Cell Culture Bioreactor Research Articles

Quality by Design-Driven Process Development of Cell Culture in Bioreactor for the Production of Foot-And-Mouth Veterinary Vaccine

Quality by design (QbD) principle has been established as a guideline to emphasize the understanding of the relationship of product quality with process control. Vaccine product have characteristics of security and high efficiency, but it also has features such as complexity and rigorous regulatory for production. This case study describes an example of QbD-driven process development for manufacturing a veterinary vaccine produced with baby hamster kidney-21 cells. The study revealed that cell culture duration was the most significant factor affecting 50% tissue culture infectious doses (TCID50) and antigenic titer, and the factors of culture temperature and pH at infection phase exhibited less effect. Culture temperature at infection phase was the only significant factor for total protein. Through the Monte Carlo simulation, the design spaces of process parameters were determined. Meanwhile, the excellent and robust performance in manufacturing scale (4000-L) validated the effectiveness of this strategy. A reliable and robust multivariate process parameter range, that is, design space, was identified by this systematic approach. Our investigation presents a successful case of QbD principle, which encourages other researchers to combine the methodology into other biopharmaceutical manufacturing process.

Adiponectin Relieves Human Adult Cardiac Myocytes Injury Induced by Intermittent Hypoxia.

BackgroundObstructive sleep apnea (OSA) is associated with many cardiovascular disorders. Intermittent hypoxia (IH) is a key pathological hallmark of OSA. This study was conducted to evaluate the potential therapeutic effects and the associated mechanisms of adiponectin (APN) on IH induced human adult cardiac myocytes (HACMs) injury.Material/MethodsHACMs were exposed to normoxia or IH (1% to 21% O2) using a novel cell culture bio-reactor with gas-permeable membranes. Cell viability was detected by Cell Counting Kit-8 assay. Cell membrane integrity was assessed by the detection of lactate dehydrogenase (LDH) release. Cell apoptosis was analyzed by flow cytometry. Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) levels were determined using specific assay kits. P-AMPK (AMP-activated protein kinase), p-LKB1, and p-p65 protein levels were measured by western blotting. Pro-inflammatory factors including interleukin (IL)-1β, IL-6, IL-8 expressions were detected by enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction.ResultsThe results showed that APN had no cytotoxic to HACMs. Compared with the control group, HACMs cell viability significantly decreased, LDH release increased and cell apoptosis increased in the IH group. The levels of IL-1β, IL-6, IL-8, MDA, and p-p65 were higher, while the levels of SOD, GSH-Px, p-AMPK, and p-LKB1 were lower in HACMs cells in the IH group than that in the control group. However, APN treatment significantly rescued these effects compared with the IH group in a dose-dependent manner.ConclusionsIn conclusion, these results indicated that APN protected against IH induced HACMs injury possibly mediated by AMPK and NF-κB pathway.

Quality Control and Downstream Processing of Therapeutic Enzymes.

Therapeutic enzymes are a commercially minor but clinically important area of biopharmaceuticals. An array of therapeutic enzymes has been developed for a variety of human diseases, including leukaemia and enzyme-deficiency diseases such as Gaucher's disease. Production and testing of therapeutic enzymes is strictly governed by regulatory bodies in each country around the world, and batch-to-batch consistency is crucially important. Manufacture of a batch starts with the fermentation or cell culture stage. After expression of the therapeutic enzyme in a cell culture bioreactor, robust and reproducible protein purification, or downstream processing (DSP) of the target product, is critical to ensuring safe delivery of these medicines. Modern processing technology, including the use of disposable processing equipment, has greatly improved the DSP development pathway in terms of robustness and speed to clinic. Once purified, the drug substance undergoes rigorous quality control (QC) testing according to current regulatory guidance, to enable release to the clinic and patient. QC testing is conducted to ensure the safety, purity, identity, potency and strength of the medicinal product, requiring multiple analytical methods that are rigorously validated and monitored for robust performance. Several case studies, including L-asparaginase and asfotase alfa, are discussed to illustrate the methods described herein.

Avian Bioreactor Systems: A Review.

Animal bioreactors are genetically modified animal systems that have the potential to reduce production cost, and improve production efficiency, of pharmaceutically relevant recombinant proteins. Several species including goats, cattle, rabbits, and avians have been genetically modified to secrete target proteins into milk, egg whites, blood, or other bodily fluids. There are several advantages associated with the use of avians as bioreactor systems. Avians have a short generation time, leading to the quick establishment of a transgenic line and high egg production. Transgenic avian systems allow for appropriate post-translational modification, as opposed to prokaryotic cell culture bioreactors, and have higher productivity than mammalian cell culture systems. Furthermore, recombinant proteins can be incorporated into egg whites and easily collected from the sterile environment of the egg. Magnum-specific expression of target genes has been achieved by use of the ovalbumin promoter, leading to a localization of the target protein into the avian egg. In this review, we discuss the current advancements, future potential, and limitations of avian bioreactor systems.

A systematic mass-transfer modeling approach for mammalian cell culture bioreactor scale-up

A mathematical mass-transfer model was developed based on the mass balance of gas exchange in cell culture bioreactors. The model combines the bioreactor mass transfer properties and mammalian cell metabolic parameters to predict the cell culture process oxygen demands and carbon dioxide accumulation in bioreactors. With the guidance of the mass-transfer model, a CHO cell culture process to produce a therapeutic antibody was successfully scaled up from 2-L to 1500-L bioreactor directly without intermediate steps. The process performances and product quality attributes demonstrate good robustness and consistency between small and large scales. This mass-transfer model provides a more systematic and reliable approach for mammalian cell culture bioreactor scaling up.

Effect of cell culture medium additives on color and acidic charge variants of a monoclonal antibody.

This manuscript summarizes the effect of certain cell culture medium additives on antibody drug substance coloration and acidic charge variants. It has been shown previously that B-vitamins and iron in the cell culture medium could significantly impact color intensity. In this manuscript, we detail the effect of several other cell culture components that have been shown to impact coloration. It is shown that if cystine is used instead of cysteine in the cell culture medium, coloration was reduced. Hydrocortisone has been shown to reduce coloration and boost specific productivity. The effect of a peptone/hydrolysate on coloration was investigated in cell culture experiments, which showed its use can lead to reduced coloration. Mechanisms by which these compounds influence coloration will be briefly discussed. Since it has been previously shown that antibody oxidation could potentially lead to coloration, the current effort was focused on screening for specific antioxidant additives to the culture medium to reduce coloration. An in-vitro incubation model was used to screen antioxidant compounds, several of which were found to significantly reduce antibody color, while some led to significantly increased color. Hypotaurine and carboxymethylcysteine, which had the most significant color reducing effect in the incubation study, were further tested in small-scale bioreactor cell culture experiments. These studies demonstrated that these compounds lead to reduced coloration in cell culture without affecting cell growth and titer. Hypotaurine, hydrocortisone, peptone, and cystine were also shown to reduce the acidic charge variant levels, which was previously shown to correlate with color. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1298-1307, 2018.

Design and Manufacturing of a Uniform, Massively Parallel Fluid Distributor for Automated Cell Culture Bioreactor

In this study, we present a design methodology that enables the automation of cell culture using a fluidic bioreactor module in a uniform, massively parallel fashion. To demonstrate our proof-of-concept validation, we designed the channel lengths that can uniformly distribute the fluid from one inlet to two to the power of n outlets. In our experiments, the module was composed of four layers which can hold eight petri dishes on each layer. The channel outlets were connected to the petri dishes, respectively. Cells and media were distributed into each petri dish by compensating the channel lengths with different potential energy of each layer in a controlled manner. The results show that the module was well-designed for the parallel supply of medium in automatic cell culture. Overall, the process can be automated by controlling the on/off events temporally, such as injecting a new cell media and removing the waste.

Billion-scale production of hepatocyte-like cells from human induced pluripotent stem cells

Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells are expected to be utilized in drug screening and regenerative medicine. However, hepatocyte-like cells have not been fully used in such applications because it is difficult to produce such cells on a large scale. In this study, we tried to establish a method to mass produce hepatocyte-like cells using a three-dimensional (3D) cell culture bioreactor called the Rotary Cell Culture System (RCCS). RCCS enabled us to obtain homogenous hepatocyte-like cells on a billion scale (>109 cells). The gene expression levels of some hepatocyte markers (alpha-1 antitrypsin, cytochrome (CYP) 1A2, CYP2D6, and hepatocyte nuclear factor 4alpha) were higher in 3D-cultured hepatocyte-like cells than in 2D-cultured hepatocyte-like cells. This result suggests that RCCS could provide more suitable conditions for hepatocyte maturation than the conventional 2D cell culture conditions. In addition, more than 90% of hepatocyte-like cells were positive for albumin and could uptake low-density lipoprotein in the culture medium. We succeeded in the large-scale production of homogenous and functional hepatocyte-like cells from human iPS cells. This technology will be useful in drug screening and regenerative medicine, which require enormous numbers of hepatocyte-like cells.

Product Attribute Forecast: Adaptive Model Selection Using Real-Time Machine Learning

A real-time machine learning framework is developed to forecast product concentration in mammalian cell culture bioreactors. In real-time, the framework evaluates several machine learning algorithms and chooses the most representative algorithm based on current dynamics of the system. Data from multiple sources is combined and only subset of features are fed to the model based on a pre-selection criteria. The model performance is tested using two small-scale bioreactors run. The performance improved towards the end of the process with accumulating data and results for 1 day ahead prediction is presented.

Impact of media and antifoam selection on monoclonal antibody production and quality using a high throughput micro-bioreactor system.

Monoclonal antibody production in commercial scale cell culture bioprocessing requires a thorough understanding of the engineering process and components used throughout manufacturing. It is important to identify high impact components early on during the lifecycle of a biotechnology‐derived product. While cell culture media selection is of obvious importance to the health and productivity of mammalian bioreactor operations, other components such as antifoam selection can also play an important role in bioreactor cell culture. Silicone polymer‐based antifoams were known to have negative impacts on cell health, production, and downstream filtration and purification operations. High throughput screening in micro‐scale bioreactors provides an efficient strategy to identify initial operating parameters. Here, we utilized a micro‐scale parallel bioreactor system to study an IgG1 producing CHO cell line, to screen Dynamis, ProCHO5, PowerCHO2, EX‐Cell Advanced, and OptiCHO media, and 204, C, EX‐Cell, SE‐15, and Y‐30 antifoams and their impacts on IgG1 production, cell growth, aggregation, and process control. This study found ProCHO5, EX‐Cell Advanced, and PowerCHO2 media supported strong cellular growth profiles, with an IVCD of 25‐35 × 106 cells‐d/mL, while maintaining specific antibody production (Qp > 2 pg/cell‐d) for our model cell line and a monomer percentage above 94%. Antifoams C, EX‐Cell, and SE‐15 were capable of providing adequate control of foaming while antifoam 204 and Y‐30 noticeably stunted cellular growth. This work highlights the utility of high throughput micro bioreactors and the importance of identifying both positive and negative impacts of media and antifoam selection on a model IgG1 producing CHO cell line. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:262–270, 2018

Automation and control system for fluid dynamic stability in hollow‐fiber membrane bioreactor for cell culture

AbstractBACKGROUNDIn recent years, biochemical and biotechnological engineering has been applied to the culture of human and animal tissue cells, which requires the design, operation and control of complex bioreactors. Hollow fibre membrane bioreactors provide favourable conditions for cellular function and metabolism. To develop bioartificial systems ensuring stable and long‐term operation, fluid dynamics and transport phenomena require careful automation strategies.RESULTSStarting from a crossed hollow‐fiber membrane bioreactor for the culture of complex cell systems configured to operate manually, a 2×2 liquid level/flow‐rate control system is experimentally developed and thoroughly tested for its robustness against liquid level or flow‐rate set‐point changes and disturbances arising from loop interaction. The automation system is shown to be fast for flow‐rate and sufficiently reliable for liquid level (response times of minutes). Limitations are mostly owing to flow‐rate difference constraints and level sensor noise, both originating from cell culture requirements. Prolonged operation (27 days) of the bioreactor in maintaining human hepatocytes in a three‐cell co‐culture system is presented and discussed.CONCLUSIONThe results shown in the present work allow improving the understanding of the dynamic behaviour of a membrane bioreactor for biomedical application and examining the possibility to run the bioreactor under fully automated pre‐set conditions smoothly and for extended periods of time. © 2017 Society of Chemical Industry

Production of Recombinant Rabies Virus Glycoprotein by Insect Cells in a Single-Use Fixed-Bed Bioreactor.

A single-use fixed-bed bioreactor (iCELLis nano) can be used for cultivating non adherent insect cells, which can be then recovered for scaling up or for harvesting a membrane-associated viral glycoprotein with high quality in terms of preserved protein structure and biological function. Here, we describe the procedures for establishing genetically modified Drosophila melanogaster Schneider 2 (S2) cell cultures in the iCELLis nano bioreactor and for quantifying by ELISA the recombinant rabies virus glycoprotein (rRVGP) synthesized. By using the described protocol of production, the following performance can be regularly achieved: 1.7±0.6 × 1E10 total cells; 2.4±0.8 × 1E7 cells/mL and 1.2±0.9μg of rRVGP/1E7 cells; 1.5±0.8mg of total rRVGP.

Oxygen transport in hollow fibre membrane bioreactors for hepatic 3D cell culture: A parametric study

Hepatic cell cultures are exploited for different applications in liver disease studies, drug toxicity testing and bioartificial liver (BAL) devices. Despite the development of various 3D cell culture systems, mass transfer properties and limitations, especially oxygen supply, still remain a controversial topic. In this paper, oxygen transport in a convection-enhanced, crossed-configuration hollow fibre membrane bioreactor hosting hepatocyte spheroids has been investigated through coupled free/porous media flow hydrodynamics and mass transfer modelling using finite-element simulations. Starting from experimental measurements of the oxygen concentration in the bioreactor, a systematic parametric study was performed to evaluate the effect of different parameters – oxygen partial pressure, perfusion rate, hollow fibre spacing, spheroid size, Michaelis-Menten kinetics for oxygen uptake and porosities of the spheroid and the membrane – on dissolved oxygen concentration profile. These parameters were studied in relation to dimensionless groups and variables. Among the operational conditions, oxygen concentration was much more influential than perfusion rate. As for the Michaelis-Menten parameters, oxygen concentration profile inside the spheroids was strongly affected by the maximum consumption rate (Vmax) and weakly dependent on the constant KM. Culturing smaller spheroids minimized the exposure of cells to hypoxic conditions, owing to the short diffusive penetration depth of oxygen especially at low porosities (0.2). An in vivo-like microenvironment in terms of physiological oxygen concentration range was achieved in large spheroids (400µm diameter) at spheroid porosity of 0.47. Membrane porosity also affected the intra-spheroid oxygen concentration and an improvement in oxygen supply to the spheroids could be achieved by reducing the corresponding transfer resistances. The mechanical stress on the spheroid surfaces was found to be within the tolerated ranges. Overall, the most important parameters for optimization of the bioreactor were identified.

The role of nuclear mechanics in cell deformation under creeping flows

Despite the relevant regulatory role that nuclear deformation plays in cell behaviour, a thorough understanding of how fluid flow modulates the deformation of the cell nucleus in non-confined environments is lacking. In this work, we investigated the dynamics of cell deformation under different creeping flows as a general simulation tool for predicting nuclear stresses and strains. Using this solid–fluid modelling interaction framework, we assessed the stress and strain levels that the cell nucleus experiences as a function of different microenvironmental conditions, such as physical constraints, fluid flows, cytosol properties, and nucleus properties and size. Therefore, the simulation methodology proposed here allows the design of deformability-based experiments involving fluid flow, such as real-time deformability cytometry and dynamic cell culture in bioreactors or microfluidic devices.

From MFA to FBA: Defining linear constraints accounting for overflow metabolism in a macroscopic FBA-based dynamical model of cell cultures in bioreactor

Macroscopic dynamical models of cell cultures in bioreactor are made of sets of ODEs representing the mass balances of the main macroscopic species (biomass, main substrates and metabolites). They can be coupled to a Flux Balance Analysis (FBA) linear program whose solution gives metabolic flux values at each time instant. The linear constraints used in this linear program are made of positivity constraints for the fluxes, equalities which link the fluxes to simple macroscopic models of the substrate uptake rates and a set of equalities and inequalities corresponding to some biological assumptions. In this paper, a methodology is proposed to help defining this latter set of linear constraints, with a special focus on overflow metabolism description. It is shown how these linear constraints, together with the objective cost function to be used in the linear program, can be derived from a preliminary Metabolic Flux Analysis (MFA) and a careful comparison of the admissible metabolic flux intervals obtained either using both input and output flux measurements or using only input flux measurements. Finally, a method is proposed for estimating the dynamical model output uncertainties resulting from the intervals of admissible fluxes. In a case study made of hybridoma cell batch and fed-batch cultures, it is shown how to model overflow metabolism on both glucose and glutamine thanks to inequality constraints. The linear program corresponding to a FBA problem is then used in a macroscopic dynamical model which is able to reproduce biomass, substrates and metabolites concentration time profiles in direct and cross validation.

Optical biosensor optimized for continuous in-line glucose monitoring in animal cell culture.

Biosensors for continuous glucose monitoring in bioreactors could provide a valuable tool for optimizing culture conditions in biotechnological applications. We have developed an optical biosensor for long-term continuous glucose monitoring and demonstrated a tight glucose level control during cell culture in disposable bioreactors. The in-line sensor is based on a commercially available oxygen sensor that is coated with cross-linked glucose oxidase (GOD). The dynamic range of the sensor was tuned by a hydrophilic perforated diffusion membrane with an optimized permeability for glucose and oxygen. The biosensor was thoroughly characterized by experimental data and numerical simulations, which enabled insights into the internal concentration profile of the deactivating by-product hydrogen peroxide. The simulations were carried out with a one-dimensional biosensor model and revealed that, in addition to the internal hydrogen peroxide concentration, the turnover rate of the enzyme GOD plays a crucial role for biosensor stability. In the light of this finding, the glucose sensor was optimized to reach a long functional stability (>52days) under continuous glucose monitoring conditions with a dynamic range of 0-20mM and a response time of t 90≤10min. In addition, we demonstrated that the sensor was sterilizable with beta and UV irradiation and only subjected to minor cross sensitivity to oxygen, when an oxygen reference sensor was applied. Graphical abstract Measuring setup of a glucose biosensor in a shake flask for continuous glucose monitoring in mammalian cell culture.

Facile fabrication of poly(ε-caprolactone)/graphene oxide membranes for bioreactors in tissue engineering

Promising polymer membranes of blended biocompatible poly(ε-caprolactone) and graphene oxide (PCL/GO) and PCL and partially reduced graphene oxide (PCL/rGO) with outstanding water and nutrient transport properties for cell culture bioreactors were prepared using phase inversion at mild temperatures. Some of the prepared PCL/GO membranes were subjected to a ‘chemical-free’ GO post-reductive process using UV (PCL/GO/UV) irradiation. The PCL/rGO membranes exhibited 2.5 times higher flux than previously reported biocompatible polymer membranes for cell culture bioreactors, which was attributed to the highly interconnected porosity. On the other hand, the formation of PCL-graphene oxide composites in the PCL/GO and PCL/GO/UV membranes was not conclusive according to spectroscopic analyses, thermal analyses and mechanical characterization, probably due to the low graphene oxide loading in the membranes (0.1%w/w). The presence of graphene oxide-based nanomaterials in the polymer matrix slightly reduced the mechanical properties of the PCL-graphene oxide membranes by limiting the polymer chain mobility in comparison to that of the plain PCL membranes. However, their mechanical stability was sufficient for the applications pursued. Finally, the biocompatibility assay indicated that the incorporation of GO and rGO into the PCL matrix enhanced the uniform distribution and morphology of the glioblastoma cells on the surface of the PCL-graphene oxide membranes.

Development and Characterization of a Parallelizable Perfusion Bioreactor for 3D Cell Culture.

The three dimensional (3D) cultivation of stem cells in dynamic bioreactor systems is essential in the context of regenerative medicine. Still, there is a lack of bioreactor systems that allow the cultivation of multiple independent samples under different conditions while ensuring comprehensive control over the mechanical environment. Therefore, we developed a miniaturized, parallelizable perfusion bioreactor system with two different bioreactor chambers. Pressure sensors were also implemented to determine the permeability of biomaterials which allows us to approximate the shear stress conditions. To characterize the flow velocity and shear stress profile of a porous scaffold in both bioreactor chambers, a computational fluid dynamics analysis was performed. Furthermore, the mixing behavior was characterized by acquisition of the residence time distributions. Finally, the effects of the different flow and shear stress profiles of the bioreactor chambers on osteogenic differentiation of human mesenchymal stem cells were evaluated in a proof of concept study. In conclusion, the data from computational fluid dynamics and shear stress calculations were found to be predictable for relative comparison of the bioreactor geometries, but not for final determination of the optimal flow rate. However, we suggest that the system is beneficial for parallel dynamic cultivation of multiple samples for 3D cell culture processes.

Facile microfluidic production of composite polymer core-shell microcapsules and crescent-shaped microparticles

HypothesisCore-shell microcapsules and crescent-shaped microparticles can be used as picolitre bioreactors for cell culture and microwells for cell trapping/immobilisation, respectively. ResultsMonodisperse polylactic acid (PLA) core-shell microcapsules with a diameter above 200μm, a shell thickness of 10μm, and 96% water entrapment efficiency were produced by solvent evaporation from microfluidically generated W/O/W emulsion drops with core-shell structure, and used to encapsulate Saccharomyces cerevisiae yeast cells in their aqueous cores. The morphological changes of the capsules stained with Nile red were studied over 14days under different osmotic pressure and pH gradients. FindingsThe shell retained its integrity under isotonic conditions, but buckling and particle crumbling occurred in a hypertonic solution. When the capsules containing 5wt% aqueous Eudragit® S 100 solution in the core were incubated in 10−4M HCl solution, H+ diffused through the PLA film into the core causing an ionic gelation of the inner phase and its phase separation into polymer-rich and water-rich regions, due to the transition of Eudragit from a hydrophilic to hydrophobic state. Crescent-shaped composite microparticles with Eudragit cores and PLA shells were fabricated by drying core-shell microcapsules with gelled cores, due to the collapse of PLA shells encompassing water-rich crescent regions.

Enhancing enterovirus A71 vaccine production yield by microcarrier profusion bioreactor culture

Hand, foot and mouth diseases (HFMD) are mainly caused by Enterovirus A71 (EV-A71) infections. Clinical trials in Asia conducted with formalin-inactivated EV-A71 vaccine candidates produced from serum-free Vero cell culture using either roller bottle or cell factory technology, are found to be safe and highly efficacious. To increase vaccine yields and reduce the production costs, the bioprocess improvement for EV-A71 vaccine manufacturing is currently being investigated. The parameters that could affect and enhance the production yields of EV-A71 virus growth in the microcarrier bioreactor were investigated. The medium replacement culture strategy included a multi-harvested semi-batch process and perfusion technology and was found to increase the production yields more than 7–14 folds. Based on the western blot and cryo-EM analyses of the EV-A71 virus particles produced from either the multi-harvested semi-batch (MHSBC) or perfusion cultures were found to be similar to those virus particles obtained from the single batch culture. Mouse immunogenicity studies indicate that the EV-A71 vaccine candidates produced from the perfusion culture have similar potency to those obtained from single batch bioprocess. The physical structures of the EV-A71 particles revealed by the cryo-EM analysis were found to be spherical capsid particles. These results provide feasible technical bioprocesses for increasing virus yields and the scale up of EV-A71 vaccine manufacturing using the bioreactor cell culture methods.