RESUMEN
Cultivating cells in shake flasks is a routine operation that is largely unchanged since its inception. A glass or plastic Erlenmeyer vessel with the primary gas exchange taking place across various porous plugs is used with media volumes typically ranging from 100 mL to 2 L. Oxygen limitation and carbon dioxide accumulation in the vessel is a major concern for studies involving shake flask cultures. In this study, we enhance mass transfer in a conventional shake flask by replacing the body wall with a permeable membrane. Naturally occurring concentration gradient across the permeable membrane walls facilitates the movement of oxygen and carbon dioxide between the flask and the external environment. The modified flask called the breathable flask, has shown a 40% improvement in mass transfer coefficient (kLa) determined using the static diffusion method. The prokaryotic cell culture studies performed with Escherichia coli showed an improvement of 28%-66% in biomass and 41%-56% in recombinant product yield. The eukaryotic cell culture study performed with Pichia pastoris expressing proinsulin exhibited a 40% improvement in biomass and 115% improvement in protein yield. The study demonstrates a novel approach to addressing the mass transfer limitations in conventional shake flask cultures. The proposed flask amplifies its value by providing a membrane-diffusion-based sensing platform for the integration of low-cost, noninvasive sensing capabilities for real-time monitoring of critical cell culture parameters like dissolved oxygen and dissolved carbon dioxide.
Asunto(s)
Reactores Biológicos , Escherichia coli , Escherichia coli/metabolismo , Fermentación , Pichia/metabolismo , Pichia/crecimiento & desarrollo , Dióxido de Carbono/metabolismo , Oxígeno/metabolismo , Técnicas de Cultivo Celular por Lotes/métodos , BiomasaRESUMEN
Rapid and accurate bioburden detection has become increasingly necessary for food, health, pharmaceutical and environmental applications. To detect bioburden accurately, and in a highly sensitive manner, we have fabricated a novel microfluidic device with an integrated filter to trap the cells. Bioburden is detected on the filter paper in situ using the redox reaction of fluorescent label resorufin and a portable multichannel fluorometer is used for fluorescence measurement. The microfluidic device was fabricated in a facile, low-cost, and rapid way with microwave-induced thermally assisted bonding. To characterize the bonding quality of the microfluidic cassettes, different tests were performed, and the filter paper material and size were optimized. Primary Bacillus subtilis culture bacterial samples were filtered through the device to validate and investigate the performance parameters. Our results show that a limit of detection (LOD) of 0.037 CFU/mL can be achieved through this microfluidic device whereas the LOD in a normal microfluidic cassette in the fluorometer and the golden standard spectrophotometer are 0.378 and 0.128 CFU/mL respectively. The results depict that three to ten times LOD improvement is possible through this microfluidic cassette and more sensitive detection is possible depending on the volume filtered within a rapid 3 min. This novel microfluidic device along with the fluorometer can be used as a rapid portable tool for highly sensitive, accurate and high-throughput bacterial detection for different applications.
Asunto(s)
Técnicas Analíticas Microfluídicas , Microfluídica/métodos , Límite de Detección , Dispositivos Laboratorio en un ChipRESUMEN
Disposable small-scale vessels are commonly used in cell culture studies in academia as well as early stages of bioprocess development. These types of research are crucial for our understanding about cells and bioprocesses as they provide important information regarding different parameters affecting cells. Dissolved carbon dioxide (DCO2) is one main parameter affecting cell metabolism. It is also an indicator of cell culture well-being. Despite CO2 being a critical process parameter, there is a lack of appropriate monitoring system for CO2 in small-scale vessels. Here, we present a membrane-based noninvasive method for measuring DCO2 in cell culture medium. The idea was achieved by modifying a T-flask and replacing a small area of it with CO2 permeable silicone membrane. In the proposed method, the concentration of CO2 dissolved in the cell culture medium is determined by measuring the initial diffusion rate of CO2 through a silicone membrane attached to the bottom wall of the T-flask. The measurement method was validated previously, and the efficacy of the noninvasive method was evaluated by growing E.coli, Pichia pastoris, and CHO cells in the proposed prototype. The results obtained from this method were verified with other quantitative data obtained from the process such as optical density (OD), cell density, dissolved oxygen (DO) and pH. The results show that the proposed membrane-based method is an effective way for completely noninvasive monitoring of DCO2 in small-scale cell culture processes. Additional diffusing species such as oxygen could also be measured using the same approach.
RESUMEN
Contamination detection often requires lengthy culturing steps to detect low-level bioburden. To increase the rate of detection and decrease the limit of detection (LOD), a system featuring microfluidics and a multichannel fluorometer has been developed. The eight-channel fluorometer enables parallel testing of multiple samples with the LOD as low as <1 cfu/mL. This low-cost system utilizes the slope of fluorescence intensity that serves as the criterion for bioburden detection. The redox indicator dye resazurin is used to monitor the presence of viable cells in this study and is reduced to resorufin with a high quantum yield at 585 nm. The sample under investigation is spiked with resazurin and loaded in a special-design microfluidic cassette, and the rate of change is observed via the fluorometer. The method was validated using primary Escherichia coli culture in comparison with a spectrophotometer which served as the gold standard. An optimized assay based on Luria-Bertani medium was developed. The impact on the assay sensitivity based on incubation and filtration steps was also explored. The assay is shown to pick up inadvertent contamination from test tubes and pipette tips showing its applicability in real-world settings. The data analysis demonstrated a comparable performance of the multichannel fluorometer vis-a-vis the conventional plate reader. The multichannel system is shown to detect bioburden presence in as low as 20 s for bacterial concentrations ≥5 cfu/mL after 6 h of incubation. Considering its portability, low cost, simplicity of operation, and relevant assay sensitivity, the system is well positioned to detect low-level bioburden in the laboratory, pharmaceutical, and field settings.
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Filtración , Microfluídica , Contaminación de Medicamentos , Escherichia coli , Límite de DetecciónRESUMEN
Airborne spread of coronavirus disease 2019 (COVID-19) by infectious aerosol is all but certain. However, easily implemented approaches to assess the actual environmental threat are currently unavailable. We present a simple approach with the potential to rapidly provide information about the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the atmosphere at any location. We used a portable dehumidifier as a readily available and affordable tool to collect airborne virus in the condensate. The dehumidifiers were deployed in selected locations of a hospital ward with patients reporting flu-like symptoms which could possibly be due to COVID-19 over three separate periods of one week. Samples were analyzed frequently for both virus envelope protein and SARS-CoV-2 RNA. In several samples across separate deployments, condensate from dehumidifiers tested positive for the presence of SARS-CoV-2 antigens as confirmed using two independent assays. RNA was detected, but not attributable to SARS-CoV-2. We verified the ability of the dehumidifier to rapidly collect aerosolized sodium chloride. Our results point to a facile pool testing method to sample air in any location in the world and assess the presence and concentration of an infectious agent to obtain quantitative risk assessment of exposure, designate zones as "hot spots" and minimize the need for individual testing which may often be time consuming, expensive, and laborious.
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COVID-19/genética , ARN Viral , SARS-CoV-2 , Manejo de Especímenes , COVID-19/epidemiología , COVID-19/transmisión , Humanos , ARN Viral/química , ARN Viral/genética , SARS-CoV-2/química , SARS-CoV-2/genéticaRESUMEN
Mass transfer is known to play a critical role in bioprocess performance and henceforth monitoring dissolved O2 (DO) and dissolved CO2 (dCO2 ) is of paramount importance. At bioreactor level these parameters can be monitored online and can be controlled by sparging air/oxygen or stirrer speed. However, traditional small-scale systems such as shake flasks lack real time monitoring and also employ only surface aeration with additional diffusion limitations imposed by the culture plug. Here we present implementation of intensifying surface aeration by sparging air in the headspace of the reaction vessel and real-time monitoring of DO and dCO2 in the bioprocesses to evaluate the impact of intensified surface aeration. We observed that sparging air in the headspace allowed us to keep dCO2 at low level, which significantly improved not only biomass growth but also protein yield. We expect that implementing such controlled smart shake flasks can minimize the process development gap which currently exists in shake flask level and bioreactor level results.
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Reactores Biológicos/microbiología , Dióxido de Carbono/metabolismo , Oxígeno/metabolismo , Proteínas Recombinantes/metabolismo , Técnicas de Cultivo Celular por Lotes , Biomasa , Escherichia coli/metabolismo , Fermentación , Yarrowia/metabolismoRESUMEN
Dissolved carbon dioxide (dCO2 ) is a well-known critical parameter in bioprocesses due to its significant impact on cell metabolism and on product quality attributes. Processes run at small-scale faces many challenges due to limited options for modular sensors for online monitoring and control. Traditional sensors are bulky, costly, and invasive in nature and do not fit in small-scale systems. In this study, we present the implementation of a novel, rate-based technique for real-time monitoring of dCO2 in bioprocesses. A silicone sampling probe that allows the diffusion of CO2 through its wall was inserted inside a shake flask/bioreactor and then flushed with air to remove the CO2 that had diffused into the probe from the culture broth (sensor was calibrated using air as zero-point calibration). The gas inside the probe was then allowed to recirculate through gas-impermeable tubing to a CO2 monitor. We have shown that by measuring the initial diffusion rate of CO2 into the sampling probe we were able to determine the partial pressure of the dCO2 in the culture. This technique can be readily automated, and measurements can be made in minutes. Demonstration experiments conducted with baker's yeast and Yarrowia lipolytica yeast cells in both shake flasks and mini bioreactors showed that it can monitor dCO2 in real-time. Using the proposed sensor, we successfully implemented a dCO2 -based control scheme, which resulted in significant improvement in process performance.
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Reactores Biológicos , Biotecnología , Dióxido de Carbono , Biotecnología/instrumentación , Biotecnología/métodos , Calibración , Dióxido de Carbono/análisis , Dióxido de Carbono/metabolismo , Diseño de Equipo , Glucosa/metabolismo , Oxígeno/metabolismo , Saccharomyces cerevisiae/metabolismo , Yarrowia/metabolismoRESUMEN
Cell-Free Protein Synthesis (CFPS) offers many advantages for the production of recombinant therapeutic proteins using the CHO cell-free system. However, many complex proteins are still difficult to express using this method. To investigate the current bottlenecks in cell-free glycoprotein production, we chose erythropoietin (40% glycosylated), an essential endogenous hormone which stimulates the development of red blood cells. Here, we report the production of recombinant erythropoietin (EPO) using CHO cell-free system. Using this method, EPO was expressed and purified with a twofold increase in yield when the cell-free reaction was supplemented with CHO microsomes. The protein was purified to near homogeneity using an ion-metal affinity column. We were able to analyze the expressed and purified products (glycosylated cell-free EPO runs at 25-28 kDa, and unglycosylated protein runs at 20 kDa on an SDS-PAGE), identifying the presence of glycan moieties by PNGase shift assay. The purified protein was predicted to have â¼2,300 IU in vitro activity. Additionally, we tested the presence and absence of sugars on the cell-free EPO using a lectin-based assay system. The results obtained in this study indicate that microsomes augmented in vitro production of the glycoprotein is useful for the rapid production of single doses of a therapeutic glycoprotein drug and to rapidly screen glycoprotein constructs in the development of these types of drugs. CFPS is useful for implementing a lectin-based method for rapid screening and detection of glycan moieties, which is a critical quality attribute in the industrial production of therapeutic glycoproteins.
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Biotecnología/métodos , Sistema Libre de Células , Eritropoyetina/metabolismo , Microsomas/metabolismo , Proteínas Recombinantes/metabolismo , Animales , Células CHO , Cromatografía de Afinidad , Cricetulus , Electroforesis en Gel de Poliacrilamida , Eritropoyetina/química , Eritropoyetina/genética , Eritropoyetina/aislamiento & purificación , Expresión Génica , Glicosilación , Humanos , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The use of cell-free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell-free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation, and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFP) and streptokinase in a cell-free system using instrumented mini-bioreactors for highly reproducible protein production. We achieved recombinant protein production (â¼600 µg/ml of tGFP and 500 µg/ml streptokinase) in 2.5 hr of expression time, comparable to previously reported yields for cell-free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare those to a tag-free self-cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose-level production of therapeutic proteins at the point-of-care.
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Sistema Libre de Células , Mezclas Complejas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Estreptoquinasa/biosíntesis , Estreptoquinasa/aislamiento & purificación , Animales , Células CHO , Cricetulus , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/aislamiento & purificación , Proteínas Recombinantes/genética , Estreptoquinasa/genéticaRESUMEN
Manufacturing technologies for biologics rely on large, centralized, good-manufacturing-practice (GMP) production facilities and on a cumbersome product-distribution network. Here, we report the development of an automated and portable medicines-on-demand device that enables consistent, small-scale GMP manufacturing of therapeutic-grade biologics on a timescale of hours. The device couples the in vitro translation of target proteins from ribosomal DNA, using extracts from reconstituted lyophilized Chinese hamster ovary cells, with the continuous purification of the proteins. We used the device to reproducibly manufacture His-tagged granulocyte-colony stimulating factor, erythropoietin, glucose-binding protein and diphtheria toxoid DT5. Medicines-on-demand technology may enable the rapid manufacturing of biologics at the point of care.
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Productos Biológicos/química , Proteínas/química , Animales , Células CHO , Línea Celular , Cricetulus , ADN Ribosómico/química , Eritropoyetina/química , Factor Estimulante de Colonias de Granulocitos/química , Humanos , Sistemas de Atención de PuntoRESUMEN
Cell-free protein synthesis (CFPS) is an ideal platform for rapid and convenient protein production. However, bioreactor design remains a critical consideration in optimizing protein expression. Using turbo green fluorescent protein (tGFP) as a model, we tracked small molecule components in a Chinese Hamster Ovary (CHO) CFPS system to optimize protein production. Here, three bioreactors in continuous-exchange cell-free (CECF) format were characterized. A GFP optical sensor was built to monitor the product in real-time. Mass transfer of important substrate and by-product components such as nucleoside triphosphates (NTPs), creatine, and inorganic phosphate (Pi) across a 10-kDa MWCO cellulose membrane was calculated. The highest efficiency measured by tGFP yields were found in a microdialysis device configuration; while a negative effect on yield was observed due to limited mass transfer of NTPs in a dialysis cup configuration. In 24-well plate high-throughput CECF format, addition of up to 40 mM creatine phosphate in the system increased yields by up to â¼60% relative to controls. Direct ATP addition, as opposed to creatine phosphate addition, negatively affected the expression. Pi addition of up to 30 mM to the expression significantly reduced yields by over â¼40% relative to controls. Overall, data presented in this report serves as a valuable reference to optimize the CHO CFPS system for next-generation bioprocessing. Biotechnol. Bioeng. 2017;114: 1478-1486. © 2017 Wiley Periodicals, Inc.
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Células CHO/metabolismo , Sistema Libre de Células/metabolismo , Biosíntesis de Proteínas/fisiología , Ingeniería de Proteínas/instrumentación , Ingeniería de Proteínas/métodos , Animales , Células CHO/química , Cricetulus , Control de CalidadRESUMEN
Every year, an unacceptably large number of infant deaths occur in developing nations, with premature birth and asphyxia being two of the leading causes. A well-regulated thermal environment is critical for neonatal survival. Advanced incubators currently exist, but they are far too expensive to meet the needs of developing nations. We are developing a thermodynamically advanced low-cost incubator suitable for operation in a low-resource environment. Our design features three innovations: (1) a disposable baby chamber to reduce infant mortality due to nosocomial infections, (2) a passive cooling mechanism using low-cost heat pipes and evaporative cooling from locally found clay pots, and (3) insulated panels and a thermal bank consisting of water that effectively preserve and store heat. We developed a prototype incubator and visited and presented our design to our partnership hospital site in Mysore, India. After obtaining feedback, we have determined realistic, nontrivial design requirements and constraints in order to develop a new prototype incubator for clinical trials in hospitals in India.