Studies of Protein Oxidation as a Product Quality Attribute on a Scale-Down Model for Cell Culture Process Development
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Of importance to the biological properties of proteins produced in cell culture systems are the complex post-translational modifications that are affected by variations in process conditions. Protein oxidation, oxidative modification to intracellular proteins that involves cleavage of the polypeptide chain, and modifications of the amino acid side chains can be affected by such process variations. Dissolved oxygen is a parameter of increasing interest since studies have shown that despite the necessity of oxygen for respiration, there may also be some detrimental effects of oxygen to the cell. Production and accumulation of reactive oxygen species can cause damage to proteins as a result of oxidation of the cell and cellular components. Variation, or changes to cell culture products, can affect function, clearance rate, immunogenicity, and specific activity, which translates into clinical implications. The effect of increasing dissolved oxygen on protein oxidation in immunoglobulin G3–producing mouse hybridoma cells was studied using 50 mL high-throughput mini-bioreactors that employ non-invasive optical sensor technology for monitoring and closed feedback control of pH and dissolved oxygen. Relative protein carbonyl concentration of proteins produced under varying levels of dissolved oxygen was measured by enzyme-linked immunosorbent assay and used as an indicator of oxidative damage. A trend of increasing protein carbonyl content in response to increasing dissolved oxygen levels under controlled conditions was observed. LAY ABSTRACT: Protein oxidation, oxidative modification to intracellular proteins that involves cleavage of the polypeptide chain, and modifications of the amino acid side chains can be affected by variations in dissolved oxygen levels in cell culture systems. Studies have shown that despite the necessity of oxygen for respiration, there may be detrimental effects of oxygen to the cell. Production and accumulation of reactive oxygen species can cause damage to proteins as a result of oxidation of the cell and cellular components, affecting function, clearance rate, immunogenicity, and specific activity, which translates into clinical implications. The effect of increasing dissolved oxygen on protein oxidation in immunoglobulin G3–producing mouse hybridoma cells was studied using 50 mL high-throughput mini-bioreactors that employ non-invasive optical sensor technology for monitoring and closed feedback control of pH and dissolved oxygen. Protein carbonyl concentration of proteins produced under varying levels of dissolved oxygen was measured by enzyme-linked immunosorbent assay and used as an indicator of oxidative damage. A trend of increasing protein carbonyl content in response to increasing dissolved oxygen levels under controlled conditions was observed.Due to the short half-life of reactive oxygen species (ROS) such as a superoxide radical, controlled and localized generation of ROS is challenging. Here, we report a rationally designed small-molecule 1c that generates ROS only when triggered by a bacterial enzyme. We provide evidence for 1c predictably enhancing the intracellular superoxide radical in a model bacterium. Spatiotemporal control over ROS generation offered by 1c should help better understand stress responses in bacteria to increased ROS.
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Uncoupling Agents
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Amino Acid Analysis
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Reactive oxygen species (ROS) have a crucial role in human physiological and pathophysiological processes. Prolonged exposure to high ROS concentrations may lead to cardiovascular, neurodegenerative, etc. diseases. In this study, gallocyanine has been proposed to register the ROS production. The gallocyanine spectral properties changes under ROS (•О2ˉ, H2O2) and reactive halogen (HOCl) species are analyzed. It is shown that the dye is oxidized in solution, both under the action of ROS and reactive halogen species. Based on the data obtained, it is possible to suggest that superoxide anion radicals make a major contribution to chemical conversion of the dye in suspensions of activated neutrophils. It is that gallocyanine can be used to assess the functional activity of neutrophils, namely, the NADPH-oxidase, as well as to design and test novel therapeutic agents for diseases associated with developing oxidative stress.
Reactive nitrogen species
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3T3 cells
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Mitochondria of young seedlings of a wheat hybrid 31MS × 28 exhibited high efficiency of oxidative phosphorylation. The ADP:O ratios estimated polarographically approached or were equivalent to 6, 4, and 5 with α-ketoglutarate, succinate, and malate, respectively. The respiratory control values with these substrates were about 7.5, 4.5, and 2.9. When assayed manometricafly, the P:O ratios with α-ketoglutarate as substrate and 10 min of reaction time were between 5.4 and 5.8. ADP was utilized almost exclusively in oxidative phosphorylation; microbial contamination in phosphorylating reaction mixtures had no measurable effect on oxidative phosphorylation. A concentration of 1.7 × 10 −5 M 2,4-dinitrophenol had no appreciable effect on stimulation of respiration by ADP. ATPase activity was increased about 13% by dinitrophenol. It appears that high efficiency of oxidative phosphorylation is a true characteristic of the mitochondria used in this study.
Dinitrophenol
2,4-Dinitrophenol
Uncoupling Agents
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Intracellular pH of the turtle bladder was measured with fluorescent probe 6-carboxyfluorescein (6-CF) diacetate. In isolated cells this probe provides reliable, reproducible and fast measurements of intracellular pH. The probe was mainly located in the cytosol and thus the values of intracellular pH mainly reflect cytosolic pH. The values of intracellular pH obtained with 6-CF were very similar to those measured with 14C-methylamine and 'null point' technique. The 6-CF technique was capable of detecting small changes in intracellular pH induced by acetazolamide. The intracellular pH of the mitochondrial-rich and granular cell fraction was not different. In addition to assessing intracellular pH of isolated cells, it was possible to monitor the intracellular pH of whole bladders continuously with 6-CF. Addition of CO2 to serosal solution decreased intracellular pH while perfusion with NH3 increased intracellular pH. Thus, 6-CF provides reliable and accurate measurements of intracellular pH in isolated cells and in whole bladders. This technique is capable of detecting rapid changes in intracellular pH and provides continuous monitoring of intracellular pH and thus should allow correlation of changes in urinary acidification with intracellular H+ concentration.
Intracellular pH
Intracellular Fluid
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Reactive oxygen species (ROS) are highly reactive intermediates of oxygen formed during successive reduction ofoxygen. Most common ROS include singlet oxygen 1O2, Superoxide (O2•−), Hydrogen Peroxide (H2O2), and Hydroxylradical (•OH). ROS are produced as by-products of normal aerobic metabolism in plants. However, during biotic orabiotic stress conditions ROS greatly increase in number causing oxidative stress. The highly reactive nature of thesespecies poses a problem for cellular metabolism. Oxidative damage caused by ROS can even lead to cell death. As theproduction of ROS is inevitable, plants have evolved means to eliminate ROS and maintain a reducing environment inthe cell. These strategies involve antioxidant enzymes as well as non-enzymatic antioxidant compounds. Two systemswork together to neutralise reactive species and protect the cell from oxidative injury. A useful aspect of ROS has alsobeen observed in that they act as second messengers in signalling. Ultimately, it is important to maintain equilibriumbetween the production and removal of ROS for a healthy cellular environment. This review deals with mechanisms ofproduction of ROS and strategies of a plant cell to scavenge them.
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