Protons dictate the charge and structure of macromolecules and are used as energy currency by eukaryotic cells. The unique function of individual organelles therefore depends on the establishment and stringent maintenance of a distinct pH. This, in turn, requires a means to sense the prevailing pH and to respond to deviations from the norm with effective mechanisms to transport, produce or consume proton equivalents. A dynamic, finely tuned balance between proton-extruding and proton-importing processes underlies pH homeostasis not only in the cytosol, but in other cellular compartments as well.

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Sensors and regulators of intracellular pH

Regulation of mitochondrial dehydrogenases by calcium ions

In the biopharmaceutical industry, mammalian cell culture systems, especially Chinese hamster ovary (CHO) cells, are predominantly used for the production of therapeutic glycoproteins. Glycosylation is a critical protein quality attribute that can modulate the efficacy of a commercial therapeutic glycoprotein. Obtaining a consistent glycoform profile in production is desired due to regulatory concerns because a molecule can be defined by its carbohydrate structures. An optimal profile may involve a spectrum of product glycans that confers a desired therapeutic efficacy, or a homogeneous glycoform profile that can be systemically screened for. Studies have shown some degree of protein glycosylation control in mammalian cell culture, through cellular, media, and process effects. Studies upon our own bioprocesses to produce fusion proteins and monoclonal antibodies have shown an intricate relationship between these variables and the resulting protein quality. Glycosylation optimization will improve therapeutic efficacy and is an ongoing goal for researchers in academia and industry alike. This review will focus on the advancements made in glycosylation control in a manufacturing process, as well as the next steps in understanding and controlling protein glycosylation.

https://www.actip.org/wp-content/uploads/2015/03/Optimal-and-consistent-protein-glycosylation-in-mammalian-cell-culture.pdf

Optimal and consistent protein glycosylation in mammalian cell culture.

We have seen that there is no simple answer to the question 'what controls respiration?' The answer varies with (a) the size of the system examined (mitochondria, cell or organ), (b) the conditions (rate of ATP use, level of hormonal stimulation), and (c) the particular organ examined. Of the various theories of control of respiration outlined in the introduction the ideas of Chance & Williams (1955, 1956) give the basic mechanism of how respiration is regulated. Increased ATP usage can cause increased respiration and ATP synthesis by mass action in all the main tissues. Superimposed on this basic mechanism is calcium control of matrix dehydrogenases (at least in heart and liver), and possibly also of the respiratory chain (at least in liver) and ATP synthase (at least in heart). In many tissues calcium also stimulates ATP usage directly; thus calcium may stimulate energy metabolism at (at least) four possible sites, the importance of each regulation varying with tissue. Regulation of multiple sites may occur (from a teleological point of view) because: (a) energy metabolism is branched and thus proportionate regulation of branches is required in order to maintain constant fluxes to branches (e.g. to proton leak or different ATP uses); and/or (b) control over fluxes is shared by a number of reactions, so that large increases in flux requires stimulation at multiple sites because each site has relatively little control. Control may be distributed throughout energy metabolism, possibly due to the necessity of minimizing cell protein levels (see Brown, 1991). The idea that energy metabolism is regulated by energy charge (as proposed by Atkinson, 1968, 1977) is misleading in mammals. Neither mitochondrial ATP synthesis nor cellular ATP usage is a unique function of energy charge as AMP is not a significant regulator (see for example Erecinska et al., 1977). The near-equilibrium hypothesis of Klingenberg (1961) and Erecinska & Wilson (1982) is partially correct in that oxidative phosphorylation is often close to equilibrium (apart from cytochrome oxidase) and as a consequence respiration and ATP synthesis are mainly regulated by (a) the phosphorylation potential, and (b) the NADH/NAD+ ratio. However, oxidative phosphorylation is not always close to equilibrium, at least in isolated mitochondria, and relative proximity to equilibrium does not prevent the respiratory chain, the proton leak, the ATP synthase and ANC having significant control over the fluxes. Thus in some conditions respiration rate correlates better with [ADP] than with phosphorylation potential, and may be relatively insensitive to mitochondrial NADH/NAD+ ratio.(ABSTRACT TRUNCATED AT 400 WORDS)

/pdf/control-of-respiration-and-atp-synthesis-in-mammalian-3o3oqgvske.pdf

Control of respiration and ATP synthesis in mammalian mitochondria and cells.

BACKGROUND The decreased incidence of coronary artery disease observed in postmenopausal women given estrogen (E2) replacement demonstrates an atheroprotective effect of E2 that is generally believed to be mediated by indirect, E2-induced changes in cardiovascular risk factor profiles We hypothesized that the atheroprotective effect of E2 may be in part mediated by a direct effect of E2 on vascular smooth muscle cells (VSMCs) Therefore, a series of experiments was performed to determine whether human VSMCs contain a competent E2 receptor, a ligand-activated transcription factor known to mediate E2-induced effects in nonvascular cells METHODS AND RESULTS Ribonuclease protection assays, with a probe derived from the human E2 receptor, were used to demonstrate E2-receptor mRNA in human saphenous vein VSMCs To show that VSMCs contain E2-receptor protein as well as message, immunoblotting and immunofluorescence studies with a monoclonal anti-E2-receptor antibody were performed, and E2-receptor protein was detected by both methods Transient transfection assays using a specific E2-responsive reporter system were used next to determine whether the VSMC E2 receptor is capable of E2-induced transcriptional transactivation Initial studies using mammary artery-derived VSMCs resulted in a 24-fold increase in reporter activity in response to 10(-7) mol/L E2 Subsequent studies using saphenous vein VSMCs demonstrated increasing levels of reporter activation as the concentration of E2 was increased from 10(-9) mol/L (13-fold increase; SEM, 007; P = 05, n = 3) to 10(-7) mol/L (16-fold increase; SEM, 004; P = 002, n = 6) The specificity of the E2-induced transactivation of the reporter gene was shown by dose-dependent inhibition of transactivation by the pure E2 antagonist ICI 164,384 and by enhancement of the transactivation by simultaneous overexpression of the E2 receptor CONCLUSIONS We have demonstrated for the first time that human VSMCs express E2-receptor mRNA and protein and that the E2 receptor in VSMCs is capable of estrogen-dependent gene activation These data suggest a mechanism by which estrogen may directly alter VSMC function

Human vascular smooth muscle cells contain functional estrogen receptor

The enhancement of recombinant protein expression of a transfected cell line is essential for the development of an efficient large-scale bioprocess. The effect of various media additives and temperature conditions were studied in an attempt to optimize protein production, stability, and protein glycosylation from a Chinese hamster ovary (CHO) cell line producing human beta-interferon (Hu-beta-IFN). We observed a decrease in the ELISA response of the glycoprotein in the later stages of batch cultures, which was attributed to molecular aggregation. Cells were subjected to various concentrations of glycerol, dimethyl sulfoxide (DMSO), and sodium butyrate (NaBu) in a variety of culture systems and conditions. The addition of both NaBu and DMSO resulted in higher specific productivities but reduced growth rates that resulted in a net reduction of interferon produced. Glycerol appeared to stabilize the secreted beta-IFN, resulting in reduced aggregation, despite a decrease in cell growth rate. Glycosylation analysis of isolated beta-IFN showed a time-dependent decrease in sialylation in batch culture that was ameliorated by the presence of glycerol. Low-temperature conditions (30 degrees C) had the greatest effect on productivity with a significant increase in beta-IFN titer as well as a reduction in the degree of molecular aggregation.

Enhanced Production of Monomeric Interferon‐β by CHO Cells through the Control of Culture Conditions

Human recombinant erythropoietin (rHuEPO) was produced from Chinese hamster ovary (CHO) cells transfected with the human EPO gene. The cells were grown in batch cultures in controlled bioreactors in which the set-points for dissolved oxygen varied between 3% and 200%. The cell-specific growth rate and final cell yield was significantly lower under hyperoxic conditions (200% DO). However, there was no significant difference in growth rates at other oxygen levels compared to control cultures run under a normoxic condition (50% DO). The specific productivity of EPO was significantly lower at a DO set-point of 3% and 200% but maintained a consistently high value between 10% to 100% DO. The EPO produced under all conditions as analyzed by two-dimensional electrophoresis showed a molecular weight range of 33 to 37 kDa and a low isoelectric point range of 3.5 to 5.0. This corresponds to a highly glycosylated and sialylated protein with a profile showing at least seven distinct isoforms. The glycan pattern of isolated samples of EPO was analyzed by weak anion exchange (WAX) HPLC and by normal-phase HPLC incorporating sequential digestion with exoglycosidase arrays. Assigned structures were confirmed by mass spectrometry (MALDI-MS). The most prominent glycan structures were core fucosylated tetranntenary with variable sialylation. However, significant biantennary, triantennary, and non-fucosylated glycans were also identified. Detailed analysis of these glycan structures produced under variable dissolved oxygen levels did not show consistently significant variations except for the ratio of fucosylated to non-fucosylated isoforms. Maximum core fucosylation (80%) was observed at 50% and 100% DO, whereas higher or lower DO levels resulted in reduced fucosylation. This observation of lower fucosylation at high or low DO levels is consistent with previous data reported for glycoprotein production in insect cells.

The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells

A Chinese hamster ovary (CHO) cell line that expresses human erythropoietin (huEPO) was in a 2-L Cytopilot fluidized-bed bioreactor with 400 mL macroporous Cytoline-1 microcarriers and a variable perfusion rate of serum-free and protein-free medium for 48 days. The cell density increased to a maximum of 23 x 10(6) cells/mL, beads on day 27. The EPO concentration increased to 600 U/mL during the early part of the culture period (on day 24) and increased further to 980 U/mL following the addition of a higher concentration of glucose and the addition of sodium butyrate. The EPO concentration was significantly higher (at least 2x than that in a controlled stirred-tank bioreactor, in a spinner flask, or in a stationary T-flask culture. The EPO accumulated to a total production of 28,000 kUnits over the whole culture period. The molecular characteristics of EPO with respect to size and pattern of glycosylation did not change with scale up. The pattern of utilization and production of 18 amino acids was similar in the Cytopilot culture to that in a stationary batch culture in a T-flask. The concentration of ammonia was maintained at a low level (< 2 mM) over the entire culture period. The specific rate of consumption of glucose, as well as the specific rates of production of lactate and ammonia, were constant throughout the culture period indicating a consistent metabolic behavior of the cells in the bioreactor. These results indicate the potential of the Cytopilot bioreactor culture system for the continuous production of a recombinant protein over several weeks.

Erythropoietin production from CHO cells grown by continuous culture in a fluidized-bed bioreactor.

https://www.jbc.org/content/263/23/11498.full.pdf

The calcium-binding ATPase inhibitor protein from bovine heart mitochondria. Purification and properties.

The murine B-lymphocyte hybridoma, CC9C10 was grown at steady state under serum-free conditions in continuous culture at dissolved oxygen (DO) concentrations in the range of 10% to 150% of air saturation. Cells could be maintained with this range at high viability in a steady state at a dilution rate of 1 d−1, although with lower cell concentrations at higher DO. A higher specific antibody production measured at higher DO was matched by a decrease in the viable cell concentration at steady state, so that the volumetric antibody titre was not changed significantly. An attempt to grow cells at 250% of air saturation was unsuccessful but the cells recovered to normal growth once the DO was decreased.

There was a requirement for cellular adaptation at each step-wise increase in dissolved oxygen. Adaptation to a DO of 100% was associated with an increase in the specific activities of glutathione peroxidase (×18), glutathione S-transferase (×11) and superoxide dismutase (×6) which are all known antioxidant enzymes. At DO above 100%, the activities of GPX and GST decreased possibly as a result of inactivation by reactive oxygen radicals.

The increase in dissolved oxygen concentration caused changes in energy metabolism. The specific rate of glucose uptake increased at higher dissolved oxygen concentrations with a higher proportion of glucose metabolized anaerobically. Short-term radioactive assays showed that the relative flux of glucose through glycolysis and the pentose phosphate pathway increased whereas the flux through the tricarboxylic acid cycle decreased at high DO. Although the specific glutamine utilization rate increased at higher DO, there was no evidence for a change in the pattern of metabolism. This indicates a possible blockage of glycolytic metabolites into the TCA cycle, and is compatible with a previous suggestion that pyruvate dehydrogenase is inhibited by high oxygen concentrations.

Analysis of the oxygen uptake rate of cell suspensions at steady state under all conditions showed a pronounced Crabtree effect which was manifest by a decrease (up to 40%) in oxygen consumption on addition of glucose. This indicates that the degree of aerobic metabolism in these cultures is highly sensitive to the glucose concentration. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 153–164, 1997.

N. Huzel

Papers

Enhanced Production of Monomeric Interferon‐β by CHO Cells through the Control of Culture Conditions

The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells

Erythropoietin production from CHO cells grown by continuous culture in a fluidized-bed bioreactor.

The calcium-binding ATPase inhibitor protein from bovine heart mitochondria. Purification and properties.

The effect of dissolved oxygen on the metabolic profile of a murine hybridoma grown in serum-free medium in continuous culture.