Mammalian Cell Bioreactors Research Articles

Single cell analysis of Chinese hamster ovary cells during a bioprocess using a novel dynamic imaging system.

Reliable monitoring of mammalian cells in bioreactors is essential to biopharmaceutical production. Trypan blue exclusion is a method of determining cell density and viability that has been used for over one hundred years to monitor cells in culture and is the current standard method in biomanufacturing. This method has many disadvantages however and there is a growing demand for more detailed and in-line measurements of cell growth in bioreactors. This article assesses a novel dynamic imaging system for single cell analysis. This data shows that comparable total cell density, viable cell density and percentage viability data shown here, generated by the imaging system, aligned well with conventional trypan blue counting methods for an industrially relevant Chinese Hamster Ovary (CHO) cell line. Furthermore, detailed statistical analysis shows that the classification system used by the PharmaFlow system can reveal trends of interest in monitoring the health of mammalian cells over a 6-day bioreactor culture. The system is also capable of sampling at-line, removing the necessity for taking samples off-line and enabling real time monitoring of cells in a bioreactor culture.

Model Identification and Model Predictive Control of Biopharmaceutical and Biomedical Systems

This paper demonstrates the use of model predictive control (MPC) formulations for uncertain time-varying biopharmaceutical and biomedical systems implemented using measured data without prior knowledge of an accurate model. Furthermore, we demonstrate how prior knowledge can be incorporated in the identification of the model either through constraints or as regularization of the system identification procedure. We demonstrate the use of system identification to develop a model of the fed-batch Chinese hamster ovary mammalian cell bioreactor process and the implementation of model-based control to maximize therapeutic product yields. We also use a time-varying nonlinear biomedical system to demonstrate improvements due to incorporating prior information in the learning of the models and reidentification of the models when prediction accuracy deteriorates. We propose a new partial least squares algorithm that incorporates regularization from prior knowledge and can handle missing data in the independent covariates. Simulation case studies involving a biopharmaceutical production process and automated drug delivery demonstrate the capabilities of the proposed techniques.

Continuous optical in-line glucose monitoring and control in CHO cultures contributes to enhanced metabolic efficiency while maintaining darbepoetin alfa product quality.

Great efforts are directed towards improving productivity, consistency and quality of biopharmaceutical processes and products. One particular area is the development of new sensors for continuous monitoring of critical bioprocess parameters by using online or in-line monitoring systems. Recently, we developed a glucose biosensor applicable in single-use, in-line and long-term glucose monitoring in mammalian cell bioreactors. Now, we integrated this sensor in an automated glucose monitoring and feeding system capable of maintaining stable glucose levels, even at very low concentrations. We compared this fed-batch feedback system at both low (<1mM) and high (40mM) glucose levels with traditional batch culture methods, focusing on glycosylation and glycation of the recombinant protein darbepoetin alfa (DPO) produced by a CHO cell line. We evaluated cell growth, metabolite and product concentration under different glucose feeding strategies and show that continuous feeding, even at low glucose levels, has no harmful effects on DPO quantity and quality. We conclude that our system is capable of tight glucose level control throughout extended bioprocesses and has the potential to improve performance where constant maintenance of glucose levels is critical.

Critical Quality Predictive Control of Fed-Batch Mammalian Cell Bioreactors

Abstract A model predictive control (MPC) formulation for a mammalian cell fed-batch bioreactor processes is developed. A nonlinear fundamental model for the bioreactor is used to generate a database of historical runs comprising of the measurement variables and the manipulated input feed flow rate to the bioreactor. The database is used with subspace identification methods to develop a state-space model of the process. The identified model is used to design various MPC formulations with different objective criteria, including the conventional trajectory-tracking objective function and a novel terminal objective for maximizing the product yield at completion of a run. Case studies involving the simulated bioreactor process demonstrate the efficacy of the MPC algorithms subject to unknown disturbances, random variations in the inlet feed glucose and glutamine concentrations, and measurement noise. Compared to the traditional proportional-integral control algorithm, the trajectory-tracking predictive control algorithm is able to better track the reference glucose concentration set-point with an improvement of 5.1% in the tracking error. The critical quality attribute predictive control algorithm designed to maximize the product yield results in a 3.9% increase in the product concentration at the completion of the run.

Scale-down cultivation in mammalian cell bioreactors—The effect of bioreactor mixing time on the response of CHO cells to dissolved oxygen gradients

Scale-down simulators have been widely applied to investigate the impact of various concentration gradients on the efficiency of industrial-scale fed-batch bioprocesses involving microbial cell factories. However, due to the general fragility and shear sensitivity of mammalian cells, these simulators, especially those with multiple compartments, have not been widely adopted to investigate the effects of similar concentration gradients on cell culture processes. In this work, we developed a multi-compartment scale-down simulator comprising a stirred tank bioreactor and a plug-flow reactor (PFR). CHO cells expressing a recombinant protein were recycled through the PFR whilst being subjected to dissolved oxygen gradients at varying residence times from 70 s to 120 s. The results show a clear cut switch in the metabolism of CHO cells, such that until a residence time of 90s was exceeded, the cells did not respond to the presence of dissolved oxygen gradients in the PFR. Beyond this residence time threshold, there was a marked accumulation of lactate and up to 15 % decline in viable cell density in the stationary phase, compared to the reference fed-batch process. Interestingly, the recombinant protein productivity was not affected by the dissolved oxygen gradients, although the purity of the product, measured as the concentration of host cell proteins in the supernatant was adversely affected. The response time threshold in the metabolism of the cells was used to propose a scale-up technique based on similar physiological indices between bioreactor scales. The demonstrated method is a possible tool for assessing the suitability of a process in a given large scale bioreactor with known mixing times.

AGC to buy an AstraZeneca facility

AGC Biologics is acquiring an idle AstraZeneca biologic drug manufacturing facility in Boulder, Colorado. The plant includes two 20,000 L stainless steel mammalian cell bioreactors and sits on 8 he...

S-Trap Eliminates Cell Culture Media Polymeric Surfactants for Effective Proteomic Analysis of Mammalian Cell Bioreactor Supernatants.

Proteomic analysis of bioreactor supernatants can inform on cellular metabolic status, viability, and productivity, as well as product quality, which can in turn help optimize bioreactor operation. Incubating mammalian cells in bioreactors requires the addition of polymeric surfactants such as Pluronic F68, which reduce the sheer stress caused by agitation. However, these surfactants are incompatible with mass spectrometry proteomics and must be eliminated during sample preparation. Here, we compared four different sample preparation methods to eliminate polymeric surfactants from filtered bioreactor supernatant samples: organic solvent precipitation; filter-assisted sample preparation (FASP); S-Trap; and single-pot, solid-phase, sample preparation (SP3). We found that SP3 and S-Trap substantially reduced or eliminated the polymer(s), but S-Trap provided the most robust cleanup and highest quality data. Additionally, we observed that SP3 sample preparation of our samples and in other published data sets was associated with partial alkylation of cysteines, which could impact the confidence and robustness of protein identification and quantification. Finally, we observed that several commercial mammalian cell culture media and media supplements also contained polymers with similar mass spectrometry profiles, and we suggest that proteomic analyses in these media will also benefit from the use of S-Trap sample preparation.

Probable Scenarios of Process Contamination with Cutibacterium (Propionibacterium) acnes in Mammalian Cell Bioreactor.

Mammalian cell lines constitute an important part in the manufacture of therapeutic proteins. A contamination-free operation of a mammalian cell bioreactor demands constant attention to details. Cutibacterium acnes, a slow-growing bacterium, is a common skin flora and is often associated with mammalian cell bioreactor contaminations. This paper reviews the literature published about such C. acnes contaminations and presents three hypothetical contamination scenarios based on the author's experience in the hope of fostering early detection of contamination events. Bioreactor process parameters such as unusual fluctuation in dissolved oxygen, high levels of ammonia, and microscopic examination have been identified as useful tools to detect slow-growing bacteria contamination.

AGC invests in US, European biologics

AGC will invest a total of almost $20 million in biologics contract manufacturing facilities in the US and Denmark. The Japanese firm will add a 500 L mammalian cell bioreactor in Seattle and a purification line in Copenhagen. The projects are set to be completed next year. AGC, the former Asahi Glass, got the facilities in 2017, when it bought the Danish firm CMC Biologics as part of a push into the life sciences.

A Framework for the Development of Integrated and Computationally Feasible Models of Large-Scale Mammalian Cell Bioreactors

Industrialization of bioreactors has been achieved by applying several core concepts of science and engineering. Modeling has deepened the understanding of biological and physical phenomena. In this paper, the state of existing cell culture models is summarized. A framework for development of dynamic and computationally feasible models that capture the interactions of hydrodynamics and cellular activities is proposed. Operating conditions are described by impeller rotation speed, gas sparging flowrate, and liquid fill level. A set of admissible operating states is defined over discretized process parameters. The burden on a dynamic solver is reduced by assuming hydrodynamics at its fully developed state and implementation of compartmental modeling. A change in the conditions of operation is followed by hydrodynamics switching instantaneously to the steady state that would be reached under new conditions. Finally, coupling the model with optimization solvers leads to improvements in operation.

Fujifilm grows biomanufacturing

Fujifilm will invest $37 million to expand its biomanufacturing business in the U.S. and the U.K. In the U.S., Fujifilm Diosynth Biotechnologies will spend $28 million on mammalian cell bioreactors at a recently opened facility in College Station, Texas. Construction of the $92 million plant was partly financed by the U.S. Biomedical Advanced Research & Development Authority. In Billingham, England, Fujifilm Diosynth will spend $9 million to expand process development capabilities. Fujifilm acquired the Texas facility in 2014 and the Billingham plant in 2011.

A simplified model of glycoprotein production within cell culture

Complex biological products, such as those used to treat various forms of cancer, are typically produced by mammalian cells in bioreactors. The most important class of such biological medicines is proteins. These typically bind to sugars (glycans) in a process known as glycosylation, creating glycoproteins, which are more stable and effective medicines. The glycans are large polymers that are formed by a long sequence of enzyme catalysed reactions. This sequence is not always completed, thus leading to a heterogeneous glycoprotein distribution. A better comprehension of this distribution could lead to more efficient production of high quality drugs. To understand how the manufacturing process can affect the extent of glycosylation of protein, a non-linear ODE model of glycoprotein production is developed which describes the bioreactor configuration as well as the protein production and glycosylation reactions within the cell. The entire system evolves eventually to a stable steady state. The earlier evolution is critical however, as the amount of product produced and its quality varies over time. The model is considered as two coupled systems: the bioreactor submodel and the glycosylation submodel. To investigate the early time evolution within the bioreactor submodel, analytical and numerical properties are derived using matched asymptotic expansions and a finite difference scheme for a range of initial conditions. This leads to qualitatively different regimes for aglycosylated protein production, which affect the glycosylation submodel. The discrete glycoprotein distribution is approximated as continuous and written as a first-order PDE, with good agreement between the discrete and continuous models. The PDE is found to admit shocks, but the existence of these shocks is dependent on the early time evolution within the bioreactor submodel and leads to higher levels of glycosylation at early time. This suggests that changing the bioreactor configuration can lead to higher quality product at certain times.

Review of the important challenges and opportunities related to modeling of mammalian cell bioreactors

Industrialization of mammalian cell culture has been achieved by integrating knowledge from several applying core concepts of chemical engineering, cellular and molecular biology, and biochemistry. Modeling has been applied to biological and physical processes to gain additional insights into such processes. This article covers modeling of the bioreactor and metabolic processes as it applies to bioprocess. Hydrodynamics of a bioreactor is briefly described while additional focus is given to gas‐liquid mass transfer. Biological modeling is presented in the order of increasing complexity. First steady state models are presented followed by dynamic models, cybernetic models, and finally bioreactor integrated models. The closing discussion summarizes challenges of implementation of model‐based approaches in the biopharmaceutical industry. © 2016 American Institute of Chemical Engineers AIChE J, 63: 398–408, 2017

Performance characteristics of qualified cell lines for isolation and propagation of influenza viruses for vaccine manufacturing

Cell culture is now available as a method for the production of influenza vaccines in addition to eggs. In accordance with currently accepted practice, viruses recommended as candidates for vaccine manufacture are isolated and propagated exclusively in hens’ eggs prior to distribution to manufacturers. Candidate vaccine viruses isolated in cell culture are not available to support vaccine manufacturing in mammalian cell bioreactors so egg-derived viruses have to be used. Recently influenza A (H3N2) viruses have been difficult to isolate directly in eggs. As mitigation against this difficulty, and the possibility of no suitable egg-isolated candidate viruses being available, it is proposed to consider using mammalian cell lines for primary isolation of influenza viruses as candidates for vaccine production in egg and cell platforms.To investigate this possibility, we tested the antigenic stability of viruses isolated and propagated in cell lines qualified for influenza vaccine manufacture and subsequently investigated antigen yields of such viruses in these cell lines at pilot-scale. Twenty influenza A and B-positive, original clinical specimens were inoculated in three MDCK cell lines. The antigenicity of recovered viruses was tested by hemagglutination inhibition using ferret sera against contemporary vaccine viruses and the amino acid sequences of the hemagglutinin and neuraminidase were determined. MDCK cell lines proved to be highly sensitive for virus isolation. Compared to the virus sequenced from the original specimen, viruses passaged three times in the MDCK lines showed up to 2 amino acid changes in the hemagglutinin. Antigenic stability was also established by hemagglutination inhibition titers comparable to those of the corresponding reference virus. Viruses isolated in any of the three MDCK lines grew reasonably well but variably in three MDCK cells and in VERO cells at pilot-scale. These results indicate that influenza viruses isolated in vaccine certified cell lines may well qualify for use in vaccine production.

Application of spectroscopic methods for monitoring of bioprocesses and the implications for the manufacture of biologics

The ability to monitor and control bioreactor processes is an integral component to the implementation of Process Analytical Technology and Quality by Design principles. Desirable attributes of monitoring methods include the ability to monitor multiple analytes in real time with little to no sample processing. Spectroscopic methods fit these criteria and significant advancements in their application have been made. However, implementation of these systems has been hampered by their complexity. Here, we present an overview of near IR, mid-IR, Raman and fluorescence spectroscopy technologies, and the steps taken to enable their implementation as effective bioprocess monitoring tools. Specific applications for monitoring of microbial and mammalian cell bioreactors, and screening and classification of raw materials are discussed.

Robust on-line sampling and analysis during long-term perfusion cultivation of mammalian cells

In an attempt to support robust automated sampling and analysis of mammalian cell bioreactors, an integrated platform, BaychroMAT®, was developed which includes an innovative sterile sampling device, automated sample transport, a sample preparation module, online analyzers, and communication interfaces to process automation systems. The robustness of this platform was verified by applying it to a laboratory-scale perfusion bioreactor that was operated for over 100 days. Both manual and automated samples were collected over the course of the run and a comparison was made for cell density, viability, glucose, and lactate concentrations. The highest variability (14.4%) was seen for cell density estimates while those for viability, glucose, and lactate were 0.7, 12.9, and 8.2%, respectively. In addition, cell density set-point changes were made towards the end of the perfusion culture and the high frequency automated samples provided a higher resolution description of the dynamics of cell density change compared to less frequent manual sampling. Overall, our results indicate stable and robust operation of the BaychroMAT® platform in a long-term perfusion culture. This success should readily translate to shorter duration fed-batch cultures thereby enabling feed-back control based on real-time nutrient measurements.

Effective solution chemistry instruction by integrating live pC–pH diagrams into PowerPoint

AbstractpC–pH diagrams provide a graphical illustration of chemical speciation as a function of pH and are hence a very effective tool for understanding the chemical system being studied. This understanding can lead to assumptions that simplify problem solving. However, manual construction of pC–pH diagrams for multiprotic systems is cumbersome and is further complicated by non‐standard temperature and ionic strength conditions. As a result, construction of pC–pH diagrams can become the focus of classroom teaching rather than their application to solving real‐world problems. Students tend to get lost in the complexities of pC–pH diagram construction and often fail to appreciate their value in problem solving. To address this limitation, we have developed interactive pC–pH diagrams for mono, di, and triprotic systems that are all bundled into a single PowerPoint file. The governing equilibrium equations are presented adjacent to the pC–pH diagram which makes the entire process transparent. The diagrams update in real‐time when the total concentration and pKa are changed by the user making them universally applicable to any mono, di, or triprotic system. Temperature impact on pKa and the pC–pH diagram can also be examined for some commonly encountered systems. Because of PowerPoint integration, there is no learning curve and specification of pKa is typically all that is required to use these diagrams. A real‐world application of these interactive pC–pH diagrams is presented by examining the solution chemistry in therapeutic protein‐producing mammalian cell bioreactors. The cause of high bioreactor CO2 concentration and strategies to overcome it were determined just by using the interactive pC–pH diagrams without resorting to numerical calculations. We believe these easy to use and interactive pC–pH diagrams could be more extensively used for problem solving in the classroom simultaneously enhancing student understanding of the chemical system being studied. The PowerPoint file with the pC–pH diagrams is available free of charge for academic use. © 2010 Wiley Periodicals, Inc. Comput Appl Eng Educ 20: 741–749, 2012

Links between metabolism and apoptosis in mammalian cells: Applications for anti-apoptosis engineering

Production of complex recombinant proteins requires the culture of mammalian cells in bioreactors. Inherent in these cultures is the problem of cell death, which can result from nutrient depletion, byproduct accumulation, and other bioreactor stresses which signal the cell to die through apoptosis, or programmed cell death. Apoptosis is a highly regulated pathway of both pro- and anti-apoptotic proteins that promote cell survival or death, and cell engineering efforts to inhibit the apoptosis pathway have led to increased culture viability and recombinant protein production. Originally, the exclusive function of many of these pathway proteins was believed to be binding at the mitochondria and regulating apoptosis through modulation of the mitochondria permeability. While this protein functionality does still hold true, it is now evident that these proteins also include roles in the metabolic processes of the mitochondria. Furthermore, apoptosis pathway proteins in other organelles within the cell may also both modulate apoptosis and metabolism. This review first details the known links that exist between apoptosis proteins and metabolic functions in the cytosol, mitochondria, and endoplasmic reticulum. Second, the review turns to look at potentially new cell engineering strategies that are linked to metabolism for improving cell culture viability and protein production.

Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems

BackgroundAberrant signaling by ErbB-2 (HER 2, Neu), a member of the human Epidermal Growth Factor (EGF) receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen.MethodsHerein, we describe a multi-platform approach for the production of recombinant Single chain Fragments of antibody variable regions (ScFvs) to ErbB-2 that involves their functional expression in (a) bacteria, (b) transient as well as stable transgenic tobacco plants, and (c) a newly developed cell-free transcription-translation system.ResultsAn ScFv (ScFv800E6) was selected by cloning immunoglobulin sequences from murine hybridomas, and was expressed and fully functional in all the expression platforms, thereby representing the first ScFv to ErbB-2 produced in hosts other than bacteria and yeast. ScFv800E6 was optimized with respect to redox synthesis conditions. Different tags were introduced flanking the ScFv800E6 backbone, with and without spacer arms, including a novel Strep II tag that outperforms conventional streptavidin-based detection systems. ScFv800E6 was resistant to standard chemical radiolabeling procedures (i.e. Chloramine T), displayed a binding ability extremely similar to that of the parental monovalent Fab' fragment, as well as a flow cytometry performance and an equilibrium binding affinity (Ka approximately 2 × 108 M-1) only slightly lower than those of the parental bivalent antibody, suggesting that its binding site is conserved as compared to that of the parental antibody molecule. ScFv800E6 was found to be compatible with routine reagents for immunohistochemical staining.ConclusionScFv800E6 is a useful reagent for in vitro biochemical and immunodiagnostic applications in oncology, and a candidate for future in vivo studies.

Morphology of spheroidal hepatocytes within injectable, biodegradable, and thermosensitive poly(organophosphazene) hydrogel as cell delivery vehicle

An aggregate of specific cells is often regarded as a better form of single-cell in artificial organs and mammalian cell bioreactors for improved cell-specific functions. In this study, freshly harvested primary rat hepatocytes, cultivated as spheroids and entrapped in a synthetic thermoreversible extracellular matrix, were examined for differentiation morphology and enhanced liver-specific functions and compared with a control set (single-cell hepatocytes). A copolymer of poly(organophsophazene) hydrogel, an injectable, biodegradable, and thermosensitive matrix, was used to entrap hepatocytes as spheroids or single cells. In a 28-d culture period, the spheroidal hepatocytes maintained a higher viability and produced albumin and urea at constant rates, whereas the single hepatocytes showed a reduction in the level of albumin secretion in the hydrogel. The culturing of spheroidal hepatocytes is a potentially useful three-dimensional cell system for application in bioartificial liver devices and bioreactors.