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.

The advent of plant cells in bioreactors.

Ever since agriculture started, plants have been bred to obtain better yields, better fruits, or sustainable products under uncertain biotic and abiotic conditions. However, a new way to obtain products from plant cells emerged with the development of recombinant DNA technologies. This led to the possibility of producing exogenous molecules in plants. Furthermore, plant chemodiversity has been the main source of pharmacological molecules, opening a field of plant biotechnology directed to produce high quality plant metabolites. The need for different products by the pharma, cosmetics agriculture and food industry has pushed again to develop new procedures. These include cell production in bioreactors. While plant tissue and cell culture are an established technology, beginning over a hundred years ago, plant cell cultures have shown little impact in biotechnology projects, compared to bacterial, yeasts or animal cells. In this review we address the different types of bioreactors that are currently used for plant cell production and their usage for quality biomolecule production. We make an overview of Nicotiana tabacum, Nicotiana benthamiana, Oryza sativa, Daucus carota, Vitis vinifera and Physcomitrium patens as well-established models for plant cell culture, and some species used to obtain important metabolites, with an insight into the type of bioreactor and production protocols.

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.

Development and scale-up of rVSV-SARS-CoV-2 vaccine process using single use bioreactor.

The outbreak of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the Coronavirus Disease 2019 (COVID-19) has spread through the globe at an alarming speed. The disease has become a global pandemic affecting millions of people and created public health crises worldwide. Among many efforts to urgently develop a vaccine against this disease, we developed an industrial-scale closed, single use manufacturing process for V590, a vaccine candidate for SARS-CoV-2. V590 is a recombinant vesicular stomatitis virus (rVSV) genetically engineered to express SARS-CoV-2 glycoprotein. In this work, we describe the development and optimization of serum-free microcarrier production of V590 in Vero cells in a closed system. To achieve the maximum virus productivity, we optimized pH and temperature during virus production in 3 liters (L) bioreactors. Virus productivity was improved (by ∼1 log) by using pH 7.0 and temperature at 34.0 °C. The optimal production condition was successfully scaled up to a 2000 L Single Use Bioreactor (SUB), producing a maximum virus titer of ∼1.0e+7 plaque forming units (PFU)/mL. Further process intensification and simplification, including growing Vero cells at 2 gs per liter (g/L) of Cytodex-1 Gamma microcarriers and eliminating the media exchange (MX) step prior to infection helped to increase virus productivity by ∼2-fold.

Cell Culture Process Scale-Up Challenges for Commercial-Scale Manufacturing of Allogeneic Pluripotent Stem Cell Products.

Allogeneic cell therapy products, such as therapeutic cells derived from pluripotent stem cells (PSCs), have amazing potential to treat a wide variety of diseases and vast numbers of patients globally. However, there are various challenges related to manufacturing PSCs in single-use bioreactors, particularly at larger volumetric scales. This manuscript addresses these challenges and presents potential solutions to alleviate the anticipated bottlenecks for commercial-scale manufacturing of high-quality therapeutic cells derived from PSCs.

CFD-Based and Experimental Hydrodynamic Characterization of the Single-Use Bioreactor XcellerexTM XDR-10.

Understanding the hydrodynamic conditions in bioreactors is of utmost importance for the selection of operating conditions during cell culture process development. In the present study, the two-phase flow in the lab-scale single-use bioreactor XcellerexTM XDR-10 is characterized for working volumes from 4.5 L to 10 L, impeller speeds from 40 rpm to 360 rpm, and sparging with two different microporous spargers at rates from 0.02 L min-1 to 0.5 L min-1. The numerical simulations are performed with the one-way coupled Euler-Lagrange and the Euler-Euler models. The results of the agitated liquid height, the mixing time, and the volumetric oxygen mass transfer coefficient are compared to experiments. For the unbaffled XDR-10, strong surface vortex formation is found for the maximum impeller speed. To support the selection of suitable impeller speeds for cell cultivation, the surface vortex formation, the average turbulence energy dissipation rate, the hydrodynamic stress, and the mixing time are analyzed and discussed. Surface vortex formation is observed for the maximum impeller speed. Mixing times are below 30 s across all conditions, and volumetric oxygen mass transfer coefficients of up to 22.1 h-1 are found. The XDR-10 provides hydrodynamic conditions which are well suited for the cultivation of animal cells, despite the unusual design of a single bottom-mounted impeller and an unbaffled cultivation bioreactor.

TCA Cycle and Its Relationship with Clavulanic Acid Production: A Further Interpretation by Using a Reduced Genome-Scale Metabolic Model of Streptomyces clavuligerus.

Streptomyces clavuligerus (S. clavuligerus) has been widely studied for its ability to produce clavulanic acid (CA), a potent inhibitor of ß-lactamase enzymes. In this study, S. clavuligerus cultivated in 2D rocking bioreactor in fed-batch operation produced CA at comparable rates to those observed in stirred tank bioreactors. A reduced model of S. clavuligerus metabolism was constructed by using a bottom-up approach and validated using experimental data. The reduced model was implemented for in silico studies of the metabolic scenarios arisen during the cultivations. Constraint-based analysis confirmed the interrelations between succinate, oxaloacetate, malate, pyruvate, and acetate accumulations at high CA synthesis rates in submerged cultures of S. clavuligerus. Further analysis using shadow prices provided a first view of the metabolites positive and negatively associated with the scenarios of low and high CA production.

Applicability of a single-use bioreactor compared to a glass bioreactor for the fermentation of filamentous fungi and evaluation of the reproducibility of growth in pellet form.

The implementation of single-use technologies offers several major advantages, e.g. prevention of cross-contamination, especially when spore-forming microorganisms are present. This study investigated the application of a single-use bioreactor in batch fermentation of filamentous fungus Penicillium sp. (IBWF 040-09) from the Institute of Biotechnology and Drug Research (IBWF), which is capable of intracellular production of a protease inhibitor against parasitic proteases as a secondary metabolite. Several modifications to the SU bioreactor were suggested in this study to allow the fermentation in which the fungus forms pellets. Simultaneously, fermentations in conventional glass bioreactor were also conducted as reference. Although there are significant differences in the construction material and gassing system, the similarity of the two types of bioreactors in terms of fungal metabolic activity and the reproducibility of fermentations could be demonstrated using statistic methods. Under the selected cultivation conditions, growth rate, yield coefficient, substrate uptake rate, and formation of intracellular protease-inhibiting substance in the single-use bioreactor were similar to those in the glass bioreactor.

Evaluation of the Single-Use Fixed-Bed Bioreactors in Scalable Virus Production.

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.

Numerical Methods for the Design and Description of In Vitro Expansion Processes of Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (hMSCs) are a valuable source of cells for clinical applications (e.g., treatment of acute myocardial infarction or inflammatory diseases), especially in the field of regenerative medicine. However, for autologous (patient-specific) and allogeneic (off-the-shelf) hMSC-based therapies, in vitro expansion is necessary prior to the clinical application in order to achieve the required cell numbers. Safe, reproducible, and economic in vitro expansion of hMSCs for autologous and allogeneic therapies can be problematic because the cell material is restricted and the cells are sensitive to environmental changes. It is beneficial to collect detailed information on the hydrodynamic conditions and cell growth behavior in a bioreactor system, in order to develop a so called "Digital Twin" of the cultivation system and expansion process. Numerical methods, such as Computational Fluid Dynamics (CFD) which has become widely used in the biotech industry for studying local characteristics within bioreactors or kinetic growth modelling, provide possible solutions for such tasks.In this review, we will present the current state-of-the-art for the in vitro expansion of hMSCs. Different numerical tools, including numerical fluid flow simulations and cell growth modelling approaches for hMSCs, will be presented. In addition, a case study demonstrating the applicability of CFD and kinetic growth modelling for the development of an microcarrier-based hMSC process will be shown.

Efficient propagation of suspended HL-60 cells in a disposable bioreactor supporting wave-induced agitation at various Reynolds number.

Growth of human nonadherent HL-60 cell cultures performed in disposable bioreactor under various hydrodynamic conditions of 2-D wave-assisted agitation has been compared and discussed. Influence of Reynolds number for liquid (ReL) and the kLa coefficient, as key parameters characterized the bioprocessing of HL-60 cells in ReadyToProcess WAVETM 25 system, on reached values of the apparent maximal specific growth rate (µmax) and the specific yield of biomass (Y*X/S) has been identified. The values of ReL (i.e., 510-10,208), as well as kLa coefficient (i.e., 2.83-13.55 h-1), have been estimated for the cultures subjected to wave-induced mixing, based on simplified dimensionless correlation for various presents of WAVE 25 system. The highest values of apparent µmax = 0.038 h-1 and Y*X/S = 25.64 × 108 cells gglc-1 have been noted for cultures independently performed at wave-induced agitation characterized by ReL equaled to 5104 and 510, respectively. The presented results have high applicability potential in scale-up of bioprocesses focused on nonadherent animal cells, or in the case of any application of disposable bioreactors presenting similitude.

Challenges and Solutions for Commercial Scale Manufacturing of Allogeneic Pluripotent Stem Cell Products.

Allogeneic cell therapy products, such as therapeutic cells derived from pluripotent stem cells (PSCs), have amazing potential to treat a wide variety of diseases and vast numbers of patients globally. However, there are various challenges related to the manufacturing of PSCs in large enough quantities to meet commercial needs. This manuscript addresses the challenges for the process development of PSCs production in a bioreactor, and also presents a scalable bioreactor technology that can be a possible solution to remove the bottleneck for the large-scale manufacturing of high-quality therapeutic cells derived from PSCs.

Development of a scale-up strategy for Chinese hamster ovary cell culture processes using the kL a ratio as a direct indicator of gas stripping conditions.

Herein, we described a scale-up strategy focused on the dissolved carbon dioxide concentration (dCO2 ) during fed-batch cultivation of Chinese hamster ovary cells. A fed-batch culture process for a 2000-L scale stainless steel (SS) bioreactor was scaled-up from similarly shaped 200-L scale bioreactors based on power input per unit volume (P/V). However, during the 2000-L fed-batch culture, the dCO2 was higher compared with the 200-L scale bioreactor. Therefore, we developed an alternative approach by evaluating the kL a values of O2 (kL a[O2 ]) and CO2 [kL a(CO2 )] in the SS bioreactors as a scale-up factor for dCO2 reduction. The kL a ratios [kL a(CO2 )/kL a(O2 )] were different between the 200-L and 2000-L bioreactors under the same P/V condition. When the agitation conditions were changed, the kL a ratio of the 2000-L scale bioreactor became similar and the P/V value become smaller compared with those of the 200-L SS bioreactor. The dCO2 trends in fed-batch cultures performed in 2000-L scale bioreactors under the modified agitation conditions were similar to the control. This kL a ratio method was used for process development in single-use bioreactors (SUBs) with shapes different from those of the SS bioreactor. The kL a ratios for the SUBs were evaluated and conditions that provided kL a ratios similar to the 200-L scale SS bioreactors were determined. The cell culture performance and product quality at the end of the cultivation process were comparable for all tested SUBs. Therefore, we concluded that the kL a ratio is a powerful scale-up factor useful to control dCO2 during fed-batch cultures.

Hydrocyclones as cell retention device for CHO perfusion processes in single-use bioreactors.

In this study, a hydrocyclone (HC) especially designed for mammalian cell separation was applied for the separation of Chinese hamster ovary cells. The effect of key features on the separation efficiency, such as type of pumphead in the peristaltic feed pump, use of an auxiliary pump to control the perfusate flow rate, and tubing size in the recirculation loop were evaluated in batch separation tests. Based on these preliminary batch tests, the HC was then integrated to 50-L disposable bioreactor bags. Three perfusion runs were performed, including one where perfusion was started from a low-viability late fed-batch culture, and viability was restored. The successive runs allowed optimization of the HC-bag configuration, and cultivations with 20-25 days duration at cell concentrations up to 50 × 106 cells/ml were performed. Separation efficiencies up to 96% were achieved at pressure drops up to 2.5 bar, with no issues of product retention. To our knowledge, this is the first report in literature of high cell densities obtained with a HC integrated to a disposable perfusion bioreactor.

How to Produce mAbs in a Cube-Shaped Stirred Single-Use Bioreactor at 200 L Scale.

Single-use bioreactors have increasingly been used in recent years, for both research and development as well as industrial production, especially in mammalian cell-based processes. Among the numerous single-use bioreactors available today, wave-mixed bags and stirred systems dominate. Wave-mixed single-use bioreactors are the system of choice for inoculum production, while stirred single-use bioreactors are most often preferred for antibody expression. For this reason, the present chapter describes protocols instructing the reader to use the wave-mixed BIOSTAT® RM 50 for cell expansion and to produce a monoclonal antibody (mAb) in Pall's Allegro™ STR 200 at pilot scale for the first time. All methods described are based on a Chinese hamster ovary (CHO) suspension cell line expressing a recombinant immunoglobulin G (IgG).

Large-scale microcarrier culture of HEK293T cells and Vero cells in single-use bioreactors.

Gene therapy and viral vaccine are becoming attractive therapeutic options for the treatment of different malignant diseases. Viral vector productions are often using static culture vessels and small volume stainless steel bioreactors (SSB). However, the yield of each vessel can be relatively low and multiple vessels often need to be operated simultaneously. This significantly increases labor intensity, production costs, contamination risks, and limits its ability to be scaled up, thus, creating challenges to meet the quantities required once the gene therapy or viral vaccine medicine goes into clinical phases or to market. Single-use bioreactor combining with microcarrier provides a good option for viral vector and vaccine production. The goal of the present studies was to develop the microcarrier bead-to-bead expansion and transfer process for HEK293T cells and Vero cells and scale-up the cultures to 50-200 l single-use bioreactors. Following microcarrier bead-to-bead transfer, the peak cell concentration of HEK293T cells reached 1.5 × 106 cells/ml in XDR-50 bioreactor, whereas Vero cells reached 3.1 × 106 cells/ml and 3.3 × 106 cells/ml in XDR-50 bioreactor and XDR-200 bioreactor, respectively. The average growth rates reached 0.61-0.68/day. The successful microcarrier-based scaleup of these two cell lines in single-use bioreactors demonstrates potential large-scale production capabilities of viral vaccine and vector for current and future vaccines and gene therapy.

Novel Cell-Ess ® supplement used as a feed or as an initial boost to CHO serum free media results in a significant increase in protein yield and production.

Many metrics, including metabolic profiles, have been used to analyze cell health and optimize productivity. In this study, we investigated the ability of a lipid supplement to increase protein yield. At a concentration of 1% (v/v) the lipid supplement caused a significant increase in protein titer (1118 ± 65.4 ng 10(5) cells(- 1) days(- 1)) when compared to cultures grown in the absence of supplementation (819.3 ± 38.1 ng 10(5) cells(- 1) days(- 1); p < 0.05). This equated to a 37% increase in productivity. Furthermore, metabolic profiles of ammonia, glutamate, lactate, and glucose were not significantly altered by the polar lipid supplement. In a separate set of experiments, using the supplement as a feed resulted in 2 notable effects. The first was a 25% increase in protein titer. The second was an extension of peak protein production from 1 day to 2 days. These results suggest that lipid supplementation is a promising avenue for enhancing protein production. In addition, our results also suggest that an increase in protein production may not necessarily require a change in the metabolic state of the cells.

Production of oncolytic adenovirus and human mesenchymal stem cells in a single-use, Vertical-Wheel bioreactor system: Impact of bioreactor design on performance of microcarrier-based cell culture processes.

Anchorage-dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large-scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage-dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single-use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical-Wheel™ technology was evaluated for its potential to support scalable cell culture process development. Two anchorage-dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow-derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical-Wheel bioreactors (PBS-VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS-VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS-VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA-DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS-VW, and scale-up was successfully carried out for two different microcarrier-based cell cultures. Ultimately, the data herein generated demonstrate the potential of Vertical-Wheel bioreactors as a new scalable biomanufacturing platform for microcarrier-based cell cultures of complex biopharmaceuticals.

Extractables analysis of single-use flexible plastic biocontainers.

UNLABELLED: Studies of the extractable profiles of bioprocessing components have become an integral part of drug development efforts to minimize possible compromise in process performance, decrease in drug product quality, and potential safety risk to patients due to the possibility of small molecules leaching out from the components. In this study, an effective extraction solvent system was developed to evaluate the organic extractable profiles of single-use bioprocess equipment, which has been gaining increasing popularity in the biopharmaceutical industry because of the many advantages over the traditional stainless steel-based bioreactors and other fluid mixing and storage vessels. The chosen extraction conditions were intended to represent aggressive conditions relative to the application of single-use bags in biopharmaceutical manufacture, in which aqueous based systems are largely utilized. Those extraction conditions, along with a non-targeted analytical strategy, allowed for the generation and identification of an array of extractable compounds; a total of 53 organic compounds were identified from four types of commercially available single-use bags, the majority of which are degradation products of polymer additives. The success of this overall extractables analysis strategy was reflected partially by the effectiveness in the extraction and identification of a compound that was later found to be highly detrimental to mammalian cell growth. LAY ABSTRACT: The usage of single-use bioreactors has been increasing in biopharmaceutical industry because of the appealing advantages that it promises regarding to the cleaning, sterilization, operational flexibility, and so on, during manufacturing of biologics. However, compared to its conventional counterparts based mainly on stainless steel, single-use bioreactors are more susceptible to potential problems associated with compound leaching into the bioprocessing fluid. As a result, extractable profiling of the single-use system has become essential in the qualification of such systems for its use in drug manufacturing. The aim of this study is to evaluate the effectiveness of an extraction solvent system developed to study the extraction profile of single-use bioreactors in which aqueous-based systems are largely used. The results showed that with a non-targeted analytical approach, the extraction solvent allowed the generation and identification of an array of extractable compounds from four commercially available single-use bioreactors. Most of extractables are degradation products of polymer additives, among which was a compound that was later found to be highly detrimental to mammalian cell growth.