the BPSA Technology Committee

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This work aims to evaluate the benefits of the continuous process in biopharmaceutical manufacturing of monoclonal antibodies (mAbs). An integrated continuous process is designed and built using a process simulator and compared with a fed-batch process production line. The comparisons are based on cost of goods (COG/g) calculation and sensitivity analysis. The fed-batch process results in operating COG/g of $99/g in mAbs production, whereas the continuous process accounts for $51/g. Because of the smaller footprint and fewer storage tanks required in the continuous process, the facility cost reduced by 66%, compared to the fed-batch process. Learn More

As the biopharmaceutical industry continues to grow at a rapid pace, companies are realizing that they may need to transform their biomanufacturing networks to respond to changing market dynamics and anticipate customer requirements. A major factor behind the growth of the biopharmaceutical industry has been the huge and continued success of the monoclonal antibodies segment. Within the industry, however, companies have started to diversify their pipelines, developing products such as antibody-drug conjugates, nonantibody recombinant proteins, gene therapies, and autologous and allogenic cell therapies

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Authors: Ganesh Kumar, Michael Koch, and Gerben Zijlstra, Ph.D.

Process chromatography forms the core of purification of biotherapeutics. It is typical to have three to five chromatography steps in a purification process for a biotherapeutic. Generally, these steps offer different modes of separation such as ion-exchange, reversed phase, size exclusion, and hydrophobic interaction. In the past decade, multimodal chromatography has emerged as an alternative to the traditional modes. It involves use of more than one mode of separation and typically combines ion-exchange and hydrophobic interactions to achieve selectivity and sensitivity. This review aims to present key recent developments that have occurred on this topic together with a perspective on future applications of multimodal chromatography. Learn More