The use of temporary immersion systems (TIS) for plant micropropagation is an efficient technique for plant production, and we have applied it for the production of alstroemerias. This method ...involves the cultivation of explants such as rhizomes and axillary buds in a nutrient medium to stimulate shoot growth. TIS offer advantages such as accelerated multiplication processes, uniform production, and cost reduction. This process has shown promise in meeting the growing demand for alstroemeria plants in the market. This chapter describes a specific protocol for temporary immersion bioreactor micropropagation of the "Albatroz" cultivar, with the potential for large-scale automation.
Monoclonal antibodies are widely used as diagnostic reagents and for therapeutic purposes, and their demand is increasing extensively. To produce these proteins in sufficient quantities for ...commercial use, it is necessary to raise the output by scaling up the production processes. This review describes recent trends in high-density cell culture systems established for monoclonal antibody production that are excellent methods to scale up from the lab-scale cell culture. Among the reactors, hollow fiber bioreactors contribute to a major part of high-density cell culture as they can provide a tremendous amount of surface area in a small volume for cell growth. As an alternative to hollow fiber reactors, a novel disposable bioreactor has been developed, which consists of a polymer-based supermacroporous material, cryogel, as a matrix for cell growth. Packed bed systems and disposable wave bioreactors have also been introduced for high cell density culture. These developments in high-density cell culture systems have led to the monoclonal antibody production in an economically favourable manner and made monoclonal antibodies one of the dominant therapeutic and diagnostic proteins in biopharmaceutical industry.
Volatile organic compounds (VOCs) and odorous compounds discharged into the environment create ecological and health hazards. In the recent past, biological waste air treatment processes using ...bioreactors have gained popularity in control of VOCs and odour, since they offer a cost effective and environment friendly alternative to conventional air pollution control technologies. This review provides an overview of the various bioreactors that are used in VOC and odour abatement, along with details on their configuration and design, mechanism of operation, insights into the microbial biodegradation process and future R&D needs in this area.
Extensive research in recent years has explored numerous new features in the forward osmosis membrane bioreactor (FOMBR) process. However, there is an aspect, which is revolutionary but not yet been ...investigated. In FOMBR, FO membrane shows high rejection for a wide range of soluble contaminants. As a result, hydraulic retention time (HRT) does not correctly reflect the nominal retention of these dissolved contaminants in the bioreactor. This decoupling of contaminants retention time (CRT, i.e. the nominal retention of the dissolved contaminants) from HRT endows FOMBR a potential in significantly reducing the HRT for wastewater treatment. In this work, we report our results in this unexplored treatment potential. Using real municipal wastewater as feed, both a hybrid microfiltration-forward osmosis membrane bioreactor (MF-FOMBR) and a newly developed hybrid biofilm-forward osmosis membrane bioreactor (BF-FOMBR) achieved high removal of organic matter and nitrogen under HRT of down to 2.0 h, with significantly enhanced phosphorus recovery capacities. In the BF-FOMBR, the used of fixed bed biofilm not only obviated the need of additional solid/liquid separation (e.g. MF) to extract the side-stream for salt accumulation control and phosphorus recovery, but effectively quarantined the biomass from the FO membrane. The absence of MF in the side-stream further allowed suspended growth to be continuously removed from the system, which produced a selection pressure for the predominance of attached growth. As a result, a significant reduction in FO membrane fouling (by 24.7–54.5%) was achieved in the BF-FOMBR due to substantially reduced bacteria deposition and colonization.
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•FO membrane decouples CRT from HRT; HRT can thus be significantly reduced.•Both BF-FOMBR and MF-FOMBR achieved high pollutant removal with HRT of 2 h.•Reduced HRT resulted in enhanced phosphorus recovery capacity in both systems.•The use of fixed-bed biofilm overcame the need for MF in side-stream extraction.•The use of biofilm also significantly lowered FO membrane fouling.
The accelerating development of gene therapy from research towards clinical trials and beyond has elevated the demand for practical viral vector‐manufacturing solutions. The use of disposable ...upstream technology is gaining traction in clinical manufacturing. Packed‐bed or fixed‐bed reactors, where column is packed with immobilized biocatalyst particles providing surface to constrain the cells in a particular region of the reactor, have been widely used in bioprocessing applications since mid‐1900s. However, the world's first single‐use, fully integrated, high cell density, fixed‐bed bioreactor was launched only approximately a decade ago. By now, several single‐use, fixed‐bed technology solutions have been developed in a small scale. Scaling‐up the manufacturing can be challenging and for commercial‐scale manufacturing, there is practically only one single‐use, good manufacturing practice‐compliant option available. This study reviews the latest, fully disposable, fixed‐bed bioreactors; compares the virus production in the different systems; and discusses important manufacturing cost‐related topics. It is predicted that single‐use, fixed‐bed bioreactors will receive even more attention in the field of viral vector manufacturing and commercialization, especially with the need for higher virus titers and virus yields.
Adherent cells can attach and grow in three‐dimensional fixed‐bed matrix. The single use disposable upstream technology has been implemented in many different viral vector and vaccine productions. Main process steps in viral production contain cell expansion phase, induction of the virus production and harvest.
Scalable and efficient expansion of cell cultures are an important need for pluripotent stem cell (PSC)-derived allogeneic cell therapies. The large-scale production of high-quality PSCs can be ...achieved by three-dimensional (3D) suspension culture, wherein PSCs are cultured as aggregates or spheroids. However, adoption of 3D PSC suspension culture in clinical workflows is limited by the lack of commercial options for PSC suspension culture media. To address this, we developed the new GMP manufactured Gibco™ Cell Therapy Systems (CTS) StemScale™ PSC Suspension Medium to support PSC-based clinical manufacturing workflows. CTS StemScale is xeno-free and enables single cells to self-aggregate into 3D spheroids for efficient cell expansion.
CTS StemScale supports both induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), with cell line-dependent growth in the range of 5X – 10X expansion per passage. When cultured over multiple consecutive passages, these spheroids have been demonstrated to maintain pluripotency, genomic stability, and trilineage differentiation potential. This suspension culture approach enables easy scale-up in various cell culture vessel sizes, from small-scale (<100 mL) culture vessels to large-scale (>1L) culture systems inclusive of bioreactors. Notably, 450 million cells were expanded to 5 billion cells in 5 days by using this PSC culture medium to grow spheroids within a bioreactor. To better enable future scale-up or other downstream applications, we also cryopreserved these cells at high densities which would minimize the number of cryovials required to thaw. Cells thawed from these vials showed high viability and were able to form spheroids which were capable of expanding at normal rates. Ultimately, cells grown in CTS StemScale have the flexibility to differentiate as 3D spheroids, dissociate into single cells and be utilized in downstream applications, or be cryopreserved as single cells for future use.
The need for scalable and reproducible methodologies in cell culture processes has increased with the advancement of biopharmaceutical research. HEK293T cell line is versatile and widely used in gene ...therapy applications such as viral production for the generation of CAR-T treatment. To meet the growing demand for point-of-care manufacturing of this therapy, efficient scale-up of these cells is crucial. A common strategy for this is the use of microcarriers, such as Cytodex 1, which is a popular choice for HEK293T culture. Using design-of-experiments (DoE) methodology, we investigated the influence of rocking speed, seeding density, and time of initial pause on HEK293T growth in the SCINUS Cell Expansion System (SCES), to understand factor interactions and their influence on cell proliferation.
The SCES (Figure 1A) is a closed bioreactor that provides controlled cell culture for research and GMP processes. It offers precise temperature, pH, and DO control, together with a unique roller/clamp system for scalability. A bag-in-bag system (Figure 1B) was developed using Permalife bags, to test different conditions in the same device on a small scale. Limits for the design space parameters (rocking speed, seeding density, and static pause) were evaluated based on individual experiments. A two-level factorial design was created for three different rocking speeds within these limits: 60°/sec, 90°/sec and 320°/sec. HEK293T cells were seeded in the corresponding density (range 5 ×104 – 2×105 cells/mL) and with an initial static pause of 1-4 h, being in culture for 3 days. The optimal outcome was based on the population doubling time (PDT) on harvest day.
The study did not reveal any significant interaction between the evaluated parameters. Nevertheless, the response surface models (Figure 2) indicated that a lower seeding density (p=0.0002) and a lower initial static pause (p=0.0162) resulted in faster cell growth, while the rocking speed did not affect the cell growth rate. Furthermore, the experiment confirmed that the distribution of HEK293T cultured in SCES was comparable to the spinner flask cultures (Figure 3), despite using three different seeding densities.
The outcomes of this investigation have enhanced our understanding of the dynamics governing HEK293T cell growth and offer a foundation for developing refined bioprocessing strategies based on microcarriers for large-scale cell culture applications, such as viral production in gene therapy.
The number of applications for expanded induced pluripotent stem cells (iPSCs) have increased notably in recent years. iPSCs can provide the raw material required for differentiated cell types, a ...tailored substrate for drug development and testing, or produce the therapeutic agent themselves in the case of iPSCs derived exosomes. As the range of applications for iPSCs grows, so does the need for reliable methods for large-scale expansion of this versatile cell type. The Quantum Flex hollow fiber bioreactor (HFB) provides such an option for the scale-up and manufacture of iPSCs for a range of applications.
In this work, human iPSCs from a single donor were expanded in the small (2000 cm2) HFB of Quantum Flex. HFBs were coated with either 1 mg recombinant laminin (LN; 17µg/mL; 0.5µg/cm2) or 2 mg recombinant vitronectin (VN; 34µg/mL; 1.0µg/cm2) prior to cell seeding using a method designed specifically for the small bioreactor system to maximize the evenness of cell distribution in that system. Regardless of coating, iPSCs were seeded at 7500 iPSCs/cm2 (1.5 x 107 total cells/HFB) and expanded for ∼4 days following methods that were kept as aligned as possible with typical manual culture methods to facilitate ease of process transfer. In that time, iPSCs expanded on LN coated HFBs yielded a mean of 5.0 x 108 iPSCs (n=4; SD=7.1 x 107) and iPSCs expanded on VN coated HFBs yielded a mean of 2.5 x 108 iPSCs (n=3; SD=9.4 x 106). Of those yields, an average of 12% (LN) or 10% (VN) were lost to the system washout that occurs prior to the introduction of 60 mL Accutase that was used to harvest the cells. Mean iPSC viability upon harvest was 96.8% (LN) or 97.9% (VN). Cells recovered from the system were assayed via flow cytometry for 4 common iPSC markers (SOX2, OCT4, TRA-1-60, SSEA4) and all cells from the system harvest displayed all 4 markers at rates >90%. Interestingly, when cells recovered from the pre-harvest washout were also interrogated for the same markers, these cells displayed those markers at frequencies that were 10-19% (LN) or 4-8% (VN) lower than that seen in the cells recovered from the harvest only. This may indicate that the preharvest washout can help prune less desirable (potentially more differentiated) iPSCs prior to harvest and leave a less differentiated population behind for the final harvest.
Ex-vivo gene manipulation in T cells is delivering spectacular results in haematological malignancies, and currently evaluated in several other indications. However, the development and production of ...these ‘living drugs’ remain complex, leading to treatments that are largely unaffordable and therefore often inaccessible to patients. New technologies supporting manufacturing at scale are needed to overcome the limitations of current manual methods that are labour intensive, prone to human error, and require high-grade clean room facilities.
Limula SA is developing a closed and automated manufacturing platform based on a novel design combining a bioreactor and a centrifuge into one single system. This unique technology provides a ‘one pot’ solution that allows for keeping the cells in the same container for every step of the manufacturing process, while offering unprecedented flexibility on the volume of the cell sample being processed. Our solution is composed of a mechatronic device for process automation, single-use consumable containing the core bioreactor technology and software encoding the process sequence.
We collected proof of concept data on the feasibility of a clinically compliant process in Limula's bioreactor for the manufacture of T cells encoding chimeric antigen receptors (CAR). While the expansion potential and phenotype of the resulting cell product were comparable with existing procedures, we could confirm that the unique design of the bioreactor/centrifuge allows for precise control over process unit operations across a wide range of volumes (from 2 mL to 450 mL) and cell numbers (from millions to billions of T cells).
The ability to process volumes smaller than any bioreactor system currently available enables new cell therapy modalities and next-generation manufacturing approaches that require lower cell number and/or short expansion times and that not amenable to automation until now. In addition, scalability within the same device has the potential to drastically reduce the costs, time and efforts currently invested in technology transfer during transition from pre-clinical to commercial stage.
The device used to scale up and manufacture an immunotherapeutic agent is a highly relevant factor in ensuring the most optimal product in terms of the time it takes to produce a dose of the therapy, ...the risk encountered in processing, the final quality of that product, and the necessary inputs required for production, e.g. cells, reagents, and hands-on time (HoT). In this work, three types of devices for Tcell expansion were compared: a static culture perfusion membrane device (SCPM) device, a rocking bag device (RB), and two sizes of hollow-fiber perfusion bioreactor (HFB-Large and HFB-Small). The Tcell yield, time to dose, fold-expansion (FE) from the initial seed, number of open events (OE), and HoT required for each device were measured.
Tcells from three donors were selected from leukopaks and cryopreserved. Upon thaw, Tcells were activated as a pool in a T-flask overnight using a soluble activator. For three donors, 5×106 Tcells were seeded into the HFB-Small and 5×107 Tcells were seeded into the HFB-Large, the RB, and the SCPM. For Donors 2-3, an additional arm seeding 5×107 Tcells into the HFB-Small was also conducted. Both HFB devices and the SCPM were seeded directly, while Tcells for the RB were maintained in T-flasks until the manufacturer's minimum recommended seeding number of 1.5×108 was reached. Tcells were cultured according to each manufacturer's guidance and harvested when the total number of cells was ≥ 2×109, an estimated autologous dose of a Tcell product. Results from each platform were as follows. RB: 2.9×109 Tcells at 98% viability in 8.0 days with 234 min HoT, 6 OE, and a FE of 57.6; SCPM: 2.4×109 Tcells at 98% viability in 9.0 days with 22 min HoT, 4 OE, and a FE of 47.0; HFB-Large: 1.5×1010 Tcells at 98% viability in 4.9 days with 57 min HoT, 1 OE, and a FE of 303.6; HFB-Small bioreactor: 3.0x109 Tcells at 96% viability in 7.8 days with 57 min HoT, 1 OE, and a FE of 613.7. The arms seeding 5×107 Tcells into the HFB-Small yielded an average of 4.9× 109 Tcells at 94% viability in 6.5 days with 1 OE, and a FE of 98.5. When harvested cells were interrogated by flow cytometry, no notable difference was seen between Tcells harvested from all platforms in terms of the expression of TIM-3 and PD-1. The TSCM phenotype (CCR7+/CD45RA+) was displayed in 30% of cells harvested from the RB, 8% of cells from the SCPM, 16% of cells from the HFB-Large, 31% of the cells from the HFB-Small (5M), and 49% of cells from the HFB-Small (50M).
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