In situ pH maintenance for mammalian cell cultures in shake flasks and tissue culture flasks

pH in animal cell cultures decreases due to production of metabolites like lactate. pH control via measurement and base addition is not easily possible in small‐scale culture formats like tissue‐culture flasks and shake flasks. A hydrogel‐based system is reported for in situ pH maintenance without pH measurement in such formats, and is demonstrated to maintain pH between 6.8 and 7.2 for a suspension CHO cell line in CD CHO medium and between 7.3 and 7.5 for adherent A549 cells in DMEM:F12 containing 10% FBS. This system for pH maintenance, along with our previous report of hydrogels for controlled nutrient delivery in shake flasks can allow shake flasks to better mimic bioreactor‐based fed batch operation for initial screening during cell line and process development for recombinant protein production in mammalian cells. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

pH measurement and a rational and practical pH control strategy for high throughput cell culture system

The number of therapeutic proteins produced by cell culture in the pharmaceutical industry continues to increase. During the early stages of manufacturing process development, hundreds of clones and various cell culture conditions are evaluated to develop a robust process to identify and select cell lines with high productivity. It is highly desirable to establish a high throughput system to accelerate process development and reduce cost. Multiwell plates and shake flasks are widely used in the industry as the scale down model for large‐scale bioreactors. However, one of the limitations of these two systems is the inability to measure and control pH in a high throughput manner. As pH is an important process parameter for cell culture, this could limit the applications of these scale down model vessels. An economical, rapid, and robust pH measurement method was developed at Eli Lilly and Company by employing SNARF‐4F 5‐(‐and 6)‐carboxylic acid. The method demonstrated the ability to measure the pH values of cell culture samples in a high throughput manner. Based upon the chemical equilibrium of CO₂, HCO, and the buffer system, i.e., HEPES, we established a mathematical model to regulate pH in multiwell plates and shake flasks. The model calculates the required %CO₂ from the incubator and the amount of sodium bicarbonate to be added to adjust pH to a preset value. The model was validated by experimental data, and pH was accurately regulated by this method. The feasibility of studying the pH effect on cell culture in 96‐well plates and shake flasks was also demonstrated in this study. This work shed light on mini‐bioreactor scale down model construction and paved the way for cell culture process development to improve productivity or product quality using high throughput systems. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Studies on normal human bone marrow cultures in controlled environment thin-film culture systems

Summary Two thin film culture systems, the controlled environment steady state system (SS) and the rocker tube configuration of that system (RT), were used to identify some of the conditions that appear to maintain morphologic and functional characteristics of cells of human bone marrow explants in vitro. The systems configuration assured continual gassing, control and easy monitoring of the cultures. Cytocentrifuge preparations of media of specimens cultured in RT disclosed, though in decreasing numbers, various hematopoietic cells for periods exceeding one month. Hematopoietic cells shed from specimens cultured in the SS system were retained in the culture tubes; cells of the myelocytic series predominated for the first 2 weeks while an increasing number of monocytes and macrophages appeared in the media of older cultures. Histologic examination of cultured explants disclosed preservation of the marrow architecture and the persistence of hematopoietic cells. Specimens cultured in RT tubes tended to be less cellular than similar cultures placed in dialysis bags or as cultured in the SS system. Immunoglobulins (Ig) were released into the culture media at a constant rate throughout the period of culture. Specimens that were cultured at a controlled pH of 7.4 released 2 to more than 4 times as much Ig as similar specimens maintained at a pH level of 7.1. There were no definitive differences in Ig levels in the cultures maintained at comparable pH levels and overlaid with various CO₂ concentrations, i.e. 2%, 5%, 10%; similarly, no differences in Ig levels were found in specimens cultured in media containing fetal bovine sera as opposed to horse sera. Supported by U.S.P.H.S. Grant CA-5834 from the National Cancer Institute. Department of Medicine A. Department of Cell Physiology Department of Immunology and Immunochemistry.     

Studying the drift of in line pH measurements in cell culture

The culture of Chinese hamster ovary (CHO) cells to produce monoclonal antibodies (MAb) requires accurate measurement and control of pH. Unwanted pH drifts in cell culture can adversely affect process performance, product quality, and product yield. To measure and control pH throughout the length of a culture, most cell culture processes use traditional glass pH probes. Several variables can affect the design and performance of glass pH electrodes and lead to drift in the measurement. Understanding these variables and their effects on pH performance can lead to design improvements and potentially reduce the drift. In this study, a set of Rosemount Analytical glass pH probes was investigated in cell culture operations. Electrochemical properties of the probes were monitored throughout the experiments. Experimental results show that the glass membrane potential experiences the biggest change during cell culture operations. Changes in the reference electrode potential are small compared with the changes in glass membrane potential. The glass membranes are affected by the steam sterilization process and this is the main cause for drift in the probe sensing signal during cell culture operations. Steam sterilization can cause the potential of glass membranes to change by up to 15 mV (～ 0.25 pH units). This change in membrane potential can be observed as an undesirable pH drift in bioreactors.     

Influence of nutrient solution pH on N2O and N2 emissions from a soilless culture system

The influence of nutrient solution pH on the emission of N2O and N2 was investigated during cultivation of cucumbers in a closed-loop rockwool system. Between pH 4 and 7 these gaseous nitrogen losses increased from 1.6 to 21.1% of the N fertilizer input. This was equivalent to average flux rates of 0.06 and 0.85 kg nitrogen per hectare greenhouse area and day, respectively. The N2O/N2 ratio was inversely related to the total gaseous nitrogen losses. At neutral pH dinitrogen was the main emission product, whereas more acidic conditions favoured the emission of nitrous oxide. The pH effects were probably not indirectly affected by root respiration or exudation as much as by a direct inhibition of the activity of denitrifying microorganisms due to high H+ concentrations since similar results were obtained in unplanted nutrient solution systems with the addition of glucose as carbon source. Despite the low microbial denitrification activity under acidic conditions, nitrogen balance deficits of up to one-fifth of the N input still occurred. It is suggested these losses were predominantly caused by chemodenitrification.     

A comparative study of ammonium toxicity at different constatn pH of the nutrient solution

Seedlings of 14 species were grown for 14–28 days on nutrient solution with 6 mmol.l⁻¹ NH₄ as the sole nitrogen source. Solutions acidity was were kept constant at pH 4.0, 5.0, 6.0 and 7.0 by continuous titration with diluted KOH. The following species were used: barley, maize, oats sorghum, yellow and white lupin, pea, soybean, carrot, flax, castor-oil, spinach, sugarbeet and sunflower. Most plant species grew optimally at pH 6.0 with slight reductions at pH 5.0. Growth of many species was severely inhibited at pH 4.0, but this inhibition was not observed with the legume and cereal species. Yield depressions at pH 4.0 relative to pH 6.0 were well correlated with the respective relative decreases of the K concentration in their roots (P<0.002). In the roots of two species (sunflower and flax) total N concentrations were also strongly reduced at pH 4.0. apparently, the interactions between uptake of K, NH₄ and H ions become the prevalent problem at suboptimal pH. At pH 7.0, yields were also considerably decreased, with the exception of the lupines. At this pH, the roots of the growth inhibited plants were characterized by increased levels of total N and free NH₄. It is thought that the binding capacity of the roots for NH₄ is an important factor in preventing NH₄/NH₃ toxicity at supraoptimal pH.     

Use of high culture pH for the decreased proteolysis of human parathyroid hormone secreted by Saccharomyces cerevisiae

Human parathyroid hormone (hPTH) was expressed and secreted in Saccharomyces cerevisiae. In batch fermentations performed at pH = 5.6, 6.5, 7.2 and 7.5, optimal production of hPTH (12.1 mg/l) was obtained at pH 7.2 after 24 h of culture. At pH 5.6, most of secreted hPTH was degraded. Proteolysis of hPTH was significantly decreased by increasing the culture pH.     

pH optima for crop growth

Summary Ginger, cassava, maize, wheat, french bean and tomato were grown for periods up to six weeks in continuously flowing nutrient solutions at seven constant pH values ranging from 3.3 to 8.5. All species achieved maximum or near-maximum growth in the pH range 5.5 to 6.5. However, there were substantial differences in the ability of species to grow outside this range. Ginger and cassava were the most tolerant species to low solution pH, while ginger and tomato were the only species to show no yield depression at the highest solution pH. Roots of all species at pH 3.3 and some species at pH 4.0 exhibited symptoms of hydrogen ion injury. In addition, the concentrations of magnesium in the tops of all six species, of nitrogen in the tops of tomato and cassava, and of manganese in the tops of maize at these pH values were inadequate for optimal growth. Growth depression at high solution pH was associated with iron deficiency in maize and wheat and with nitrogen and/or copper deficiency in cassava.The relevance of the present results to crop growth under field conditions is discussed. The complex interplay of plant and soil characteristics militates against precise definition of an optimum pH range for the growth of a particular crop unless the soil is also specified.     

pH控制下绿色木霉（Trichoderma viride）流加发酵生产纤维素酶

绿色木霉（Trichodermaviride）在pH控制发酵条件下,采用流加葡萄糖发酵策略,可显著提高综合滤纸酶活力（FPA）和内切酶（endo—β—1,4-glucanase,EG）、外切酶exo—β-1,4-glucanase,CBH）、纤维二糖酶（cellobiase,CB）酶活。在5L发酵罐中采用pH控制和流加葡萄糖工艺,可提高CB酶含量,改变酶组分之间的比例,使得FPA、EG、CB和CBH酶活分别达到50．0U／mL,210．0U／mL,4．0U／mL和2．5U／mL,比摇瓶发酵分别提高了6．7．4．2、19、2．5倍。     

Monitoring pH and dissolved oxygen in mammalian cell culture using optical sensors

Here, we have studied two parameters critical to process control in mammalian cell culture; dissolved oxygen (dO₂) and pH, measured with fluorescent sensors thus allowing the study of the metabolic state of cells in culture without removing or damaging cells during cultivation. Two cell lines, namely, NS0 and CHO were batch-grown in 24-well plates at different serum concentrations with the sensors implemented in the bottom of each well. The data showed a good relationship between the dO₂ and pH data obtained from fluorescent probes and the growth and death characteristics of cells. The method has provided a high throughput on-line multi-parametric analysis of mammalian cell cellular activity.     

Product shifting by controlling medium pH in immobilised Streptomyces coelicolor A3(2) culture

Actinorhodin production by Streptomyces coelicolor A3(2) immobilised naturally in a porous support material was investigated in a 20 l bioreactor. The effect of pH-control on actinorhodin fermentation was conducted by performing different strategies. The combination of pH-control and immobilisation of the cells produced a different result than pH-control with freely suspended cells. The control of pH at 7.2 in an immobilised cell system altered the physiology of the cells shifting the product, actinorhodin, to another pigmented secondary metabolite, undecylprodigiosin. Although the cells were biologically active in such an environment, they were unable to produce actinorhodin. In the freely suspended cell system, however, actinorhodin biosynthesis was not hindered by controlling the medium pH.     

分批培养时pH和温度对重组大肠杆菌生产谷胱甘肽合成酶系的影响

研究了2．5L罐分批培养时pH和温度对重组大肠杆菌生产谷胱甘肽合成酶系的影响,确定了分批培养时生产谷胱甘肽合成酶系的最佳pH和最佳温度。研究结果表明：在发酵液的pH为7．2和温度为37℃时,谷胱甘肽合成酶系产量和细胞干重达到最大,分别为690．6U／L和3．77g／L。采用变温控制对菌体的生长和谷胱甘肽合成酶系的合成并没有明显的优点。     

Phytotoxicity of aluminium-fluoride complexes and their uptake from solution culture by Avena sativa and Lycopersicon esculentum

Avena sativa (oats) and Lycopersiconesculentum (tomatoes) were grown in dilute nutrientsolutions supplemented with Al only, F only or acombination of both. In solutions containing Al andF, shoot growth was limited when predicted Al_r(Al³⁺, AlOH²⁺ and Al(OH) )activities were < 0.1 M, activities three orders ofmagnitude lower than the critical value determinedwith Al only. The data suggest that at the activitiesused in these experiments, Al_r is most toxic,AlF²⁺ and AlF are toxic to a lesserextent, and AlF₃, AlF and F^-are least toxic. Fluoride concentrations in shootsgrown in solutions correlated best with positivelycharged AlF species (i.e.AlF²⁺, AlF ) and the molar ratio ofF:Al in most plants shoots was about 3:2. However,when activities of positively chargedAlF species were low (< 50 M)and theactivity of AlF₃ species high (500 M) the molarratio of F:Al in plant shoots suggested AlF₃ wastaken up. These findings are discussed in relation toplant uptake and toxicity. Measured concentrations of Al_r and F^- insolutions containing Al and F were compared withconcentrations predicted by a computer model(GEOCHEM-PC). The method for measuring F^-concentrations, using a non-complexing buffer and Fion-selective electrode, gave good agreement withpredicted F^- concentrations. The8-hydroxyquinoline method for measuring Al_rconcentrations did not agree with predictedconcentrations, highlighting the limitations of thismethod when measuring Al_r in the presence of F ina multi-ligand system with high concentrations of Fand Al.     

Raman spectroscopy as a method to replace off-line pH during mammalian cell culture processes

Raman spectroscopy is a robust, well-established tool utilized for measuring important cell culture process variables for example, feed, metabolites, and biomass in real-time. This study further expands the functionality of in-line Raman spectroscopy coupled with partial least squares (PLS) regression modelling to develop a pH measurement tool. Cell line specific models were developed to enhance the robustness for processes with different pH setpoints, deadbands, and cellular metabolism. The modelling strategy further improved robustness by reducing the temporal complexity of pH shifts by splitting data sets into two time zones reflective of major changes in pH. In addition, models were developed to assess if lactate and partial pressure of carbon dioxide (pCO₂) could be used in a PLS model for pH. Splitting the data sets into early and late for the process resulted in errors of 0.035 pH and 0.034 pH for the two respective Raman cell lines models which was within acceptance criteria. The lactate and pCO₂ PLS model with values provided by Raman models had a further 0.001 pH error reduction. This study illustrates the potential to eliminate off-line samples to correct for in-line measurements of pH and further illustrates the capabilities of Raman to measure additional process variables.     

台湾哈密瓜营养液栽培的生育特性

以台湾哈密瓜(新世纪)为供试品种,通过温室营养液栽培试验,观测其生育特性。结果表明,全生育期为97d;出叶速度呈"S"曲线;叶面积与叶长宽呈线性正相关,单果重与果径呈线性正相关;单株留瓜数与单果重呈负相关。文中还讨论了不同生育时期的栽培要点。     

Continuous-flow NMR culture system for mammalian cells

A continuous-flow NMR culture system for mammalian cells has been developed on which ³¹P-NMR experiments under complete and strictly physiologic conditions have been performed. Observations on the response of the cellular metabolism to stresses such as starvation, low temperature and changes in environmental pH monitored by ³¹P-NMR are reported. The response of the intracellular pH relative to the external pH of the growth medium is studied. We find that under the experimental conditions used there exists a ΔpH varying between less than 0.2 and more than 0.6 pH units. These results are compatible with those obtained using other techniques.     

pH Condition in temperature shift cultivation enhances cell longevity and specific hMab productivity in CHO culture

Controlling cell proliferation during cell culturing is an effective way to improve the production yield in mammalian cell culture. We examined the effect of temperature shifts (TS) under pH control conditions in Chinese hamster ovary cells. When we shifted the culture temperature from 37 °C to 31 °C before a stationary phase at pH 6.8 (TS/pH 6.8), cell viability remained high, and the final human monoclonal antibody (hMab) concentration increased to 2.3 times that in the culture remaining at 37 °C. However, there were no significant effects on the cell viability or production yield with the same TS at pH 7.0 (TS/pH 7.0). The average specific hMab productivity and mRNA level of TS/pH 7.0 were the same as that of TS/pH 6.8. The control of cell growth by the TS or the addition of rapamycin was effective in the maintenance of cell viability, but there was no significant increase of the average specific hMab productivity in the culture where cell proliferation was controlled with rapamycin. The hMab mRNA concentration decreased to 55%–65% at a 37 °C culture with the addition of actinomycin D. In contrast, actinomycin D did not affect the mRNA level in the TS culture. This result suggested that the increase in the mRNA level in the TS condition was caused by an increase in mRNA stability. In this study, we show that TS can produce two unrelated effects: a prolongation of cell longevity and an improvement in mRNA stability.     

老年患者24小时食管pH监测的护理

目的：探讨和总结老年人24小时食管pH监测的有效的护理方法。方法：我病区自2010年6月到2010年12月接受24小时食管pH监测的患者共50例,分为老年组（≥60岁）和非老年组,采用使用电池的动态pH监测仪进行监测。结果：本研究的50例老年患者中,46例置管一次成功,4例因咽部敏感给予2%的利多卡因10ml咽下后第二次均置管成功。本组检查后无一例造成咽部及胃部的损伤,无误吸等并发症发生。结论：24小时食管pH监测是一种安全、简便、无创伤、客观的检查技术,通过动态pH监测,可检测有无胃食管反流,并算出食管真正接触到反流胃酸的时间。正确的操作方法及细致到位的护理可减少老年患者检查时的痛苦,并有效预防并发症的发生。     

Nitrate and ammonium uptake and solution pH changes for Al-tolerant and Al-sensitive sorghum (Sorghum bicolor) genotypes grown with and without aluminium

Plant tolerance to Al toxicity has been associated with differential nitrate and ammonium uptake and solution pH changes. Sorghum [Sorghum bicolor (L.) Moench] genotypes with tolerance (SC283) and sensitivity (ICA-Nataima) to Al toxicity were grown with different nitrate/ammonium ratios (39:1, 9:1, and 3:1) at 0 and 300 μM Al to determine genotypic differences in nitrate and ammonium uptake, changes in nutrient solution pH, and relationships of these traits to Al toxicity tolerance in the genotypes. ICA-Nataima had greater reductions in nitrate and ammonium uptake than SC283 when plants were grown with Al, but SC283 had higher nitrate and ICA-Nataima had higher ammonium uptake when plants were grown without Al. Differences in nitrate and ammonium uptake were associated with changes in solution pH; pH decreased as long as ammonium was in solution and increased when ammonium was depleted from solution. Greater changes in solution pH occurred when plants were grown with 39:1 compared to 9:1 and 3:1 nitrate/ammonium ratios. Solution pH values were lower when plants were grown with than without Al. The genotypes maintained their relative differences in Al toxicity tolerance when plants were grown separately or together in the same container with Al and with different nitrate/ammonium ratios.