pH excursions impact CHO cell culture performance and antibody N-linked glycosylation

Bioprocess and Biosystems Engineering - pH excursions exist due to frequent base addition and environmental heterogeneity in large-scale bioreactors. Such excursions could lead to suboptimal...     

Calcium and pH homeostasis in neurons during hypoxia and ischemia

One of the important events during hypoxia or ischemia in the brain (or other organs for that matter, including the myocardium) is the accumulation of Ca2+ ions intracellularly. Although various studies have shown various sources of and routes for Ca2+ entry and accumulation, it is clear now that it is likely that there is a multitude rather than a single mechanism for this accumulation. In this review, we highlight this Ca2+ accumulation during low O2 states and discuss some of the mechanisms leading to accumulation for two main reasons: (a) an accumulation of Ca2+ in the cytosol has been proven to be deleterious for cell function although this accumulation of Ca2+ and consequences represent only a limited view of events that can lead to cell injury during such stress and (b) developing therapeutic strategies involving the reduction or elimination of this accumulation depends, by and large, on the mechanism of entry. In addition to reviewing some of these Ca2+ events, we will also review the relation between pH (H+) and Ca2+ since these two ions and their regulation are tied to each other in a major way. For example, extracellular acidosis, which can occur during ischemia, has a remarkable effect on the function of some of the Ca2+ entry routes.     

pH control options for hybridoma cultures

Summary An examination of some of the different methods available for pH control in mammalian cell cultures and the effect of pH on cell productivity has been carried out. We show that pH has a significant effect on the production of monoclonal antibodies by hybridoma cells and hence the importance of pH control. We discovered that the use of alkali to control pH led to increases in osmolarity which significantly reduced cell yields. Strategies designed to decrease the requirement for alkali addition have been tested and are discussed.     

Design of stability at extreme alkaline pH in streptococcal protein G

Protein G (PrtG) is widely used as an affinity-based ligand for the purification of IgG. It would be desirable to improve the resistance of affinity chromatography ligands, such as PrtG, to commercial cleaning-in-place procedures using caustic alkali (0.5 M NaOH). It has been shown that Asn residues are the most susceptible at extreme alkaline pH: here, we show that replacement of all three Asn residues within the IgG-binding domain of PrtG only improves stability towards caustic alkali by about 8-fold. Study of the effects of increasing pH on PrtG by fluorescence and CD shows that the protein unfolds progressively between pH 11.5 and 13.0. Calculation of the variation in electrostatic free energy with pH indicated that deprotonation of Tyr, Lys and Arg side-chains at high pH would destabilize PrtG. Introduction of the triple mutation Y3F/T16I/T18I into PrtG stabilized it by an extra 6.8 kcal/mol and the unfolding of the protein occurred at a pH of about 13, or 1.5 pH units higher than wild type. The results show that strategies for the stabilization of proteins at extreme alkaline pH should consider thermodynamic stabilization that will retain the tertiary structure of the protein and modification of surface electrostatics, as well as mutation of alkali-susceptible residues.     

pH Transitions in ion-exchange systems: role in the development of a cation-exchange process for a recombinant protein

Unexpected transient changes in effluent pH can occur during ion-exchange chromatography. Such changes can occur even if a column that is equilibrated with a buffer receives another solution in the same buffer and of the same pH but of a different salt concentration. An attempt is made to understand the basis for this phenomenon and apply it to the process purification of a recombinant protein on a strong cation-exchange resin. Incomplete column equilibration was eliminated as a possible cause of these effects. Various buffering species and various salt ions were studied at different solution concentrations to investigate pH transitions on strong cation-exchange resins. A further comparison was made between cation-exchange resins with different backbone chemistries. On the basis of these studies, a mechanism is proposed for these phenomena based on competitive equilibria between ions from the buffer salts and H(+)/OH(-) ions. In addition to the equilibria between these ions and the functional groups on the resins, charged groups on the resin backbone were also found to contribute to transient pH changes. The results from this study were applied to the cation-exchange step for a recombinant protein that was sensitive to pH excursions to help maintain activity of the protein during the purification process.     

Models of protein modification in Tris-glycine and neutral pH Bis-Tris gels during electrophoresis: effect of gel pH

The pH of conventional Tris-glycine SDS-PAGE gels during a run is determined to be 9.5, in contrast to Bis-Tris-Mes gels where the pH is 7.2. Concentrations of free acrylamide are determined to be less than 10mM in commercial gels of both types, and it is found that of the major components in these gels, only glycine and protein amine or sulfhydryl functions are likely to react with residual acrylamide during the time frame of typical separations. The addition of acrylamide to sulfhydryl groups on proteins is modeled using glutathione and cysteine at acrylamide concentrations found in the commercial gels. Rate constants are determined for these reactions as well as for reaction with glycine at the pH that proteins will encounter in these gel types. The half-life for glutathione sulfhydryl at 10mM acrylamide and pH 7.2 is more than 4h at room temperature. Rates are significantly lower in Bis-Tris-Mes gels than in Tris-glycine gels, reducing the risk of adventitious protein modification. Commercial Bis-Tris-Mes gels provide a sample reduction buffer at pH 8.5 versus the conventional pH 6.8 of Tris-glycine gels. It is shown that significantly less protein degradation occurs during sample preparation at the higher pH used with Bis-Tris gels.     

酸碱调控污泥厌氧发酵实现乙酸累积及微生物种群变化

摘要：【目的】通过对污泥厌氧发酵pH调控,研究挥发性脂肪酸的累积、产酸微生物种群变化及产氢产乙酸菌群对乙酸产生的贡献。【方法】测定不同pH条件下污泥厌氧发酵过程中挥发性脂肪酸的累积;分别应用末端限制性片段长度多态性（T-RFLP）和荧光原位杂交技术（FISH）分析产酸系统中微生物种群结构的变化及产氢产乙酸菌的数量。【结果】 pH为10.0时,有机酸和乙酸的产率在发酵结束时分别达到652.6 mg COD/g-VS和322.4 mg COD/g-VS,显著高于其它pH条件。T-RFLP结果表明,pH值为12     

Regulation of intracellular pH

The approach that most animal cells employ to regulate intracellular pH (pH(i)) is not too different conceptually from the way a sophisticated system might regulate the temperature of a house. Just as the heat capacity (C) of a house minimizes sudden temperature (T) shifts caused by acute cold and heat loads, the buffering power (beta) of a cell minimizes sudden pH(i) shifts caused by acute acid and alkali loads. However, increasing C (or beta) only minimizes T (or pH(i)) changes; it does not eliminate the changes, return T (or pH(i)) to normal, or shift steady-state T (or pH(i)). Whereas a house may have a furnace to raise T, a cell generally has more than one acid-extruding transporter (which exports acid and/or imports alkali) to raise pH(i). Whereas an air conditioner lowers T, a cell generally has more than one acid-loading transporter to lower pH(i). Just as a house might respond to graded decreases (or increases) in T by producing graded increases in heat (or cold) output, cells respond to graded decreases (or increases) in pH(i) with graded increases (or decreases) in acid-extrusion (or acid-loading) rate. Steady-state T (or pH(i)) can change only in response to a change in chronic cold (or acid) loading or chronic heat (or alkali) loading as produced, for example, by a change in environmental T (or pH) or a change in the kinetics of the furnace (or acid extrudes) or air conditioner (or acid loaders). Finally, just as a temperature-control system might benefit from environmental sensors that provide clues about cold and heat loading, at least some cells seem to have extracellular CO(2) or extracellular HCO(3)(-) sensors that modulate acid-base transport.     

盐、碱胁迫下小冰麦体内的pH及离子平衡

通过混合两种中性盐（NaCl和Na₂SO₄）和两种碱性盐（NaHCO₃和Na₂CO₃）分别模拟出不同强度的盐、碱胁迫条件,对小冰麦苗进行12 d胁迫处理,测定茎叶组织液的pH值及Na⁺、K⁺、Ca²⁺、Cl^-、SO₄^2-、NO₃^-、H₂PO₄-和有机酸等溶质的浓度,以探讨盐、碱两种胁迫下小冰麦体内的pH及离子平衡特点.结果表明：盐、碱胁迫下小冰麦茎叶内的pH值均稳定不变;随胁迫强度的增加,盐胁迫下小冰麦茎叶内有机酸浓度没有明显变化,Cl^-浓度大幅度增加,而碱胁迫下有机酸浓度大幅度增加,Cl^-浓度没有明显变化.盐、碱胁迫下小冰麦茎叶中的阳离子均以Na⁺和K⁺为主,但阴离子的来源明显不同.盐胁迫下无机阴离子对负电荷的贡献起主导作用,其贡献率达61.3%～66.7%;而碱胁迫下,随胁迫强度的增大,有机酸对负离子的贡献率从38.35%上升到61.60%,逐渐成为主导成分.实验结果表明,有机酸积累是小冰麦在碱胁迫下保持体内离子平衡和pH稳定的关键生理响应.     

Identification of a distinct phase during melanogenesis that is sensitive to extracellular pH and ionic strength

The cell line B16/C3 will undergo melanogenesis at a specific time after plating. We have found that this time can be modulated by varying the pH of the culture medium. At high pH levels (8.2–8.6) the onset of melanogenesis occurs in 3 or 4 days, while at lower pH (6.7–7.2) it occurs in 7 or 8 days. Furthermore, the time of onset is also sensitive to the extracellular ionic strength. The addition of sodium lactate, sodium chloride, or any other salt tested delays or blocks completely the onset of melanogenesis. These effects are not simply consequence of growth inhibition, nor can they be correlated with patterns of lactate accumulation. These cells are sensitive to pH or ionic strength after entering the stationary phase just prior to the time of onset of melanogenesis. The existence of a specific pH-and ionic-strength-sensitive phase may provide an important clue to the events responsbile for differentiation in this system.     

Anion transport regulates intracellular pH in renal cortical tissue

The regulation of cell pH by anion transport was examined in suspensions of rabbit renal proximal tubules. Values for cell pH were derived from 14C-labeled 5,5-dimethyloxazolidine-2,4-dione distribution. In buffer with 10 mM/l HCO3-- and gassed with 95% O2/5% CO2, the anion transport inhibitors, 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene and furosemide, raised the cell-to-extracellular pH gradient from 0.23 +/- 0.02 to 0.31 +/- 0.02 and 0.31 +/- 0.03, respectively, but in combination their effects were not additive. Replacement of extracellular Cl-- by NO3-- raised the pH gradient from 0.24 +/- 0.04 to 0.37 +/- 0.05. Neither inhibitor raised the pH gradient in Cl-- -free media. Incubation of suspensions in HCO3-- and CO2-free media raised the pH gradient from 0.18 +/- 0.02 to 0.29 +/- 0.03. Removal of Cl-- in addition to HCO3-- and CO2 raised the pH gradient still further, to 0.36 +/- 0.02. The results demonstrate that two different anion transport inhibitors raise cell pH and the cell-to-extracellular pH gradient in proximal tubules and are consistent with the idea that the mechanism for this effect is inhibition of alkali anion exit from the tubule cell. This process appears to depend on extracellular Cl-- and probably occurs primarily by HCO3-- transport. The results support the concept that alkali anion transport, most probably HCO3-- exit from the peritubular cell border, is an important regulator of cell pH in renal proximal tubule.     

The response of GS-NS0 myeloma cells to pH shifts and pH perturbations

The perceived sensitivity of animal cells to hydrodynamic shear has limited agitation and aeration at large-scale. This makes it difficult to ensure adequate mixing of the vessel contents and may lead to inhomogeneities in operational parameters such as temperature, dissolved oxygen concentration, and especially pH. The effect of pH shifts and pH perturbations on the cellular responses, in batch culture, of a GS-NS0 mouse myeloma cell line, expressing a recombinant antibody, was investigated. In addition, the effect of extreme pH on the structure of the purified antibody product was studied using isoelectric focusing. The fermentation pH value was shifted abruptly from pH 7.3 to pH values ranging from 6.5 to 9.0. Culture pH was maintained at this new value for the remainder of the fermentation. All pH shifts of above 0.2 units caused a transient increase in apoptosis. However, cultures shifted to pH values between 7.0 and 8.0 continued to grow and the apoptotic fraction returned to initial levels. Cultures shifted to pH values above pH 8.0 and below pH 7.0 did not recover resulting in culture death. For example, a shift to pH 8.5 caused accumulation of cells in the G(2)/M phase of the cell cycle followed by apoptotic death. After the pH shift, maximum specific growth rate was observed over the range pH 7.3 to 7.5 and maximum viable cell number was seen at pH 7.3. Maximum volumetric antibody production, resulting from increased culture longevity, was seen at pH 7.0. It was also observed that glucose consumption increased with increasing pH. In a separate set of experiments cells were subjected to a single pH perturbation ranging in duration from 0 to 600 minutes. Exposure of cells to a pH value greater than 8.5 for more than 10 minutes caused a decrease in the proportion of viable cells and induced a lag in cell growth. At very low pH (6.5) similar effects were seen, but only for extended perturbations (600 min). However, after recovery from the pH perturbation, growth, product secretion and metabolism all returned to original levels. Incubation of the antibody, at the range of pH values investigated, indicated no alterations in the structure of the antibody as determined by the isoelectric focusing pattern.     

Harpin modulates the accumulation of salicylic acid by Arabidopsis cells via apoplastic alkalization

It is reported here that salicylic acid (SA) is rapidly taken up by Arabidopsis cells, and its uptake is accompanied by media alkalization and cytosolic acidification, and it is inhibited by the ionophore nigericin, suggesting that its import is linked with that of H+ and driven by a proton gradient. Such import and accumulation declined sharply within a narrow physiological pH range (pH 5.7-6.1), corresponding to a reduction in the [H+] of the media from 1.99 micromol l(-1) to 0.79 micromol l(-1). Following the initial uptake, SA was exported back into the media as free SA against a continued [H+]-dependent import. Since the uptake and accumulation of SA declines sharply within a narrow pH range and cell wall alkalization is an early response during incompatible plant/pathogen interactions, the bacterial elicitor harpin(Pss) was used to investigate how SA transport may be modulated during defence responses. Harpin induced a rapid and sustained alkalization of the cell suspension media, reaching the critical pH (pH 5.9-6.1) at which SA import is inhibited at c. 60 min. Such media alkalization corresponded with a reduction in the SA associated with cells co-treated with harpin, and an inhibition of SA uptake in cells pretreated with harpin. Scavengers of ROS, or compounds which generate H2O2 or NO had little effect on the import or net export of SA, suggesting that media alkalization induced by harpin is sufficient to modulate the kinetics of SA transport.     

Factors affecting short-term variability in sediment pH as a function of marsh elevation in a Virginia mesohaline marsh

An analysis of 5 days of nearly continuously recorded sediment temperature, pH, and radiation measurements along a transect in a mesohaline marsh in Virginia suggests there was a shift in control over short-term pH variability from tidal inundation to radiation with height of the marsh surface at least in the surficial sediments. There was little evidence for tidal control of pH variability at depth in the sediment column. However, biological control of subsurface pH variability was evident both near the tidal creek and in the high marsh. Sediment pH means were generally highest toward the tidal creek and lower in the high marsh. The latter zone had the highest pH variability with diurnal pH excursions up to two units being observed during the experimental period. It is hypothesized that macrophyte transpiration or a series of interlocking mechanisms associated with photosynthesis and microbial activity were responsible for the pH excursions observed at depth in the marsh, since the rapid changes in pH were triggered at sunrise and sunset. The large excursions in sediment pH in the high marsh rhizosphere suggest that geochemical activity may be dynamic over the diurnal cycle.     

A multichannel pH controller for solution culture systems

A multichannelled, computer-controlled system has been developed for maintaining near-constant pH in nutrient solutions. This system will monitor, record and individually maintain pH in up to seven solutions continuously. All components except the software and pump controller were available commercially.In an experiment for which the system was developed, in which six individual solutions were controlled for 20 days, the pH was generally maintained within 0.1 pH unit of the set value. Drift outside this range was generally due to addition of nutrients required to maintain adequate levels. Such drifts were quickly and automatically corrected.This equipment has been subsequently used in several experiments in which it was imperative to maintain pH at a constant level in several solutions simultaneously with actively growing plants over a long period of time.     

Intragastric pH regulates conversion from net acid to net alkaline secretion by the rat stomach

Our previous report showed gastric mucosal surface pH was determined by alkali secretion at intragastric luminal pH 3 but by acid secretion at intragastric pH 5. Here, we question whether regulation of mucosal surface pH is due to the effect of luminal pH on net acid/base secretions of the whole stomach. Anesthetized rats with a gastric cannula were used, the stomach lumen was perfused with weakly buffered saline, and gastric secretion was detected in the gastric effluent with 1) a flow-through pH electrode and 2) a fluorescent pH-sensitive dye (Cl-NERF). During pH 5 luminal perfusion, both pH sensors reported the gastric effluent was acidic (pH 4.79). After perfusion was stopped transiently (stop-flow), net acid accumulation was observed in the effluent when perfusion was restarted (peak change to pH 4.1-4.3). During pH 3 luminal perfusion, both pH sensors reported gastric effluent was close to perfusate pH (3.0-3.1), but net alkali accumulation was detected at both pH sensors after stop-flow (peak pH 3.3). Buffering capacity of gastric effluents was used to calculate net acid/alkaline secretions. Omeprazole blocked acid secretion during pH 5 perfusion and amplified net alkali secretion during pH 3 perfusion. Pentagastrin elicited net acid secretion under both luminal pH conditions, an effect antagonized by somatostatin. We conclude that in the basal condition, the rat stomach was acid secretory at luminal pH 5 but alkaline secretory at luminal pH 3.     

Capturing Intracellular pH Dynamics by Coupling Its Molecular Mechanisms within a Fully Tractable Mathematical Model

We describe the construction of a fully tractable mathematical model for intracellular pH. This work is based on coupling the kinetic equations depicting the molecular mechanisms for pumps, transporters and chemical reactions, which determine this parameter in eukaryotic cells. Thus, our system also calculates the membrane potential and the cytosolic ionic composition. Such a model required the development of a novel algebraic method that couples differential equations for slow relaxation processes to steady-state equations for fast chemical reactions. Compared to classical heuristic approaches based on fitted curves and ad hoc constants, this yields significant improvements. This model is mathematically self-consistent and allows for the first time to establish analytical solutions for steady-state pH and a reduced differential equation for pH regulation. Because of its modular structure, it can integrate any additional mechanism that will directly or indirectly affect pH. In addition, it provides mathematical clarifications for widely observed biological phenomena such as overshooting in regulatory loops. Finally, instead of including a limited set of experimental results to fit our model, we show examples of numerical calculations that are extremely consistent with the wide body of intracellular pH experimental measurements gathered by different groups in many different cellular systems.     

Resistance of Streptococcus bovis to acetic acid at low pH: relationship between intracellular pH and anion accumulation

Streptococcus bovis JB1, an acid-tolerant ruminal bacterium, was able to grow at pHs from 6.7 to 4.5, and 100 mM acetate had little effect on growth rate or proton motive force across the cell membrane. When S. bovis was grown in glucose-limited chemostats at pH 5.2, the addition of sodium acetate (as much as 100 mM) had little effect on the production of bacterial protein. At higher concentrations of sodium acetate (100 to 360 mM), production of bacterial protein declined, but this decrease could largely be explained by a shift in fermentation products (acetate, formate, and ethanol production to lactate production) and a decline in ATP production (3 ATP per glucose versus 2 ATP per glucose). YATP (grams of cells per mole of ATP) was not decreased significantly even by high concentrations of acetate. Cultures supplemented with 100 mM sodium acetate took up [14C]acetate and [14C]benzoate in accordance with the Henderson-Hasselbalch equation and gave similar estimates of intracellular pH. As the extracellular pH declined, S. bovis allowed its intracellular pH to decrease and maintained a relatively constant pH gradient across the cell membrane (0.9 unit). The decrease in intracellular pH prevented S. bovis from accumulating large amounts of acetate anion. On the basis of these results it did not appear that acetate was acting as an uncoupler. The sensitivity of other bacteria to volatile fatty acids at low pH is explained most easily by a high transmembrane pH gradient and anion accumulation.     

Resistance of Streptococcus bovis to acetic acid at low pH: relationship between intracellular pH and anion accumulation.

Streptococcus bovis JB1, an acid-tolerant ruminal bacterium, was able to grow at pHs from 6.7 to 4.5, and 100 mM acetate had little effect on growth rate or proton motive force across the cell membrane. When S. bovis was grown in glucose-limited chemostats at pH 5.2, the addition of sodium acetate (as much as 100 mM) had little effect on the production of bacterial protein. At higher concentrations of sodium acetate (100 to 360 mM), production of bacterial protein declined, but this decrease could largely be explained by a shift in fermentation products (acetate, formate, and ethanol production to lactate production) and a decline in ATP production (3 ATP per glucose versus 2 ATP per glucose). YATP (grams of cells per mole of ATP) was not decreased significantly even by high concentrations of acetate. Cultures supplemented with 100 mM sodium acetate took up [14C]acetate and [14C]benzoate in accordance with the Henderson-Hasselbalch equation and gave similar estimates of intracellular pH. As the extracellular pH declined, S. bovis allowed its intracellular pH to decrease and maintained a relatively constant pH gradient across the cell membrane (0.9 unit). The decrease in intracellular pH prevented S. bovis from accumulating large amounts of acetate anion. On the basis of these results it did not appear that acetate was acting as an uncoupler. The sensitivity of other bacteria to volatile fatty acids at low pH is explained most easily by a high transmembrane pH gradient and anion accumulation.     

Cell cycle regulation by environmental pH

Purified populations of quiescent human tumour cells were isolated from plateau phase cultures of PMC-22 cells by centrifugal elutriation. Dilution into fresh medium resulted in these quiescent cells entering S phase exponentially with a t1/2 of 12 hr, after a 18-20-hr lag period during which cellular RNA content increased. Subsequent studies showed that recruitment of quiescent cells into the cell cycle could be regulated by extracellular pH. When exponentially growing PMC-22 cells were exposed to acidic extracellular pH levels, three growth patterns were observed: (1) Normal growth between pH 7.2 to pH 6.8; (2) A reduction in growth rate associated with accumulation of cells with a G₁ DNA content between pH 6.7 and 6.4 (this was also shown to occur in a number of other tumour cell lines); (3) Non-cell-cycle-phase-specific arrest of growth at pH levels less than 6.3. Further studies with purified quiescent cell populations showed the possible existence of a pH-dependent restriction point in the G₁ phase of these tumour cells. The implications of these observations to tumour biology are discussed.