Chapter

Upstream Processing Equipment

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Abstract

Modern bioprocesses have become quite diverse with highly specialized designs serving a broad spectrum of customers and their products. With the specialization, there has been a growth in platform technologies, starting with the cell line or organism and extending into equipment design. Despite the breadth and diversity of equipment designs, all equipment must be scalable and address the needs of the related stakeholders, from the scientist/engineer to the maintenance personnel. In this chapter, the requirements of upstream bioprocessing equipment are introduced and discussed in detail from an engineering standpoint, with the bioreactor in focus, and including examples of available bioreactor technology.

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... Stirred systems have a long tradition in biotechnological processes, especially in the biopharmaceutical industry (Birch 2010;Jossen et al. 2017;Clapp et al. 2018). It is therefore not surprising that these systems, with their distinctive agitators, are being increasingly used to primarily reduce inhomogeneities in fluids through mixing, and thus improve product quality, increase chemical and biological turnover, and accelerate heat and mass transfer (Pahl 2002;Meyer et al. 2016). ...
... While in the chemical industry, for example, a ratio of 1:1 is typical, a ratio of 2:1 is preferred for cell culture bioreactors at laboratory and pilot scales. For microbial systems, values of 3:1 dominate since this leads to longer residence times for supplied gases, such as air or oxygen, and better temperature control due to the larger surface to volume ratio (Menkel 1992;Jossen et al. 2017;Clapp et al. 2018). Nevertheless, as bioreactor size increases, H/D ratios of 5:1 (Chisti 2006) and up to 6:1 (Najafpour 2015) can also be found. ...
... However, there are some vital aspects to consider when selecting an impeller, such as the type, number, and arrangement of the impellers on the shaft, which may limit the possible application of certain seal types and influence the seal design. Based on the flow pattern, impellers can be divided into axial and radial conveying impellers (Kumaresan and Joshi 2006;Buffo et al. 2016;Zhang et al. 2017;Clapp et al. 2018). Radial pumping impellers, the most common type of which is the Rushton turbine (Nienow 2010), produce a horizontal flow. ...
Article
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No matter the scale, stirred tank bioreactors are the most commonly used systems in biotechnological production processes. Single-use and reusable systems are supplied by several manufacturers. The type, size, and number of impellers used in these systems have a significant influence on the characteristics and designs of bioreactors. Depending on the desired application, classic shaft-driven systems, bearing-mounted drives, or stirring elements that levitate freely in the vessel may be employed. In systems with drive shafts, process hygiene requirements also affect the type of seal used. For sensitive processes with high hygienic requirements, magnetic-driven stirring systems, which have been the focus of much research in recent years, are recommended. This review provides the reader with an overview of the most common agitation and seal types implemented in stirred bioreactor systems, highlights their advantages and disadvantages, and explains their possible fields of application. Special attention is paid to the development of magnetically driven agitators, which are widely used in reusable systems and are also becoming more and more important in their single-use counterparts. Key Points • Basic design of the most frequently used bioreactor type: the stirred tank bioreactor • Differences in most common seal types in stirred systems and fields of application • Comprehensive overview of commercially available bioreactor seal types • Increased use of magnetically driven agitation systems in single-use bioreactors
... They are usually placed in a heated environment on a rack that slowly revolves (ranging between 5 and 240 revolutions/hour). They are inexpensive and are a common method used for the initial scale-up of adherent cells (42). The cells attach and cover the inner surface of the bottle; hence the cells are cyclically bathing in culture medium and exposed to gases. ...
... Like static flasks, rotating flasks are also labor intensive. For high cell numbers, a further constraint of a roller bottle process through scaling-out is the limitation in the control of O 2 and CO 2 in both the gas and the liquid phase of culture (39,42). ...
Article
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Great importance is being given to the impact our food supply chain and consumers' food habits are having on the environment, human health, and animal welfare. One of the latest developments aiming at positively changing the food ecosystem is represented by cultured meat. This form of cellular agriculture has the objective to generate slaughter-free meat products starting from the cultivation of few cells harvested from the animal tissue of interest. As a consequence, a large number of cells has to be generated at a reasonable cost. Just to give an idea of the scale, there were billions of cells just in a bite of the first cultured-meat burger. Thus, one of the major challenges faced by the scientists involved in this new ambitious and fascinating field, is how to efficiently scale-up cell manufacture. Considering the great potential presented by cultured meat, audiences from different backgrounds are very interested in this topic and eager to be informed of the challenges and possible solutions in this area. In light of this, we will provide an overview of the main existing bioprocessing technologies used to scale-up adherent cells at a small and large scale. Thus, giving a brief technical description of these bioprocesses, with the main associated advantages and disadvantages. Moreover, we will introduce an alternative solution we believe has the potential to revolutionize the way adherent cells are grown, helping cultured meat become a reality.
... The depth of the bioreactor as one of the important parameters for light penetration should be less than 5 cm [58] and its range is defined between 1 and 5 cm [59]. Moreover, based on P. Clapp et al. [60], where gas flow rates are low, and mixing is gentle, a common range for height to width is 1.5:1-2.1:1. Accordingly, in the present study, the panel dimension was defined as 20 cm by 40 cm by 1 cm, whose surface area to volume is 1.1. ...
Article
As a recent trend in the energy-efficient architecture, microalgae bio-reactive façades can control buildings’ thermal loads by the responsiveness to solar radiations and adaptive variations in culture density. Although these smart systems can provide adaptable shading during the year, they cannot meet the varying thermal comfort needs of building users in a short time because the microalgae medium’s culture remains almost unchanged during the day. This paper reports on an innovative method that helps microalgae bioreactive façade respond to solar radiation and users’ thermal needs in a short time. To achieve it, a smart window panel will be introduced, which contains two remotely-controlled adjustable bioreactors which can regulate the algae medium in height based on the users’ thermal needs. This novel panel can serve as a bio-adaptable sunshade integrated with the building facade. Thus, the internal building thermal loads can be adjusted via the height of the bioreactor façade. Experimental and simulation research was conducted to compare the thermal performance of bioreactor facades at different microalgae medium height levels in the BSk climate zone. The results indicate that indoor and outdoor temperature differences for full, ¾, ½, and ¼ medium height level every 15-minute time interval are 12.55, 11.50, 10.87, and 6.53, respectively, indicating that the full-height level has the most influence to control the thermal load of the system. According to the results, the bioreactor façade with adjustable medium height greatly impacts building thermal control in a short time.
... 55,69,70 The surface-area-to-volume (A/V) ratio, which is the amount of surface area per unit volume of an object, is an important parameter of mass transfer in biochemical engineering processes. 71 Especially, in the cultivation of aerobic microorganisms, culturing under a high A/V ratio is required. 8,72,73 The effect of the A/V ratio on BC production by the static cultivation of aerobic acetic acid bacteria has been investigated, and the optimum A/V ratio for BC production by the cells used has been reported. ...
... Most STBR vessels are cylindrical with a flat bottom, but conical bottoms have also been used. Many forms of STBRs are available as reusable or single-use systems in various sizes ranging from 1 L to 1000 L, and larger cubic meters are also accessible to meet customer specifications (Clapp et al., 2018). In most industrial fermentation, stirred tank is often the preference for bioreactors, and is regarded as the workhorse, especially in batch mode of operations, as it is mainly required in enzymatic hydrolysis and fermentation (Mears et al., 2017;Pino et al., 2018), and the cost of operation is comparatively low (Humbird et al., 2017;Manikandan et al., 2021). ...
Article
An important process in ethanol bioprocesses is the mixing in a bioreactor, which is a general phenomenon. To obtain and maintain mixing efficiency in this process, one of the essential parameters in consideration is the impeller design, which depends on several factors. High sugar titer through enzyme hydrolysis of high-solid biomass has been achieved in recent years. However, there are few literatures on the effect of impeller designs on mixing efficiency of the overall process of enzyme hydrolysis and fermentation, especially simultaneous saccharification and fermentation, and its modifications. This review has provided basic information on the various impeller designs of stirred tank bioreactors and the factors to consider when selecting a suitable impeller for the purpose of increasing ethanol titer from high-solids loading. This shows that an improvement to the impellers can enhance mixing by the modification of existing impellers of stirred tank bioreactors for ethanol production.
... Microcarriers are small beads with diameters in the range of 100 -300 μm (Clapp et al., 2018) and constructed from synthetic or natural materials (Ozbolat, 2017). One of the main applications of microcarriers is to provide an attachment surface for anchorage dependant cells in suspension cultures (Jung et al., 2012;Rafiq et al., 2016). ...
Conference Paper
The majority of adoptive T-cell products are manufactured using an autologous process. Although using a patient’s own cells reduces risks of rejection, it introduces variability and the presence of cell populations, such as monocytes, that might negatively affect the success of CAR T-cell production. Furthermore, the current method of collecting T-cells, leukapheresis, requires specialist equipment and trained operators, limiting patient accessibility. This project explored the effect of donor starting material composition on CAR T-cell manufacture. To achieve this, the study was divided into three phases, with the first reviewing an alternative leukapheresis enrichment method. Bead based magnetic separation is the current gold standard for T-cell purification. However, due to differences in adherency, T-cells can also be enriched by capturing unwanted cell types, such as monocytes, on a surface. A range of commercial surface coatings were trialled in static and dynamic systems. Microfluidic platforms were explored but suffered issues with consistent manufacture and cell recovery. Although only recovering ∽20% of CD3+ cells, the most successful enrichment arose from agitated microcarrier cultures, reducing monocyte populations by ~75% and enhancing T-cell activation by up to 33%. While it was possible to enrich T-cells using surface capture, monocytes were never completely removed from culture with ~20% of the starting population remaining. It was determined that microcarrier protocols would require development to make them a viable option for CAR T-cell processing. Having established the ability to deplete monocytes, subsequent work planned to examine the relationship between donor material composition and the success of CAR T processing stages. The impact of monocytes on the level of activation, growth and transduction efficiency was monitored across well-plate and culture bag platforms using healthy donor apheresis. Removal of monocytes from leukapheresis improved the level of activation 2-fold, achieving the same level of activation as when initiating the process with a purified T-cell starting material. Two activation reagents were tested in well-plate cultures, revealing differing sensitivities to starting material composition. Monocyte depletion in culture bag systems had a significant impact on transduction efficiency, improving consistency and increasing the level of CAR expression by up to 64% compared to leukapheresis. Cytotoxicity assays revealed that CAR T-cell products produced from donor material depleted of monocytes and isolated T-cells consistently outperformed those made from unsorted leukapheresis. Analysis of memory phenotypes and gene expression indicated that CAR T-cells produced using depleted starting material displayed a more rested and naïve state, potentially contributing to their enhanced cytotoxic performance. The final phase of this project explored the potential of using whole blood collections as an alternative starting material to leukapheresis for CAR T-cell manufacture. To test its applicability in CAR T-cell processing, healthy whole blood donations were processed to recover leukocytes using density gradients (Ficoll, Sepax) and less conventional filtration techniques (Imugard, Leukotrap and Hematrate). It was thought that blood filters could provide a rapid methods for WBC purification without the need for additional reagents. A complication with using whole blood as a starting material is its high level of red blood cells (RBCs). Density gradients were able to completely isolate white blood cells (WBCs) however, filters retained approximately a sixth of the RBCs present in the starting whole blood, even with refined operation. Lymphocytes derived from an automated density gradient or newly established blood filtration processes were activated. CD4+ T-cells were stimulated to a similar level as leukapheresis from unrelated donors, achieving 43-55% CD25+CD69+%. Conversely, CD8+ T-cells exhibited a significantly lower level of CD25+CD69+% than leukapheresis, at approximately half. Retroviral transduction was poor in filtered material, achieving an efficiency of ∽7% compared to ∽56% by Sepax samples. Addition of an RBC lysis inducing freeze thaw to the process alleviated this issue, with filtered whole blood able to yield transduction efficiencies of 64 – 88%. Furthermore, CAR T-cells derived from density gradients and filtered whole blood consisted of >50% early central memory cells. Although whole blood filtration can produce CAR T-cells, a higher level of RBC depletion and review of processing techniques would be necessary to achieve higher retroviral transduction.
... Much of this expansion has been due to recent progress in genetic tool development to manipulate the microorganisms, filamentous fungi, and mammalian cells utilized in biopharmaceutical and industrial enzyme production [7][8][9]. However, much of this industry is still encumbered with large fermentation tanks, submerged fermentation, and even roller-bottle based processes [4,[10][11][12]. In the end, these processes lack much of the flexibility and portability necessary for on-demand production to meet oscillating demands for varied products as well as for small-scale products requiring on-site production due to cold-chain requirements. ...
Article
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Traditional production of industrial and therapeutic proteins by eukaryotic cells typically requires large-scale fermentation capacity. As a result, these systems are not easily portable or reusable for on-demand protein production applications. In this study, we employ Bioproduced Proteins On Demand (Bio-POD), a F127-bisurethane methacrylate hydrogel-based technique that immobilizes engineered Pichia pastoris for preservable, on-demand production and secretion of medium- and high-molecular weight proteins (in this case, SEAP, α-amylase, and anti-HER2). The gel samples containing encapsulated-yeast demonstrated sustained protein production and exhibited productivity immediately after lyophilization and rehydration. The hydrogel platform described here is the first hydrogel immobilization using a P. pastoris system to produce recombinant proteins of this breadth. These results highlight the potential of this formulation to establish a cost-effective bioprocessing strategy for on-demand protein production.
... Despite the small gas-liquid interface area, the LM system had more viable cells after incubation. The surface-area-to-volume ratio (sa/vol), which is the amount of surface area per unit volume of an object, is an important parameter on mass transfer in biochemical engineering processes [34]. The sa/vol value of the LMs in this study was roughly estimated to compare that of the tube system [Supporting Information]. ...
Article
Full-text available
Liquid marble (LM), a non-stick drop coated with micro- or nano-scale particles, has great potential in a wide range of applications. LMs have an advantageous feature in which gas or vapor can freely transport through their particle shell; therefore, it makes them an ideal candidate to be utilized as microbioreactor containing aerobic microorganisms. In this study, safer and more biocompatible LMs were successfully prepared using a food-grade calcium stearate microparticle as a stabilizer. As the volume of core liquid increased, the height of LM increased and reached a constant value, as a similar trend has been reported in conventional LMs. The drying rate curve of the LMs confirmed that the LMs have a similar pattern with the drying of typical wet powders. The drying rate depended on the salt species in the core solution and the environmental humidity. For instance, in the case of MgCl2, by changing humidity from 40 to 80% RH, the lifetime of LMs (time in which the LM dried completely) was increased to about 900 min. This is nearly three times longer than those have no salt and at 40% RH. Model aerobic bacteria Bacillus subtilis has actively proliferated inside the LM during 24-h incubation. Comparing with the test tube cultivations under O2-rich stationary or O2 rich–shaken conditions, the cultivation in the LM system showed a higher proliferation than the test tube systems. As a conclusion, we demonstrated that the calcium stearate LM system would be an ideal candidate for safer and easily available microbioreactor containing aerobic bacteria.
... Adapting a cell to SFM might be challenging, as the beneficial effect of serum on protecting cells from shear stress is lost, commonly interfering with its growth and virus production ability [18]. This becomes more evident at large-scale, with the added (negative) impact of O 2 and CO 2 gradients on cell's physiological state [19]. Despite the difficulties, Vero cells growth in SFM has been successfully demonstrated [1,[20][21][22]. ...
Article
Process intensification for Peste des Petites Ruminants Virus (PPRV) vaccine production in anchorage dependent Vero cells is challenging, involving substantial amount of bioprocess development. In this study, we describe the implementation of a new, scalable bioprocess for PPRV vaccine production in Vero cells using serum-free medium (SFM), microcarrier technology in stirred-tank bioreactors (STB), in-situ cell detachment from microcarriers and perfusion. Vero cells were successfully adapted to ProVero™-1 SFM, reaching growth rates similar to serum-containing cultures (0.030 1/h vs 0.026 1/h, respectively). An in-situ cell detachment method was successfully implemented, with efficiencies above 85%. Up to 2.5-fold increase in maximum cell concentration was obtained using perfusion when compared to batch culture. Combining perfusion with the in-situ cell detachment method enabled the scale-up to 20 L STB directly from a 2 L STB, surpassing the need for a mid-scale platform (i.e. 5 L STB) and thus reducing seed train duration. Head-to-head comparison of cell growth and PPRV production in the 2 L and 20 L STB was performed, and no significant differences could be observed. Estimated infectious PPRV titers in Tissue Culture Infection Dose (TCID50) (TCID50/mL = 5 × 106 and TCID50/cell = 5) are within the log-range reported in literature for PPRV production in STB and SFM by Silva et al. (2008), thus confirming the feasibility and scalability of the seed train designed [1]. The novel and scalable vaccine production process herein proposed has the potential to assist the upcoming Peste des Petites Ruminants (PPR) Global Eradication Program (targeted by FAAO for 2030) by providing African local and/or regional manufacturers with a platform capable of generating over 25,000 doses of Nigeria 75/1 strain in just 19 days using a 20 L STB.
... Many well-known manufacturers ofer standard stainless steel systems with volumes from 2 to 1000 L, whereby larger systems with several cubic meters are also available according to customer speciications. The smaller scale glass bioreactors are used in research and process development [8]. The single-use systems, depending on their size, are either available as lexible bags or rigid vessels. ...
Article
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Production of sponge-derived bioactive compounds in vitro has been proposed as an alternative to wild harvest, aquaculture, and chemical synthesis to meet the demands of clinical drug development and manufacture. Until recently, this was not possible because there were no marine invertebrate cell lines. Recent breakthroughs in the development of sponge cell lines and rapid cell division in improved nutrient media now make this approach a viable option. We hypothesized that three-dimensional (3-D) cell cultures would better represent how sponges function in nature, including the production of bioactive compounds. We successfully cultured sponge cells in 3-D matrices using FibraCel® disks, thin hydrogel layers, and gel microdroplets (GMDs). For in vitro production of bioactive compounds, the use of GMDs is recommended. Nutrients and sponge products rapidly diffuse into and out of the 3-D matrix, the GMDs may be scaled up in spinner flasks, and cells and/or secreted products can be easily recovered. Research on scale-up and production is in progress in our laboratory.
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