Single-use Bioreactors Research Articles

Production of phenolic compounds, flavonoids, and lupeol by Lycium schweinfurthii suspension cultures in shake flasks and a rocking-motion bioreactor

AbstractLycium schweinfurthii, a wild shrub of the Solanaceae family, has received increasing attention in the last decade for its therapeutic potential in traditional medicine due to its diverse array of secondary metabolites, including phenolic substances and terpenoids. The aim of this study was to investigate the accumulation of phenolics, flavonoids, and the terpenoid lupeol in L. schweinfurthii cell suspension shake flask cultures and a single-use 2-dimensional rocking motion bioreactor. Three different media formulations were compared for in vitro cell cultures. Various parameters, such as biomass accumulation, settled cell volume, cell viability (assessed via a 2,3,5-triphenyl tetrazolium chloride assay), and sucrose consumption were determined as indicators of cell activity and growth. Total phenolic and flavonoid contents were estimated spectrophotometrically, lupeol was quantified via High-Performance Thin Layer Chromatography (HPTLC). Although a higher fresh biomass concentration of 464 g L− 1 was obtained in MS medium supplemented with a combination of each, 1 mg L− 1 of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1-Naphthaleneacetic acid (NAA), the rocking-motion bioreactor cultivation was performed with 2 mg L− 1 NAA due to its superior reproducibility in viability, productivity, and content of bioactive compounds (e.g., phenolics, flavonoids, lupeol). A final fresh biomass concentration of 185 g L− 1 was achieved in a 16 L cultivation scale with a notable increase in the concentration of phenolics (1.4-fold) and flavonoids (1.7-fold). Most importantly, the concentration of lupeol, a pentacyclic triterpenoid known for its anti-inflammatory, antibacterial, and anti-atherogenic properties, exhibited a remarkable 5.5-fold increase in the bioreactor cultivation (585 µg g− 1) compared to shake flask cultivations (106 µg g− 1). The current study demonstrated the profound impact of media composition and non-limited fed-batch conditions in a rocking-motion bioreactor on the accumulation of bioactive compounds. The findings are also relevant to other plant cell cultures.

A new strategy in bioreactor scale-up and process transfer using a dynamic initial vvm according to different aeration pore size.

Monoclonal antibody drugs have grown into a drug category with a market size of over $100 billion since the first product was launched on the market, which naturally creates a large demand for production. At the same time, the $100 billion market is distributed among more than 200 listed drugs, which indicates that the production demand for monoclonal antibody drugs is diverse. To meet this demand, major suppliers offer single-use bioreactors of all sizes. These single-use bioreactors with different specifications, especially the inconsistency of aeration pore sizes, pose great challenges for technology transfer and scale-up production, and the conventional scale-up strategies of constant Power input/volume ratio (P/V) and constant vessel volume per minute (vvm) can no longer meet the needs. This study simplified the selection of technical parameters in bioreactors based on the differences in aeration pore size. Innovatively combined the aeration pore sizes with initial aeration vvm, and comprehensively investigated the relationship between P/V, vvm and aeration pore size by designing experiments (DoE) using the orthogonal test method. The results showed a quantitative relationship between the aeration pore size and the initial aeration vvm in the P/V range of 20 ± 5W/m3. The appropriate initial aeration was between 0.01 and 0.005m3/min for aeration pore size ranging from 1 to 0.3mm, which was the optimal incubation condition in the bioreactors. The choice of initial ventilation was most related to the final expression. Follow-up studies validated these findings in a 15L glass bioreactor and a 500L single-use bioreactor, and the results were consistent with expectations.

Concept for recycling a small-scale plastic-based bioreactor in a close-loop – Technical approach

To accelerate biotherapeutic development whilst decreasing development time and project cost, small-scale multi-parallel bioreactors have been developed to maximize product yield and quality. These systems feature single-use plastic-based bioreactors for easy connection and fast turnaround. Despite well-known advantages, the perception of plastic and the lack of recycling options are raising concerns in the scientific laboratories’ community. One of the common objections to plastic circularity in life-science is the perceived “downcycling” effect of mechanical recycling. Therefore, the aim of this work was to establish the technical feasibility of a close-loop recycling concept for the main construction material of the small-scale bioreactor. Application tests (cell compatibility and cell culture) were performed and supported by quantitative physical and mechanical tests (tensile, melt flow index, light transmission). Results show that mechanically recycled polycarbonate could be re-used in the same application. A comparative life cycle assessment (LCA), based on a theoretical framework, showed with different scenarios that recycling would have a positive impact on Climate – Carbon – total within the boundaries. Even if the technical feasibility of such a concept is demonstrated through this study, several challenges remain for such a closed-loop recycling concept to be implemented at a commercial scale.

Overcoming the inconsistent cell culture performance caused by single-use bioreactors during scaling-up the manufacturing process for a therapeutic bispecific antibody

Single use technology has been widely used in modern biopharmaceutical process development and manufacturing. However, there are concerns about the adverse effects of disposable materials on bioprocess, product quality and patient safety. Recently, we observed slow cell growth, increased lactate accumulation, and decreased production titer during scaling up a bispecific antibody (BsAb) upstream process from 3 L to 500 L bioreactor. To investigate the phenomenon, we conducted medium incubation and leachable spike-in experiments. We found that cell culture medium incubated in CX5–14 bag caused poor cell culture performance, which was correlated with the concentration of bis(2,4-di-tert-butylphenyl)-phosphate (bDtBPP), a breakdown product of the antioxidant Irgafos®168 in polyethylene film. In addition, when the media were spiked with 0.15 μg/ml or a greater concentration of bDtBPP, slow cell growth and reduced BsAb expression level were observed. In contrast, Aegis5–14 film produced bDtBPP at a level of below detection during medium incubation. Thus, the bispecific-expressing CHO cell line restores normal cell culture performance and production titer when Aegis5–14 single-use bioreactor was employed in large-scale manufacturing. Our results reveal the importance of evaluating leachable profiles from disposable materials during process development of bispecifics and other novel therapeutic modalities.

Effect of culture hydrodynamics on Arthrospira platensis production using a single-use photobioreactor system through a CFD supported approach.

Laboratory scale conventional single-use bioreactor was used to investigate the effect of different stirrer speeds on the Arthrospira platensis (Spirulina platensis) culture. Experiments were handled in two steps. First step was the selection of the stirring speeds, which was simulated via using CFD, and the second was the long term cultivation with the selected speed. During 10 days of batches as the first step, under identical culture conditions, stirrer speed of 230 rpm gave higher results, compared to 130 and 70 rpm, with respect to dry biomass weight, absorbance value (AB) and chlorophyll-a concentration. Volumetric productivity during the growth phase of the cultures were calculated as 0.39 ± 0.03, 0.28 ± 0.01, and 0.19 ± 0.02 g L-1 d-1, from the fast to the slower speeds. According to the results a 17 day batch was handled with 230 rpm in order to monitor the effects on the culture. The culture reached a volumetric productivity of 0.33 ± 0.04 g L-1 d-1. Statistical analysis showed the significance of the parameters related with the stirring speed.

Characterization Data for the Establishment of Scale-Up and Process Transfer Strategies between Stainless Steel and Single-Use Bioreactors

The reliable transfer of bioprocesses from single-use bioreactors (SUBs) of different scales to conventional stainless steel stirred-tank bioreactors is of steadily growing interest. In this publication, a scale-up study for SUBs with volumes of 200 L and 2000 L and the transfer to an industrial-scale conventional stainless steel stirred-tank bioreactor with a volume of 15,000 L is presented. The scale-up and transfer are based on a comparison of mixing times and the modeling of volumetric mass transfer coefficients kLa, measured in all three reactors in aqueous PBS/Kolliphor solution. The mass transfer coefficients are compared with the widely used correlation of van’t Riet at constant stirrer tip speeds. It can be shown that a van’t Riet correlation enables a robust and reliable prediction of mass transfer coefficients on each scale for a wide range of stirrer tip speeds and aeration rates. The process transfer from single-use bioreactors to conventional stainless steel stirred-tank bioreactors is proven to be uncritical concerning mass transfer performance. This provides higher flexibility with respect to bioreactor equipment considered for specific processes.

Evaluation of various membranes at different fluxes to enable large-volume single-use perfusion bioreactors.

The growing demand for biological therapeutics has increased interest in large-volume perfusion bioreactors, but the operation and scalability of perfusion membranes remain a challenge. This study evaluates perfusion cell culture performance and monoclonal antibody (mAb) productivity at various membrane fluxes (1.5-5 LMH), utilizing polyvinylidene difluoride (PVDF), polyethersulfone (PES), or polysulfone (PS) membranes in tangential flow filtration mode. At low flux, culture with PVDF membrane maintained higher cell culture growth, permeate titer (1.06-1.34 g/L) and sieving coefficients (≥83%) but showed lower permeate volumetric throughput and higher transmembrane pressure (TMP) (>1.50 psi) in the later part of the run compared to cultures with PES and PS membrane. However, as permeate flux increased, the total mass of product decreased by around 30% for cultures with PVDF membrane, while it remained consistent with PES and PS membrane, and at the highest flux studied, PES membrane generated 12% more product than PVDF membrane. This highlights that membrane selection for large-volume perfusion bioreactors depends on the productivity and permeate flux required. Since operating large-volume perfusion bioreactors at low flux would require several cell retention devices and a complex setup, PVDF membranes are suitable for low-volume operations at low fluxes whereas PES membranes can be a desirable alternative for large-volume higher demand products at higher fluxes.

Cultivation of Bovine Mesenchymal Stem Cells on Plant-Based Scaffolds in a Macrofluidic Single-Use Bioreactor for Cultured Meat.

Reducing production costs, known as scaling, is a significant obstacle in the advancement of cultivated meat. The cultivation process hinges on several key components, e.g., cells, media, scaffolds, and bioreactors. This study demonstrates an innovative approach, departing from traditional stainless steel or glass bioreactors, by integrating food-grade plant-based scaffolds and thermoplastic film bioreactors. While thermoplastic films are commonly used for constructing fluidic systems, conventional welding methods are cost-prohibitive and lack rapid prototyping capabilities, thus inflating research and development expenses. The developed laser welding technique facilitates contamination-free and leakproof sealing of polyethylene films, enabling the efficient fabrication of macrofluidic systems with various designs and dimensions. By incorporating food-grade plant-based scaffolds, such as rice seeded with bovine mesenchymal stem cells, into these bioreactors, this study demonstrates sterile cell proliferation on scaffolds within macrofluidic systems. This approach not only reduces bioreactor prototyping and construction costs but also addresses the need for scalable solutions in both research and industrial settings. Integrating single-use bioreactors with minimal shear forces and incorporating macro carriers such as puffed rice may further enhance biomass production in a scaled-out model. The use of food-grade plant-based scaffolds aligns with sustainable practices in tissue engineering and cultured-meat production, emphasizing its suitability for diverse applications.

An in-silico analysis of hydrodynamics and gas mass transfer characteristics in scale-down models for mammalian cell cultures

Bioprocess scale-up and technology transfer can be challenging due to multiple variables that need to be optimized during process development from laboratory scale to commercial manufacturing. Cell cultures are highly sensitive to key factors during process transfer across scales, including geometric variability in bioreactors, shear stress from impeller and sparging activity, and nutrient gradients that occur due to increasing blend times. To improve the scale-up and scale-down of these processes, it is important to fully characterize bioreactors to better understand the differences that will occur within the culture environment, especially the hydrodynamic profiles that will vary in vessel designs across scales. In this study, a comprehensive hydrodynamic characterization of the Ambr® 250 mammalian single-use bioreactor was performed using time-accurate computational fluid dynamics simulations conducted with M-Star computational fluid dynamics software, which employs lattice-Boltzmann techniques to solve the Navier-Stokes transport equations at a mesoscopic scale. The single-phase and two-phase fluid properties within this small-scale vessel were analyzed in the context of agitation hydrodynamics and mass transfer (both within the bulk fluid and the free surface) to effectively characterize and understand the differences that scale-down models possess when compared to their large-scale counterparts. The model results validate the use of computational fluid dynamics as an in-silico tool to characterize bioreactor hydrodynamics and additionally identify important free-surface transfer mechanics that need to be considered during the qualification of a scale-down model in the development of mammalian bioprocesses.

Fluid dynamics and power consumptions in a single use stirred tank adopted in the pharmaceutical industry

Single use bioreactors (SUB) are recurrently adopted within the pharmaceutical industry since they offer several advantages with respect to traditional bioreactors. Generally, they consist of a stainless-steel container with a bag and an integrated impeller for handling (mixing stage), storage (post mixing stage I) and transfer (post mixing stage II) of formulated products. In this work, one of the single use bioreactor types, the Flexel-Levmixer by Sartorius, is investigated. It is a vessel with a square section stirred with a bottom impeller, for which little information is available in the literature, thus making troubleshooting and optimization difficult. To address this issue, a combination of experimental techniques, namely Particle Image Velocimetry, and torque sensors based on strain gauges, are selected for the investigation aimed at the understanding of the fluid dynamics and power consumption with different filling levels and impeller speeds.

Large-scale smart bioreactor with fully integrated wireless multivariate sensors and electronics for long-term in situ monitoring of stem cell culture.

Achieving large-scale, cost-effective, and reproducible manufacturing of stem cells with the existing devices is challenging. Traditional single-use cell-bag bioreactors, limited by their rigid and single-point sensors, struggle with accuracy and scalability for high-quality cell manufacturing. Here, we introduce a smart bioreactor system that enables multi-spatial sensing for real-time, wireless culture monitoring. This scalable system includes a low-profile, label-free thin-film sensor array and electronics integrated with a flexible cell bag, allowing for simultaneous assessment of culture properties such as pH, dissolved oxygen, glucose, and temperature, to receive real-time feedback for up to 30 days. The experimental results show the accurate monitoring of time-dynamic and spatial variations of stem cells and myoblast cells with adjustable carriers from a plastic dish to a 2-liter cell bag. These advances open up the broad applicability of the smart sensing system for large-scale, lower-cost, reproducible, and high-quality engineered cell manufacturing for broad clinical use.

Scalable manufacture of therapeutic mesenchymal stromal cell products on customizable microcarriers in vertical wheel bioreactors that improve direct visualization, product harvest, and cost

Background aimsHuman mesenchymal stromal cells (hMSCs) and their secreted products show great promise for treatment of musculoskeletal injury and inflammatory or immune diseases. However, the path to clinical utilization is hampered by donor-tissue variation and the inability to manufacture clinically relevant yields of cells or their products in a cost-effective manner. Previously we described a method to produce chemically and mechanically customizable gelatin methacryloyl (GelMA) microcarriers for culture of hMSCs. Herein, we demonstrate scalable GelMA microcarrier-mediated expansion of induced pluripotent stem cell (iPSC)-derived hMSCs (ihMSCs) in 500 mL and 3L vertical wheel bioreactors, offering several advantages over conventional microcarrier and monolayer-based expansion strategies. MethodsHuman mesenchymal stromal cells derived from induced pluripotent cells were cultured on custom-made spherical gelatin methacryloyl microcarriers in single-use vertical wheel bioreactors (PBS Biotech). Cell-laden microcarriers were visualized using confocal microscopy and elastic light scattering methodologies. Cells were assayed for viability and differentiation potential in vitro by standard methods. Osteogenic cell matrix derived from cells was tested in vitro for osteogenic healing using a rodent calvarial defect assay. Immune modulation was assayed with an in vivo peritonitis model using Zymozan A. ResultsThe optical properties of GelMA microcarriers permit noninvasive visualization of cells with elastic light scattering modalities, and harvest of product is streamlined by microcarrier digestion. At volumes above 500 mL, the process is significantly more cost-effective than monolayer culture. Osteogenic cell matrix derived from ihMSCs expanded on GelMA microcarriers exhibited enhanced in vivo bone regenerative capacity when compared to bone morphogenic protein 2, and the ihMSCs exhibited superior immunosuppressive properties in vivo when compared to monolayer-generated ihMSCs. ConclusionsThese results indicate that the cell expansion strategy described here represents a superior approach for efficient generation, monitoring and harvest of therapeutic MSCs and their products.

The Colourimetric Method for Mixing Time Measurement in Single-Use and Multi-Use Bioreactors—Methodology Overview and Practical Recommendations

Mixing time is an important parameter for quantifying the mixing efficiency of a bioreactor system, essential for successful bioprocess development in various branches of the bioengineering sector (e.g., biopharma, biorefineries, food industry and bioreactor design). The colourimetric method is one of the ways of obtaining valuable quantitative data about the mixing process and the liquid flow inside a vessel. This review consists of a catalogue and a discussion of previously published data in which the colourimetric method has been utilised; a critical comparison between the colourimetric and other mixing time measurement methods; an explanation of practical considerations regarding the bioreactor setup and the choice of reagents; a practical guide for requirements for the acquisition of high-quality images of the mixing process; a thorough discussion of aspects connected to the computer image processing of the video material. This review is intended to thoroughly present the versatility of the colourimetric method for mixing time measurement in miscellaneous bioreactor systems, i.e., in classical tanks and modern single-use (disposable) plastic film-based bag-like containers, and to facilitate the implementation of the colourimetric method in new research setups by providing complete and valuable recommendations about each step of the methodology.

3D Printed, Single-Use Bioreactor with Integrated Inline Sensors for Microbial and Mammalian Cell Cultivation—A Case Study

The development of upstream bioprocesses necessitates small, instrumented bioreactors for investigating and optimizing production processes in a cost-effective manner. Due to advances in both the equipment and the materials used in additive manufacturing, 3D printing of customized bioreactors is now in the realm of possibilities. In this study, a small-scale 3D printed bioreactor suitable for mammalian and microbial cultivations was developed, featuring a working volume of 90 mL, inline pH and dissolved oxygen probes and a levitating magnetic stirrer. Aeration channels and a sampling port were printed directly into the vessel walls. Additionally, the vessel was equipped with a 3D printed customizable optical biomass-sensor. The bioreactor’s performance was evaluated through technical characterization and proof of concept cultivations, demonstrating that mixing time and oxygen mass transfer were sufficient for cultivating mammalian as well as microbial cells at high cell densities. Specifically, an Escherichia coli fed-batch cultivation achieved a maximum OD600 of 204. Furthermore, a fed-batch cultivation of an IgG antibody-producing Chinese hamster ovary cell line reached a peak viable cell density of 10.2 × 106 cells mL−1 and a maximum product titer of 2.75 g L−1. Using a three-parameter fit, the inline biomass signal could be correlated to the corresponding offline values with satisfactory accuracy, making it possible to monitor cell growth in real-time.

High-Titer Recombinant Adenovirus 26 Vector GMP Manufacturing in HEK 293 Cells with a Stirred Single-Use Bioreactor for COVID-19 Vaccination Purposes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 virus) pandemic has shown the importance of pursuing various vaccine manufacturing strategies. In the present study, the HEK 293 cells were infected with recombinant adenovirus serotype 26 (rAd26), and the effects of critical process parameters (CPPs) including viable cell density (VCD) at infection time (0.5 × 106, 0.8 × 106, 1.4 × 106, 1.8 × 106, and 2.5 × 106 cells/mL), the multiplicity of infection (MOI) = 3, 6, 9, 12, and 15, and two aeration strategies (high-speed agitation with a sparging system and low-speed agitation with an overlay system) were investigated experimentally. The results of small-scale experiments in 2 L shake flasks (SF 2L) demonstrated that the initial VCD and MOI could affect the cell proliferation and viability. The results at these experiments showed that VCD = 1.4 × 106 cells/mL and MOI = 9 yielded TCID50 /mL = 108.9, at 72 h post-infection (hpi), while the virus titer at VCD = 0.5 × 106 and 0.8 × 106 cells/mL was lower compared to that of VCD = 1.4 × 106 cells/mL. Moreover, our findings showed that VCDs > 1.8 × 106 cells/m with MOI = 9 did not have a positive effect on TCID50 /mL and MOI = 3 and 6 were less efficient, whereas MOI > 12 decreased the viability drastically. In the next step, the optimized CPPs in a small scale were exploited in a 200 L single-use bioreactor (SUB), with good manufacturing practice (GMP) conditions, at RPM = 25 with an overlay system, yielding high-titer rAd26 manufacturing, i.e., TCID50/mL = 108.9, at 72 hpi.

Bioprocess development for cord blood mesenchymal stromal cells on microcarriers in Vertical-Wheel bioreactors.

Equine mesenchymal stromal cells (MSCs) have been found to be beneficial for the treatment of many ailments, including orthopedic injuries, due to their superior differentiation potential and immunomodulating properties. Cell therapies require large cell numbers, which are not efficiently generated using conventional static expansion methods. Expansion of equine cord blood-derived MSCs (eCB-MSCs) in bioreactors, using microcarriers as an attachment surface, has the potential to generate large numbers of cells with increased reproducibility and homogeneity compared with static T-flask expansion. This study investigated the development of an expansion process using Vertical-Wheel (VW) bioreactors, a single-use bioreactor technology that incorporates a wheel instead of an impeller. Initially, microcarriers were screened at small scale to assess eCB-MSC attachment and growth and then in bioreactors to assess cell expansion and harvesting. The effect of different donors, serial passaging, and batch versus fed batch were all examined in 0.1 L VW bioreactors. The use of VW bioreactors with an appropriate microcarrier was shown to be able to produce cell densities of up to 1E6 cells/mL, while maintaining cell phenotype and functionality, thus demonstrating great potential for the use of these bioreactors to produce large cell numbers for cell therapies.

Scale-up analysis of geometrically dissimilar single-use bioreactors.

Cell culture scale-up is a challenging task due to the simultaneous change of multiple hydrodynamic process characteristics and their different dependencies on the bioreactor size as well as variation in the requirements of individual cell lines. Conventionally, the volumetric power input is the most common parameter to select the impeller speed for scale-up, however, it is well reported that this approach fails when there are huge differences in bioreactor scales. In this study, different scale-up criteria are evaluated. At first, different hydrodynamic characteristics are assessed using computational fluid dynamics data for four single-use bioreactors, the Mobius® CellReady 3 L, the Xcellerex™ XDR-10, the Xcellerex™ XDR-200, and the Xcellerex™ XDR-2000. On the basis of this numerical data, several potential scale-up criteria such as volumetric power input, impeller tip speed, mixing time, maximum hydrodynamic stress, and average strain rate in the impeller zone are evaluated. Out of all these criteria, the latter is found to be most appropriate, and the successful scale-up from 3to 10 L bioreactor and to 200 L bioreactor is confirmed with cell culture experiments using Chinese Hamster Ovary cell cultivation.

Glass microspargers as effective frit spargers in single use bioreactors.

For multiple-use bench scale and larger bioreactors, sintered stainless steel frit spargers are commonly used as microspargers. For bench-scale single-use bioreactors (SUBs), existing microspargers are sintered plastics, such as polyethylene. However, though plastics are readily sterilized by irradiation making them convenient for single use, these designs overlook surface energy properties of the materials of construction. For these sintered plastic spargers, forces at the water-air-surface interface cause bubble coalescence, leading to lower effective mass transfer, higher gas flow rates, and differing pCO2 profiles in cell culture. Alternative materials of construction were evaluated based on contact angle information and bubble formation observations. Sintered glass was chosen over thermoplastic polymers for higher surface wettability as described in the glass/water contact angle, its history as a commonly sintered material, and availability at costs suitable for single use applications. Glass sintered spargers and traditional stainless steel frit spargers were compared by porosity, bubble size, and kL a studies. Mass transfer (kL a) and cell culture performance equal or greater than a standard 20 μm stainless steel microsparger mass transfer efficiency was achieved by a glass frit sparger, of international porosity standard "P40" according to ISO 4793-80, which corresponds to a range of 16-40 μm.

Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture.

Therapeutic viral vectors are an emerging technology with several clinical applications in gene therapy, vaccines, and immunotherapy. Increased demand has required the redevelopment of conventional, low-throughput cell culture and purification manufacturing methods such as static cell stacks and ultracentrifugation. In this work, scalable methods were investigated for the manufacture of an oncolytic virus immunotherapy application consisting of a prototype strain of coxsackievirus A21 (CVA21) produced in adherent MRC-5 cells. Cell culture was established in stirred-tank microcarrier bioreactors, and an efficient affinity chromatography method was developed for the purification of harvested CVA21 through binding of the viral capsids to an immobilized glutathione (GSH) ligand. Bioreactor temperature during infection was investigated to maximize titer, and a decrease in temperature from 37°C to 34°C yielded a two-three-fold increase in infectivity. After purification of the 34°C harvests, the GSH affinity chromatography elution not only maintained a >two-fold increase in infectivity and viral genomes but also increased the proportion of empty capsids compared to 37°C harvests. Using material generated from both infection temperature setpoints, chromatographic parameters and mobile phase compositions were studied at the laboratory scale to maximize infectious particle yields and cell culture impurity clearance. Empty capsids that co-eluted with full capsids from 34°C infection temperature harvests were poorly resolved across the conditions tested, but subsequent polishing anion exchange and cation exchange chromatography steps were developed to clear residual empty capsids and other impurities. Oncolytic CVA21 production was scaled-up 75-fold from the laboratory scale and demonstrated across seven batches in 250L single-use microcarrier bioreactors and purified with customized, prepacked, single-use 1.5L GSH affinity chromatography columns. The large-scale bioreactors controlled at 34°C during infection maintained a three-fold increase in productivity in the GSH elution, and excellent clearance of host cell and media impurities was observed across all batches. This study presents a robust method for the manufacture of an oncolytic virus immunotherapy application that may be implemented for the scalable production of other viruses and viral vectors which interact with glutathione.

Suspension culture on microcarriers and as aggregates enables expansion and differentiation of pluripotent stem cells (PSCs)

Background aimsHuman pluripotent stem cells (PSCs) hold a great promise for promoting regenerative medical therapies due to their ability to generate multiple mature cell types and for their high expansion potential. However, cell therapies require large numbers of cells to achieve desired therapeutic effects, and traditional two-dimensional static culture methods cannot meet the required production demand for cellular therapies. One solution to this problem is scaling up expansion of PSCs in bioreactors using culture strategies such as growing cells on microcarriers or as aggregates in suspension culture. MethodsIn this study, we directly compared PSC expansion and quality parameters in microcarrier- and aggregate-cultures grown in single-use vertical-wheel bioreactors. ResultsWe showed comparable expansion of cells on microcarriers and as aggregates by day 6 with a cell density reaching 2.2 × 106 cells/mL and 1.8 × 106 cells/mL and a fold-expansion of 22- and 18-fold, respectively. PSCs cultured on microcarriers and as aggregates were comparable with parallel two-dimensional cultures and with each other in terms of pluripotency marker expression and retention of other pluripotency characteristics as well as differentiation potential into three germ layers, neural precursor cells and cardiomyocytes. ConclusionsOur study did not demonstrate a clear advantage between the two three-dimensional methods for the quality parameters assessed. This analysis adds support to the use of bioreactor systems for large scale expansion of PSCs, demonstrating that the cells retain key characteristics of PSCs and differentiation potential in suspension culture.