Single-use Stirred Tank Bioreactor Research Articles

Comparability exercise of critical quality attributes of clinical-grade human mesenchymal stromal cells from the Wharton’s jelly: single-use stirred tank bioreactors versus planar culture systems

Background aimsThe increasing demand of clinical-grade mesenchymal stromal cells (MSCs) for use in advanced therapy medicinal products (ATMPs) require a re-evaluation of manufacturing strategies, ensuring scalability from two-dimensional (2D) surfaces to volumetric (3D) productivities. Herein we describe the design and validation of a Good Manufacturing Practice–compliant 3D culture methodology using microcarriers and 3-L single-use stirred tank bioreactors (STRs) for the expansion of Wharton's jelly (WJ)-derived MSCs in accordance to current regulatory and quality requirements. MethodsMSC,WJ were successfully expanded in 3D and final product characterization was in conformity with Critical Quality Attributes and product specifications previously established for 2D expansion conditions. ResultsAfter 6 days of culture, cell yields in the final product from the 3D cultures (mean 9.48 × 108 ± 1.07 × 107 cells) were slightly lower but comparable with those obtained from 2D surfaces (mean 9.73 × 108 ± 2.36 × 108 cells) after 8 days. In all analyzed batches, viability was >90%. Immunophenotype of MSC,WJ was highly positive for CD90 and CD73 markers and lacked of expression of CD31, CD45 and HLA-DR. Compared with 2D expansions, CD105 was detected at lower levels in 3D cultures due to the harvesting procedure from microcarriers involving trypsin at high concentration, and this had no impact on multipotency. Cells presented normal karyotype and strong immunomodulatory potential in vitro. Sterility, Mycoplasma, endotoxin and adventitious virus were negative in both batches produced. ConclusionsIn summary, we demonstrated the establishment of a feasible and reproducible 3D bioprocess using single-use STR for clinical-grade MSC,WJ production and provide evidence supporting comparability of 3D versus 2D production strategies. This comparability exercise evaluates the direct implementation of using single-use STR for the scale-up production of MSC,WJ and, by extension, other cell types intended for allogeneic therapies.

Lentiviral vector bioprocess economics for cell and gene therapy commercialization

• Decisional tool presented for identifying cost-effective lentiviral vector processes. • Scalable bioreactors can achieve ∼90 % COG LV /dose savings over traditional routes. • Suspension STR is the most cost-effective technology across indications. • For adherent cell culture, fixed bed bioreactor is the most cost-effective option. • Target harvest titre values were identified to drive down lentiviral vector costs. Traditional viral vector processes rely on lab-scale methods that need to be industrialised so as to avoid viral vector supply shortages during commercialisation of cell and gene therapies such as CAR T-cell and HSC gene therapies. This paper describes the application of a decisional tool to identify the most cost-effective scalable cell culture technologies used in the manufacture of lentiviral vectors (LVs) across a range of products. The tool consists of a whole bioprocess economics model linked to an optimisation algorithm and analyses the cost of goods (COG) associated with adherent (e.g. 10-layer vessels (CF10)) and suspension (e.g. single-use stirred tank bioreactor (SUB)) cell culture technologies. The SUB was the most cost-effective technology across most scenarios when a suspension-adapted cell line was available, while the fixed bed bioreactor (FB) was the most cost-effective when adherent cell culture was preferred instead. At large scale, the COG reduction achieved by switching from CF10 to SUB or FB was at least 90 %. The SUB capacity limits were highlighted for high dose and high demand scenarios. The cost drivers were explored and the target harvest titre required to drive down LV cost contributions to cell therapy costs was identified. Finally, the tool highlighted the impact of increasing the specific productivity in the FB on COG LV /dose for transient transfection and stable producer cell line scenarios.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

A novel, single-use bioreactor system for expansion of human mesenchymal stem/stromal cells

Background & Aim There is a significant need for efficient systems that can be used to generate primary cells and stem cells which can be readily implemented in research labs and expedite process development studies, pre-clinical testing, and large-scale expansion. Although various platforms for expansion of sufficient cell numbers exist, these systems are rarely comprehensive enough to allow for rapid implementation by researchers. These systems are also not easy to use nor are they robust enough to reproducibly supply high quality cells. A complete system for the generation of sufficient cell numbers that utilizes a bioreactor platform which is robust and easy-to-use is therefore highly desirable. Methods, Results & Conclusion We have previously demonstrated efficient expansion of human mesenchymal stem/stromal (hMSC) in the PadReactor® single-use bioreactor system to the 40 L scale. This system utilized commercially-available, well-characterized cells, specially-formulated growth media and supplements, SoloHill microcarriers, and a single-use bioreactor platform that could be used to generate tens of billions of cells within 7 days of culture. Here we extend these finding by employing these components in a novel bioreactor type which contains a bottom-mounted impellor. The Allegro™ STR bioreactor is a new single-use stirred tank bioreactor platform which is scalable, compact, ergonomic, and designed to maximize usability and process assurance. hMSC and medium were cultured at 30 L scale in microcarrier-based, fed-batch cultures in the Allegro STR bioreactor for 5 days. Cells harvested from microcarriers retained critical quality attributes when examined in standard cell characterization assays. The results obtained in this study lay the groundwork for a complete system for efficient generation of high quality cells for process development studies and future large scale cell expansion. Together, the Allegro STR bioreactor and the SoloHill microcarriers provide a practical manufacturing solution for large-scale expansion of adherent cells.

Simian adenovirus vector production for early-phase clinical trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components

A variety of Good Manufacturing Practice (GMP) compliant processes have been reported for production of non-replicating adenovirus vectors, but important challenges remain. Most clinical development of adenovirus vectors now uses simian adenoviruses or rare human serotypes, whereas reported manufacturing processes mainly use serotypes such as AdHu5 which are of questionable relevance for clinical vaccine development. Many clinically relevant vaccine transgenes interfere with adenovirus replication, whereas most reported process development uses selected antigens or even model transgenes such as fluorescent proteins which cause little such interference. Processes are typically developed for a single adenovirus serotype – transgene combination, requiring extensive further optimization for each new vaccine.There is a need for rapid production platforms for small GMP batches of non-replicating adenovirus vectors for early-phase vaccine trials, particularly in preparation for response to emerging pathogen outbreaks. Such platforms must be robust to variation in the transgene, and ideally also capable of producing adenoviruses of more than one serotype. It is also highly desirable for such processes to be readily implemented in new facilities using commercially available single-use materials, avoiding the need for development of bespoke tools or cleaning validation, and for them to be readily scalable for later-stage studies.Here we report the development of such a process, using single-use stirred-tank bioreactors, a transgene-repressing HEK293 cell – promoter combination, and fully single-use filtration and ion exchange components. We demonstrate applicability of the process to candidate vaccines against rabies, malaria and Rift Valley fever, each based on a different adenovirus serotype. We compare performance of a range of commercially available ion exchange media, including what we believe to be the first published use of a novel media for adenovirus purification (NatriFlo® HD-Q, Merck). We demonstrate the need for minimal process individualization for each vaccine, and that the product fulfils regulatory quality expectations. Cell-specific yields are at the upper end of those previously reported in the literature, and volumetric yields are in the range 1 × 1013 – 5 × 1013 purified virus particles per litre of culture, such that a 2–4 L process is comfortably adequate to produce vaccine for early-phase trials. The process is readily transferable to any GMP facility with the capability for mammalian cell culture and aseptic filling of sterile products.

Multi-chamber single-use bioreactor– A proof of concept prototype

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 50L. 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.

110 - Manufacturing solutions for mesenchymal stromal/stem cell therapy expansion

There is a growing need within the regenerative medicine industry for closed cell culture systems able to support scalable manufacture of adherent cell therapies. Single-use stirred tank bioreactor systems are such a platform, having been proven effective for large scale production of allogenic microcarrier-based cell therapies. However, optimization of the bioreactor culture process is essential to maximize cell yield while maintaining cell function. We will review a mesenchymal stem/stromal cell (MSC) case study using qualified media, serum alternatives, and bioreactor process control strategies to achieve increased growth. In this study, we present a method to determine optimal bioreactor process parameters, such as agitation set-point, pH set-point, and microcarrier concentration in the Mobius® 3L single-use bioreactor for MSC expansion on microcarriers. Specifically, we have utilized Zwietering's equation for suspension of solids in stirred tanks to determine a theoretical agitation operating range for microcarrier cultures, and Kolmogorov's theory of turbulent eddy lengths to define upper agitation limits. Results from this study suggest it is critical to balance minimizing shear and maximizing cell access to nutrients, DO, and cell-to-microcarrier attachment and transfer to achieve optimal MSC growth. Specifically this work indicates an agitation rate creating a slight gradient of microcarriers, produces higher growth compared to a more traditional homogenous distribution. Additionally, MSC culture performance was found to be negatively impacted by pH values outside the range of 7.4–7.6 tested in this study. Start to finish solutions for expansion, including high quality reagents and process control strategies, are key to enabling technologies for success in commercializing cell therapies.

165 - CFD simulation of small-scale single-use stirred tank bioreactors; Comparisons and perspectives

165 - CFD simulation of small-scale single-use stirred tank bioreactors; Comparisons and perspectives

Process development for expansion of human mesenchymal stromal cells in a 50 L single-use stirred tank bioreactor

The application of cell-based therapeutics requires development of refined and scalable culture processes. Stirred tank bioreactors facilitate growth of human mesenchymal stromal cells (hMSC) while meeting these needs. A process for expansion of hMSCs in a 50L bioreactor was developed. Parameters evaluated include agitation rate, pH and dissolved oxygen (DO) control set-points, and media formulation. The pH and DO levels were determined empirically in 3L bioreactors prior to implementation at the 50L scale. The agitation operating range for microcarrier cultures in the 50L bioreactor was calculated based on theoretical and empirical analyses of solid suspension and shear limitations. Additionally, small-scale experiments demonstrated that hMSC growth was improved in αMEM supplemented with human platelet lysate in comparison to DMEM supplemented with FBS. A 43-fold expansion of harvested hMSCs was achieved in 11days in a 50L bioreactor incorporating these process improvements. Cells expanded in the bioreactor exhibited the expected surface marker expression, trilineage differentiation potential, T cell growth inhibition and indoleamine 2,3-dioxygenase inducibility. The results highlight that identification of optimal pH, DO, agitation rates and culture medium for microcarrier-based bioreactor expansion of adherent cells is paramount to developing a platform to support cell-based therapies.

Characterization and performance of the Allegro™ STR 200 single-use stirred tank bioreactor

Characterization and performance of the Allegro™ STR 200 single-use stirred tank bioreactor

Characterization and performance of the Allegro™ STR 1000 single-use stirred tank bioreactor system

Background Currently, stirred tank reactors (STR) represent the gold standard for the large scale growth of suspension cell lines. Culture performance is strongly influenced by the efficiency of mixing, as measured by the mass transfer coefficient and mixing time. The success of traditional stainless steel or glass STR systems lies in their impeller driven agitation that efficiently mixes large volumes of culture fluid. However, the transition from stainless and glass STR vessels to single use STR systems still remains a challenge, particularly in terms of replicating efficient mixing. Many single use STRs use magnetically coupled driving mechanism to eliminate the risks of biocontainer leaks from rotating mechanical seals. While this design mitigates the risks of compromising sterility, insufficient magnet strength limits the power that can be transmitted to the culture fluid, and often results in poor mixing and mass transfer performance.

A single‐use purification process for the production of a monoclonal antibody produced in a PER.C6 human cell line

Advances in single-use technologies can enable greater speed, flexibility, and a smaller footprint for multi-product production facilities, such as at a contract manufacturer. Recent efforts in the area of cell line and media optimization have resulted in bioreactor productivities that exceed 8 g/L in fed-batch processes or 25 g/L in high-density cell culture processes. In combination with the development of single-use stirred tank bioreactors with larger working volumes, these intensified upstream processes can now be fit into a single-use manufacturing setting. Contrary to these upstream advances, downstream single-use technologies have been slower to follow, mostly limited by low capacity, high cost, and poor scalability. In this study we describe a downstream process based solely on single-use technologies that meets the challenges posed by expression of a mAb (IgG(1)) in a high-density suspension culture of PER.C6 cells. The cell culture harvest was clarified by enhanced cell settling (ECS) and depth filtration. Precipitation was used for crude purification of the mAb. A high capacity chromatographic membrane was then used in bind/elute mode, followed by two membranes in flow-through (FT) mode for polishing. A proof of concept of the entire disposable process was completed for two different scales of the purification train.

Discrimination of riboflavin producing Bacillus subtilis strains based on their fed-batch process performances on a millilitre scale

Forty-eight single-use stirred tank bioreactors on a 10-mL scale operated in a magnetically inductive driven bioreaction block and automated with a liquid handler were applied for discrimination of different riboflavin producing Bacillus subtilis strains based on their performances in the parallel fed-batch processes. It was shown that a discrimination of the B. subtilis riboflavin producer strains can efficiently be achieved within one parallel fermentation run based on the integral riboflavin yield after 48 h. The possibility to perform replicates within the parallel fermentation run allows for a robust statistical analysis and is a prerequisite for the discrimination of producer strains under fed-batch process conditions. Within the estimation error, all of the riboflavin producing B. subtilis strains under study showed the same fed-batch process performances on the litre scale compared to the millilitre scale.

High-throughput fermentation process design with single-use stirred-tank bioreactors

High-throughput fermentation process design with single-use stirred-tank bioreactors

Fully automated single-use stirred-tank bioreactors for parallel microbial cultivations

Single-use stirred tank bioreactors on a 10-mL scale operated in a magnetic-inductive bioreaction block for 48 bioreactors were equipped with individual stirrer-speed tracing, as well as individual DO- and pH-monitoring and control. A Hall-effect sensor system was integrated into the bioreaction block to measure individually the changes in magnetic field density caused by the rotating permanent magnets. A restart of the magnetic inductive drive was initiated automatically each time a Hall-effect sensor indicates one non-rotating gas-inducing stirrer. Individual DO and pH were monitored online by measuring the fluorescence decay time of two chemical sensors immobilized at the bottom of each single-use bioreactor. Parallel DO measurements were shown to be very reliable and independently from the fermentation media applied in this study for the cultivation of Escherichia coli and Saccharomyces cerevisiae. The standard deviation of parallel pH measurements was pH 0.1 at pH 7.0 at the minimum and increased to a standard deviation of pH 0.2 at pH 6.0 or at pH 8.5 with the complex medium applied for fermentations with S. cerevisiae. Parallel pH-control was thus shown to be meaningful with a tolerance band around the pH set-point of +/- pH 0.2 if the set-point is pH 6.0 or lower.