Single-use Bioreactors Research Articles

179 - Development and optimization of large scale production of adenoviral vector and autologous insulin producing cell using iCELLis® 500 and Xpansion® 200 single-use bioreactors

179 - Development and optimization of large scale production of adenoviral vector and autologous insulin producing cell using iCELLis® 500 and Xpansion® 200 single-use bioreactors

Principles and approach to developing mammalian cell culture media for high cell density perfusion process leveraging established fed-batch media.

Perfusion medium was successfully developed based on our fed-batch platform basal and feed media. A systematic development approach was undertaken by first optimizing the ratios of fed-batch basal and feed media followed by targeted removal of unnecessary and redundant components. With this reduction in components, the medium could then be further concentrated by 2× to increase medium depth. The medium osmolality was also optimized where we found ∼360 mOsm/kg was desirable resulting in a residual culture osmolality of ∼300 mOsm/kg for our cell lines. Further building on this, the amino acids Q, E, N, and D were rebalanced to reduce lactate and ammonium levels, and increase the cell-specific productivity without compromising on cell viability while leaving viable cell density largely unaffected. Further modifications were also made by increasing certain important vitamin and lipid concentrations, while eliminating other unnecessary vitamins. Overall, an effective perfusion medium was developed with all components remaining in the formulation understood to be important and their concentrations increased to improve medium depth. The critical cell-specific perfusion rate using this medium was then established for a cell line of interest to be 0.075 nL/cell-day yielding 1.2 g/L-day at steady state. This perfusion process was then successfully scaled up to a 100L single-use bioreactor with an ATF6 demonstrating similar performance as a 2L bioreactor with an ATF2. Large volume handling challenges in our fed-batch facility were overcome by developing a liquid medium version of the powder medium product contained in custom totes for plug-and-play use with the bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:891-901, 2017.

Novasep to build viral vector facility

The contract manufacturer Novasep will spend $29 million to build a viral vector facility in Seneffe, Belgium. The facility will house two suites equipped with single-use bioreactors ranging in size from 200 to 2,000 L. When completed in early 2019, the site will help meet growing demand for late-clinical-trial and commercial-scale production of viral-vector-based gene and immunotherapies, Novasep says. The company already has lab-scale production at a site in nearby Gosselies.

Bioprocess integration for human mesenchymal stem cells: From up to downstream processing scale-up to cell proteome characterization

To deliver the required cell numbers and doses to therapy, scaling-up production and purification processes (at least to the liter-scale) while maintaining cells’ characteristics is compulsory. Therefore, the aim of this work was to prove scalability of an integrated streamlined bioprocess compatible with current good manufacturing practices (cGMP) comprised by cell expansion, harvesting and volume reduction unit operations using human mesenchymal stem cells (hMSC) isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC).BM-MSC and AT-MSC expansion and harvesting steps were scaled-up from spinner flasks to 2L scale stirred tank single-use bioreactor using synthetic microcarriers and xeno-free medium, ensuring high cellular volumetric productivities (50×106cellL−1day−1), expansion factors (14–16 fold) and cell recovery yields (80%). For the concentration step, flat sheet cassettes (FSC) and hollow fiber cartridges (HF) were compared showing a fairly linear scale-up, with a need to slightly decrease the permeate flux (30–50 LMH, respectively) to maximize cell recovery yield. Nonetheless, FSC allowed to recover 18% more cells after a volume reduction factor of 50. Overall, at the end of the entire bioprocess more than 65% of viable (>95%) hMSC could be recovered without compromising cell’s critical quality attributes (CQA) of viability, identity and differentiation potential.Alongside the standard quality assays, a proteomics workflow based on mass spectrometry tools was established to characterize the impact of processing on hMSC's CQA; These analytical tools constitute a powerful tool to be used in process design and development.

Process-relevant concentrations of the leachable bDtBPP impact negatively on CHO cell production characteristics.

The biopharmaceutical industry has invested considerably in the implementation of single-use disposable bioreactors in place of or in addition to their stainless steel-counterparts. This new wave of construction materials for disposable bioprocess containers encompass a plethora of uncharacterized secondary compounds that, when in contact with the culture media, can leach, contaminating the bioprocess. One such cytotoxic leachable already receiving attention is bis(2,4-di-tert-butylphenyl)-phosphate (bDtBPP), a breakdown product of the secondary antioxidant Irgafos 168 in polyethylene-film based bags. This compound has been demonstrated to inhibit cell growth at concentrations ranging from 0.12 to 0.73 mg/L across an array of cell lines. Here we demonstrate that a further two CHO cell lines exhibit sensitivity to bDtBPP exposure at concentrations lower than that previously reported (0.035-0.1 mg/L). Furthermore, these inhibitory concentrations reflect bDtBPP levels found to leach early into the bioprocess, exposing reactor inoculums to serious risk. Quantitative label-free LC-MS/MS revealed that irrespective of cell line or concentration of bDtBPP, 8 proteins were found to be commonly differentially expressed in response to exposure to the compound highlighting biological processes related to cellular stress. Although the glycoprofile of the recombinant antibody remains primarily unchanged, we demonstrate that this compound when spiked at meaningful concentrations 72 h into culture considerably reduces the maximum cell density achieved. Studies like this reinforce the requirement for the complete characterization of all potential leachable compounds from disposable materials to assess their risk not only to the patient but also to the production pipeline itself. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1547-1558, 2016.

Tetanus toxin production from Clostridium tetani, using a casein-based medium in a single-use bioreactor

Clostridium tetani, a spore-forming anaerobic bacterium, and a casein-based semisynthetic medium were used to produce tetanus toxin in this study. N-Z-Case TT (casein hydrolysate) solution and glucose stock media were mixed and autoclaved, which resulted in tetanus toxin expression. The toxin was expressed when the N-Z-Case TT solution reacted with the glucose stock at a high temperature, creating an adequate amount of Maillard reaction products (MRPs). After accumulating in C. tetani cells, tetanus toxin was secreted into the medium when cell lysis was induced by surface aeration. C. tetani was cultivated and tetanus toxin was expressed in a single-use bioreactor, which produced 80 Lf/mL of tetanus toxin in a medium with MRPs. While using the correct medium to induce tetanus toxin was important, other factors played a part in achieving the desired concentration of the toxin, including the medium processing and culture methods inside the bioreactor. Tetanus toxoid with a purity level greater than 2,500 Lf/mgPN was obtained by detoxifying and purifying the toxin recovered from the fermenter or single-use bioreactor. A single-use bioreactor could be used in a limited space without the need for constructing a large scale production facility, to produce the tetanus toxoid antigen for clinical trials.

Impact of Feeding Strategies on the Scalable Expansion of Human Pluripotent Stem Cells in Single-Use Stirred Tank Bioreactors.

Human pluripotent stem cells (hPSCs) are a unique source for the, in principle, unlimited production of functional human cell types in vitro, which are of high value for therapeutic and industrial applications. This study applied single-use, clinically compliant bioreactor technology to develop advanced, matrix-free, and more efficient culture conditions for the mass production of hPSCs in scalable suspension culture. Using extensive analytical tools to compare established conditions with this novel culture strategy, unexpected physiological features of hPSCs were discovered. These data allow a more rational process development, providing significant progress in the field of translational stem cell research and medicine.

250 - Process Optimization of Human Mesenchymal Stem Cell Manufacturing at Large Scale

Scalable manufacturing of adherence-dependent mesenchymal stem cells (MSCs) is a key need for the commercial success of allogeneic cell therapies. Single-use bioreactor systems coupled with microcarriers have proven suitable as a platform to support such scalable manufacturing, however, most studies have been limited to bioreactor volumes of 3L or less. In this study we scale the manufacturing process to 50L bioreactor volume with associated downstream processing and optimize key process parameters to maximize yield, cell identity and quality. Key process parameters for cell culture such as pH, dissolved oxygen (DO) and agitation rates for effective solid-liquid mixing were optimized. Agitation rates that are high in microcarrier-based cultures can be correlated with smaller eddy lengths that lead to cellular stress. Conversely, agitation rates that are low do not support the consistent microcarrier suspension required for cell access to nutrients, DO, and maintenance of pH. Here, we demonstrate a stepwise approach to optimizing pH, DO and agitation set-points in the Mobius® 3L single-use bioreactor and the associated application and optimization of these parameters to a 50L single-use bioreactor. Identifying optimal pH, DO and agitation rates for microcarrier-based bioreactor expansion of adherent cells is paramount to developing a robust platform for use in a controlled manufacturing environment.

237 - Expansion of Adipose Derived Stromal Cells from Stromal Vascular Fraction in a Single-Use Bioreactor: Proof of Concept in the Mobius® 3L

237 - Expansion of Adipose Derived Stromal Cells from Stromal Vascular Fraction in a Single-Use Bioreactor: Proof of Concept in the Mobius® 3L

Merck KGaA expands in U.S. and France

Germany’s Merck says it will increase capacity for its viral and gene therapy products in Carlsbad, Calif. The expansion, done under FDA’s current Good Manufacturing Practice regimen, will incorporate single-use reactors for clinical and commercial bulk drug production, and should be completed this year. Meanwhile, at its biodevelopment center in Martillac, France, the company is increasing use of single-use bioreactors with the addition of a 2000 L Mobius reactor, a technology that the company manufactures.

Novel probes for pH and dissolved oxygen measurements in cultivations from millilitre to benchtop scale

pH value and the concentration of dissolved oxygen (DO) are key parameters to monitor and control cell growth in cultivation studies. Reliable, robust and accurate methods to measure these parameters in cultivation systems in real time guarantee high product yield and quality. This mini-review summarises the current state of the art of pH and DO sensors that are applied to bioprocesses from millilitre to benchtop scale by means of a short introduction on measuring principles and selected applications. Special emphasis is placed on single-use bioreactors, which have been increasingly employed in bioprocess development and production in recent years. Working principles, applications and the particular requirements of sensors in these cultivation systems are given. In such processes, optical sensors for pH and DO are often preferred to electrochemical probes, as they allow semi-invasive measurements and can be miniaturised to micrometre scale or lower. In addition, selected measuring principles of novel sensing technologies for pH and DO are discussed. These include solid-state sensors and miniaturised devices that are not yet commercially available, but show promising characteristics for possible use in bioprocesses in the near future.

Process change evaluation framework for allogeneic cell therapies: impact on drug development and commercialization.

Some allogeneic cell therapies requiring a high dose of cells for large indication groups demand a change in cell expansion technology, from planar units to microcarriers in single-use bioreactors for the market phase. The aim was to model the optimal timing for making this change. A development lifecycle cash flow framework was created to examine the implications of process changes to microcarrier cultures at different stages of a cell therapy's lifecycle. The analysis performed under assumptions used in the framework predicted that making this switch earlier in development is optimal from a total expected out-of-pocket cost perspective. From a risk-adjusted net present value view, switching at Phase I is economically competitive but a post-approval switch can offer the highest risk-adjusted net present value as the cost of switching is offset by initial market penetration with planar technologies. The framework can facilitate early decision-making during process development.

Neue Applikationsfelder für Single-use-Bioreaktoren

Single-use bioreactor development is–compared to conventional bioreactors–a young field of research. This article aims to describe recent achievements and remaining challenges of single-use bioreactors from the microliter to the cubic meter scale. A focus is put on the application for bacteria, yeast and algae. The various design approaches of single-use bioreactors allow tailored applications to fulfil the different demands considering shear forces, power input, gas mass transfer and mixing time.

Long-Term CB.Hep-1 Monoclonal Antibody Production in a Single-Use Bioreactor on a Rocker Platform with Serum- and Protein-Free Media

Long-Term CB.Hep-1 Monoclonal Antibody Production in a Single-Use Bioreactor on a Rocker Platform with Serum- and Protein-Free Media

Comparison of CHO cell cultivation and mAbproduction in the Allegro™ XRS 20 and conventional rocker type single-use bioreactors

Background The Allegro XRS 20 bioreactor system is a single-use bioreactor system suitable for applications ranging from general life sciences research to seed train operations as well as small scale production at the 2 to 25 literscale. It features a 3D rectangular biocontainer that is rocked across two independent axes perpendicular to one another. These features allow for significant improvements in mixing and kLa when compared with a conventional rocking bioreactor that features a 2D “pillow” biocontainer that rocks across a single axis. The Allegro XRS 20 controller is able to control agitation of the biocontainer over a wide range of conditions, from gentle agitation required for shear sensitive cultures, to more vigorous conditions generating higher oxygen transfer rates and decreased mixing times needed for the high productivity systems seen in the biopharmaceutical industry today. Control of pH and DO is fully automated by the optical sensors supplied with the biocontainer. Gassing strategy is managed by three independent mass flow controllers and fluid additions can be made via three integrated pumps. All operations are managed through a user friendly touch screen interface.

Identification of performance drivers for an antibody producing CHO-S cell line culture in the Allegro™ XRS 20 single-use bioreactor utilizing historical data

Background During the development of processes for high production CHO fed batch cultures, there is usually insufficient time for thorough process optimization. Once defined, these processes often have ranges rather than specific values for many parameters, resulting in minor variations in the runs. These small process variations, combined with resultant differences in performance, can be used to further optimize the process. Over the course of one year, eleven 20 L fed batch cultures of an antibody producing CHO-S cell line were generated in the Allegro XRS 20 bioreactor. In addition to normal minor process variation, small process changes occurred due to method optimization efforts or culture timing requirements. An analysis of the combined historical data was undertaken to better understand the specific culture performance drivers, leading to further improvement in culture outputs, specifically final antibody concentration and maximum viable cell density (VCD).

Veterinary vaccine production using bioreactors: scale up study from laboratory and pd up to commercial production

Background Vaccination has proven itself as the most effective tool to control and prevent the disease and to facilitate the safe trade of live animals. Viral vaccine manufacturing processes present some specific constraints as compared to other biotech products linked to the cell substrate used and to the viral production. Multiple cell lines are used for productions such as VERO, MDCK, MRC5, BHK, and CHO cells. BHK-21 cells were selected because they are the most commonly used cells for vaccine production. As the feasibility of transferring the BHK-21 cells growth in microcarriers from a conventional bioreactor to single-use bioreactor (SU) was demonstrated, actually was time to scale-up and verify the flexibility and ease of use of these SU bioreactors enable rapid scale-up without any loss in product quality. This document describes a scaled-up production of a bovine viral vaccine. Identification of optimal parameters values, testing at intermediary bioreactors scales (2 liters 50 liters) and application of the new process settings at industrial bioreactor scale of 200 liters were performed. Taking into account that a critical feature of microcarrier-based cell culture is the homogeneity of cell adhesion to the beads, mixing and aeration performance evaluation remain mandatory to ensure robust scale-up of cell culture processes. The goal of this study was to evaluate the possibility to produce the antigens for animal vaccines manufacturing in a bioreactor SU as alternative to roller bottles without any loss in product quality. Evaluation of COG reduction was done.

Evaluation of physicochemical and biological properties of chitosan/poly (vinyl alcohol) polymer blend membranes and their correlation for Vero cell growth

The blend membranes with varying weight ratios of chitosan/poly (vinyl alcohol) (CS/PVA) (1:0, 1:1, 1:2.5, 1.5:1, 1.5: 2.5) were prepared using solvent casting method and were evaluated for their potential application in single-use membrane bioreactors (MBRs). The physicochemical properties of the prepared membranes were investigated for chemical interactions (FTIR), surface morphology (SEM), water uptake, protein sorption (qe), ammonia sorption and growth kinetics of Vero cells. CS/PVA blend membrane having weight ratio of 1.5:1 had shown enhanced membrane flexibility, reduced water uptake, less protein sorption and no ammonium sorption compared to CS membrane. This blend membrane also showed comparatively enhanced higher specific growth rate (0.82/day) of Vero cells. Improved physicochemical properties and growth kinetics obtrude CS/PVA (1.5:1) as a potential surface for adhesion and proliferation with possible application in single use membrane bioreactors. Additionally, new insight explaining correlation between water holding (%) of CS/PVA (1.5:1) blend membrane and doubling time (td) of Vero cells is proposed.

Single-use systems for flexible cell cultivation

Now, biotechnologists are not only able to choose between single-use and reusable bioreactors and fermenters in the production sector, but in process development as well, where single-use systems are making their mark with advantages like lower costs and faster run times.

Insect cell entrapment, growth and recovering using a single-use fixed-bed bioreactor. Scaling up and recombinant protein production

Insect cells are largely used for industrial production of vaccines, viral vectors and recombinant proteins as well as in research and development as an important tool for biology and bioprocess studies. They grow in suspension and are semi-adherent cells. Among the cell culture systems enabling scalable bioprocess the single-use fixed-bed iCELLis® bioreactors offer great advantages. We have established the conditions for Drosophila melanogaster Schneider 2 (S2) and Spodoptera frugiperda (Sf9) cells entrapment into the fixed-bed, cell growth and recover from the fixed-bed once high cell densities were attained. Our established protocol allowed these cells, at a cell seeding of 2×1E5 cells/microfiber carriers (MC) (3.5×1E6cells/mL; 1.7×1E4cells/cm2), to grow inside a 4m2/200mL fixed-bed attaining a concentration of 5.3×1E6 cells/MC (9.5×1E7cells/mL; 4.7×1E5 cells/cm2) for S2 cells or 4.6×1E6 cells/MC (8×1E7cells/mL; 4.1×1E5cells/cm2) for Sf9 cells. By washing the fixed-bed, entrapped cells could then be recovered from the fixed-bed at a high rate (>85%) with high viability (>95%) by increasing the agitation to 1200/1500rpm. Although the cell yields in the fixed-bed bioreactor were comparable to those obtained in a stirred tank (respectively, 1.3×1E10 and 2.5×1E10 total cells), S2 cells stably transfected with a cDNA coding for the rabies virus glycoprotein (RVGP) showed a 30% higher preserved rRVGP production (2.5±0.1 and 1.9±0.1μg/1E7 cells), as evidenced by a conformational ELISA evaluation. These findings demonstrate not only the possibility to entrap, cultivate to high densities and recover insect cells using a single-use fixed-bed bioreactor, but also that this system provides suitable physiological conditions for the entrapped cells to produce a cell membrane associated recombinant protein with higher specific biological activity as compared to classical suspension cell cultures.