Citrate Buffer Concentration Research Articles

The influence of citrate buffer molarity in mRNA-LNPs: Exploring factors beyond general critical quality attributes

Lipid nanoparticles (LNPs) are crucial in delivering mRNA vaccines and therapeutics. The properties of LNPs can be influenced by the choice of lipids and the manufacturing conditions, such as mixing parameters, lipid concentration, and the type and concentration of the aqueous buffer used. In this study, we investigated the impact of the citrate buffer molarity, the buffer commonly used to dissolve mRNA in the preparation of mRNA-LNPs. We prepared SM-102 LNPs containing firefly luciferase mRNA using citrate buffers at molarities of 50 mM, 100 mM, or 300 mM. Our findings revealed that varying the molarity of the citrate buffer did not significantly affect the particle size when considering the average diameter (z-average or Mode). All formulations exhibited low polydispersity index (PDI) and high encapsulation efficiency. Detailed analysis of particle size sub-populations (D10, D50, and D90) and morphology indicated that citrate buffer concentration might influence lipid packing during LNP production, though these differences were subtle. However, using higher citrate molarity (300 mM) to produce LNPs notably reduced cellular internalisation and in vitro transfection efficiency. This trend was also observed in vivo, where similar expression levels were noted in mice receiving the 50 mM and 100 mM LNP formulations, but lower expression was seen for the 300 mM formulation. Our study highlights the importance of buffer molarity in the aqueous phase during mRNA-based LNP preparation and that generally reported critical quality attributes (CQAs) for LNPs may not detect subtle formulation differences

Tolerance of engineered Rhodosporidium toruloides to sorghum hydrolysates during batch and fed-batch lipid production

BackgroundOleaginous yeasts are a promising candidate for the sustainable conversion of lignocellulosic feedstocks into fuels and chemicals, but their growth on these substrates can be inhibited as a result of upstream pretreatment and enzymatic hydrolysis conditions. Previous studies indicate a high citrate buffer concentration during hydrolysis inhibits downstream cell growth and ethanol fermentation in Saccharomyces cerevisiae. In this study, an engineered Rhodosporidium toruloides strain with enhanced lipid accumulation was grown on sorghum hydrolysate with high and low citrate buffer concentrations.ResultsBoth hydrolysis conditions resulted in similar sugar recovery rates and concentrations. No significant differences in cell growth, sugar utilization rates, or lipid production rates were observed between the two citrate buffer conditions during batch fermentation of R. toruloides. Under fed-batch growth on low-citrate hydrolysate a lipid titer of 16.7 g/L was obtained.ConclusionsCitrate buffer was not found to inhibit growth or lipid production in this engineered R. toruloides strain, nor did reducing the citrate buffer concentration negatively affect sugar yields in the hydrolysate. As this process is scaled-up, $131 per ton of hydrothermally pretreated biomass can be saved by use of the lower citrate buffer concentration during enzymatic hydrolysis.Graphical

Dioscorea alata L anthocyanin extract methanol as a sensitive pH active compound

Anthocyanin from Dioscorea alata L has been successfully extracted using a methanol solvent and has been tested for its sensitivity to pH changes using a UV-Vis spectrophotometer in citrate and phosphate buffer solutions. The extracted anthocyanin showed positive flavonoid and phenol tests. The total yield of crude extract was 1.63% that is related to 3.34 x10−7 M or 16.72 mg/kg dried sample. The maximum absorption against citrate buffer is at a wavelength of 600 nm with a linear range toward pH 4 to 8, sensitivity = 0.121 and R2 = 0.98. Furthermore, the maximum absorption for phosphate buffer occurs at a wavelength of 590 nm that has a response linear in the range of pH 6 to 8 and sensitivity = 0.296 (R2 = 0.97) The optimum concentration of citrate buffer is 0.1M with a linear pH of 4-8 and sensitivity = 0.121 (R2 = 0.98). The optimum buffer concentration for both citrate and phosphate buffer solutions were reached at 0.1M and 0.075 M, respectively. The extracted anthocyanin showed stable response properties against citrate and phosphate buffer for 140 minutes during the test period. This anthocyanin is potential to be used as an active substance to developed optical pH sensors and biosensors based on the determination of pH change.

Production of γ-aminobutyric acid from monosodium glutamate using Escherichia coli whole-cell biocatalysis with glutamate decarboxylase from Lactobacillus brevis KCTC 3498

γ-Aminobutyric acid (GABA), an important fine chemical in pharmacotherapy and food industries, is used as a novel material in the nylon industry and has attracted attention for its potential application in large scale production. Search for new genes and strains, development of efficient reaction systems, such as fermentation and bioconversion, and use of cheap starting material like monosodium glutamate (MSG) can make GABA production using less expensive bulk chemicals possible. Therefore, in this study, we constructed a recombinant Escherichia coli whole-cell system for GABA production that expressed glutamate decarboxylase (GAD) from Lactobacillus brevis and used MSG as the starting material. We also optimized the reaction conditions for MSG to GABA conversion, such as citrate buffer concentration, pyridoxal 5′te concentration, temperature, MSG concentration, and cell density (OD600). The optimized whole-cell system converted MSG to GABA via seven repetitive cycles resulting in an average conversion rate of 86% (71.7 mM/h) within 42 h.

Purification of Pseudomonas sp. proteases through aqueous biphasic systems as an alternative source to obtain bioactive protein hydrolysates.

Aqueous biphasic systems (ABSs) are an interesting alternative for separating industrial enzymes due to easy scale-up and low operational cost. The proteases of Pseudomonas sp. M211 were purified through ABS platforms formed by polyethylene glycol (PEG) and citrate buffer salt. Two experimental designs 23 + 4 were performed to evaluate the following parameters: molar mass of PEG (MPEG ), concentration of PEG (CPEG ), concentration of citrate buffer (CCit ), and pH. The partition coefficient (K), activity yield (Y), and purification factor (PF) were the responses analyzed. The best purification performance was obtained with the system composed of MPEG =10,000 g/mol, CPEG =22 wt%, CCit =12 wt%, pH=8.0; the responses obtained were K=4.9, Y=84.5%, PF=15.1, and tie-line length=52.74%. The purified proteases of Pseudomonas sp. (PPP) were used to obtain hydrolysates of Lupinus mutabilis (Peruvian lupin cultivar) seed protein in comparison with the commercial protease Alcalase® 2.4L. A strong correlation between hydrolysis degree and radical scavenging activity was observed, and the highest antioxidant activity was obtained with Alcalase® (1.40 and 3.47 μmol Trolox equivalent/mg protein, for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and oxygen radical absorbance capacity, respectively) compared with PPP (0.55 and 1.03 μmol Trolox/mg protein). Nevertheless, the IC50 values were lower than those often observed for antioxidant hydrolysates from plant proteins. PEG/citrate buffer system is valuable to purify Pseudomonas proteases from the fermented broth, and the purified protease could be promising to produce antioxidant protein hydrolysates.

Subcutaneous Injection of Drugs: Literature Review of Factors Influencing Pain Sensation at the Injection Site.

The subcutaneous administration route is widely used to administer different types of drugs given its high bioavailability and rapid onset of action. However, the sensation of pain at the injection site might reduce patient adherence. Apart from a direct effect of the drug itself, several factors can influence the sensation of pain: needle features, injection site, volume injected, injection speed, osmolality, viscosity and pH of formulation, as well as the kind of excipients employed, including buffers and preservatives. Short and thin needles, conveniently lubricated and with sharp tips, are generally used to minimize pain, although the anatomic injection site (abdomen versus thigh) also affects the sensation of pain. Large subcutaneous injection volumes are associated with pain. In this sense, the maximum volume generally accepted is around 1.5 ml, although volumes of up to 3 ml are well tolerated when injected in the abdomen. Injected volumes of up to 0.5–0.8 ml are not expected to increase substantially the pain produced by the needle insertion. Ideally, injectable products should be formulated as isotonic solutions (osmolality of about 300 mOsm/kg) and no more than 600 mOs/kg have to be used in order to prevent pain. A pH close to the physiological one is recommended to minimize pain, irritation, and tissue damage. Buffers are frequently added to parenteral formulations to optimize solubility and stability by adjusting the pH; however, their strength should be kept as low as possible to avoid pain upon injection. The data available recommend the concentration of phosphate buffer be limited to 10 mM and that the concentration of citrate buffer should be lower than 7.3 mM to avoid an increased sensation of pain. In the case of preservatives, which are required in multiple-dose preparations, m-cresol seems to be more painful than benzyl alcohol and phenol.Funding: Sandoz SA.

Salt-Induced Thermoresponsivity of a Cationic Phosphazene Polymer in Aqueous Solutions

Poly(ethylaminophosphazene) (PEAP) in salt-free aqueous solution does not reveal thermoresponsive properties but acquires thermoresponsivity upon addition of various poly(protic acid)s. Energetics of the salt-induced phase separation transition of the PEAP solutions upon heating was investigated by high-sensitivity differential scanning calorimetry at pH 3.5. At low concentrations of poly(carboxylic acid)s (10–100 mM), the transition temperature and enthalpy vary from 90 to 25 °C and from 1.5 to 35 J g–1, respectively, depending on the pKa1 and the apparent hydrophobicity of the acids. Dependences of the transition parameters (Tt, Δth, and Δtcp) on the citrate buffer concentration were obtained. The binding curve of citrate anions to PEAP was derived. High concentrations of sulfate anions (0.1–0.8 M) induced a marginal phase transition at high temperatures. A mechanism of the PEAP thermoresponsivity is proposed. The Okado–Tanaka model for cooperative hydration of macromolecules is used to quantitatively d...

The Effect of 3.2% and 3.8% Sodium Citrate on Specialized Coagulation Tests.

- Coagulation testing is challenging and depends on preanalytic factors, including the citrate buffer concentration used. - To better estimate preanalytic effects of the citrate buffer concentration in use, the difference between results obtained by samples with 3.2% and 3.8% citrate was evaluated. - In a prospective observational study with 76 volunteers, differences related to the citrate concentration were evaluated. For both buffer concentrations, reference range intervals were established according to the recommendations of the C28-A3 guideline published by the Clinical and Laboratory Standards Institute. - In our reagent-analyzer settings, most parameters evaluated presented good comparability between citrated samples taken with 3.2% and 3.8% trisodium buffer. The ellagic acid containing activated partial thromboplastin time reagent (aPTT-FS) indicated a systemic and proportional difference between both buffer concentrations, leading to an alteration in its reference ranges. Further, a confirmation test for lupus anticoagulant assessment (Staclot LA) showed only a moderate correlation ( rρ = 0.511) with a proportional deviation between both citrate concentrations. Further, a statistically significant difference was found in the diluted Russell viper venom time confirmation testing, coagulation factors V and VIII, and the protein C activity, which was found to be of minor clinical relevance. - With caution regarding the potential impact of the reagent-analyzer combination, our findings demonstrate the comparability of data assessed with 3.2% and 3.8% buffered citrated plasma. As an exception, the aPTT-FS and the Staclot LA assay were considerably affected by the citrate concentration used. Further studies are required to confirm our finding using different reagent-analyzer combinations.

Absorption of Sulphur Dioxide with Sodium Citrate Buffer Solution in a Rotating Packed Bed

Absorption of SO 2 from a SO 2/air mixture with sodium citrate buffer solution was investigated using a rotating packed bed (RPB) in laboratory scale. The effects of operating parameters, such as the rotation speed of RPB, liquid-gas ratio, inlet gas flow rate, inlet concentration of SO 2 in flue gas, sodium citrate buffer concentration and initial pH of absorption solution, on the SO 2 concentration in the absorption solution or removal efficiency of SO 2 were examined. Incremental rate of sulfate radical ions in the absorption solution was also examined. Experimental results indicate that the efficiency of this regenerative process will be improved by using RPB under appropriate operating conditions, and the generation of SO 2− 4 will be restrained in the process in RPB.

Optimization of key factors affecting hydrogen production from food waste by anaerobic mixed cultures

Key factors (inoculums concentration, substrate concentration and citrate buffer concentration) affecting hydrogen yield (HY) and specific hydrogen production rate (SHPR) from food waste in batch fermentation by anaerobic mixed cultures were optimized using Response Surface Methodology with Central Composite Design. The experiments were conducted in 120 ml serum bottles with a working volume of 70 mL. Under the optimal condition of 2.30 g-VSS/L of inoculums concentration, 2.54 g-VS/L of substrate concentration, and 0.11 M of citrate buffer concentration, the predicted maximum HY and SHPR of 104.79 mL H 2/g-VS added and 16.90 mL H 2/g-VSS.h, respectively, were obtained. Concentrations of inoculums, substrate and citrate buffer all had an individual effect on HY and SHPR ( P < 0.05). The substrate concentration and citrate buffer concentration had the greatest interactive effect on SHPR ( P = 0.0075) while their effects on HY ( P = 0.0131) were profound. These results were reproduced in confirmation experiments under optimal conditions and generated an HY of 104.58 mL H 2/g-VS added and an SHPR of 16.86 mL H 2/g-VSS.h. This was only 0.20% and 0.24%, respectively, different from the predicted values. Microbial community analysis by PCR-DGGE indicated that Clostridium was the pre-dominant hydrogen producer at the optimum and worst conditions. The presence of Lactobacillus sp. and Enterococcus sp. might be responsible for the low HY and SHPR at the worst condition.

Mitochondrial DNA Damage Initiates a Cell Cycle Arrest by a Chk2-associated Mechanism in Mammalian Cells

Previous work from our laboratory has focused on mitochondrial DNA (mtDNA) repair and cellular viability. However, other events occur prior to the initiation of apoptosis in cells. Because of the importance of mtDNA in ATP production and of ATP in fuel cell cycle progression, we asked whether mtDNA damage was an upstream signal leading to cell cycle arrest. Using quantitative alkaline Southern blot technology, we found that exposure to menadione produced detectable mtDNA damage in HeLa cells that correlated with an S phase cell cycle arrest. To determine whether mtDNA damage was causatively linked to the observed cell cycle arrest, experiments were performed utilizing a MTS-hOGG1-Tat fusion protein to target the hOGG1 repair enzyme to mitochondria and enhance mtDNA repair. The results revealed that the transduction of MTS-hOGG1-Tat into HeLa cells alleviated the cell cycle block following an oxidative insult. Furthermore, mechanistic studies showed that Chk2 phosphorylation was enhanced following menadione exposure. Treatment of the HeLa cells with the hOGG1 fusion protein prior to menadione exposure resulted in an increase in the rate of Chk2 dephosphorylation. These results strongly support a direct link between mtDNA damage and cell cycle arrest.

A continuous-monitoring procedure of alkaline phosphatase activity with p-acetylphenylphosphace as substrate

目的 建立以对乙酰基苯基磷酸盐(p-acetylphenylphosphace,PAP-PNa2)为底物的一种新的碱性磷酸酶(ALP)的测定方法.方法 在威图半自动生化分析仪上探讨试验条件,建立试验参数.结果 新方法测定产物对乙酰基酚(PAP)的吸收峰为325 nm,ALP的米氏常数(Km)=0.376mmol/L;340 nm PAP的摩尔消光系数为23 390 L·mol-1·cm-1,缓冲液浓度0.438 mol/L,最适pH值10.4,底物浓度5.0 mmol/L,延迟期60s,线性反应期15 min,线性范围为0～1 110 U/L;Hb＜115.62 mg/L时对测定结果无影响;胆红素对测定结果无影响;本法总ALP的参考范围为男:63.1～118.3 U/L,女:52.5～89.0 U/L.结论 此方法重复性好,线性反应期长,干扰因素少,适于自动生化分析。

Effect of Buffer Species on the Inclusion Complexation of Acidic Drug Celecoxib with Cyclodextrin in Solution

The interaction of celecoxib (Celox) with cyclodextrins (CDs) has been investigated by phase solubility techniques. In this study, the influences of CD type, pH, buffer type, buffer concentration and temperature on the tendency of Celox to form inclusion complexes with CDs were examined. The tendency of Celox to complex with CDs is in the order HP-β-CD > β-CD > γ-CD > α-CD, where the complex formation constants (K11) were 1377, 693, 126 and 60 M−1, respectively. Also ionization of the slightly acidic Celox (pKa=9.7) was found to reduce its tendency to complex (i.e., The K11 values of Celox/β-CD in 0.05 M phosphate buffer were 976 and 210 M−1 for neutral and ionized Celox, respectively). Increasing citrate and phosphate buffer concentration enhances the tendency of ionized Celox to complex with β-CD as a result of a corresponding decrease in the inherent solubility (S0) of the Celox anion. On the other hand, these two buffers interact differently with neutral Celox and β-CD, where increasing phosphate buffer concentration at low pH enhances the complexation of neutral Celox by lowering S0, while increasing citrate buffer concentration at low pH reduces complex formation as citrate buffer species, mainly citric acid, act as a solublizer and a competitor for Celox and β-CD. The contribution of Celox hydrophobicity for complex stability constitutes about 77% of the driving force for complex stability. The complex formation of neutral Celox with β-CD (ΔG0=−28.6 kJ/mol) is driven by both enthalpy (ΔH0=−21.7 kJ/mol) and entropy (ΔS0=23.3 J/mol K) changes.

Sulphur dioxide absorption in air-lift-tube absorbers by sodium citrate buffer solution

Absorption of SO 2 from SO 2/air mixture into sodium citrate buffer solution was performed using air-lift-tube (ALT) absorbers in laboratory and pilot scale. The aim of the study was to improve the efficiency of this regenerative process, to find the proper operation conditions in the air-lift-tube absorbers, and to contribute to the application of this process in the industry. The SO 2-removal efficiency was measured while the gas velocity, the inlet SO 2 concentration, the liquid hold-up, the sodium citrate buffer concentration and the liquid temperature were changed according to experimental design. Furthermore, the influence of oxidation on the SO 2-removal efficiency was measured using SO 2/N 2 mixture instead of SO 2/air mixture; the effect of stoichiometric ratio was investigated and the SO 2 removal in one and two stages of tube absorbers was measured. The performance of the laboratory and pilot scale ALT absorbers was compared with that of other laboratory and pilot size absorption columns and of some non-regenerative methods.

Desorption/ionization mass spectrometry on nanocrystalline titania sol-gel-deposited films.

This paper describes a matrix-free method for performing desorption/ionization directly from mesoporous nanocrystalline titania sol-gel thin films, which have good absorption capacity in the ultraviolet (UV) range and can act as assisting materials during UV matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis. A high concentration of citrate buffer was added into this system to provide the proton source and to reduce the presence of alkali cation adducts of the analytes. The analyte signals appear uniformly over the whole sample deposition area. Protonated molecules (MH(+) ions) of analytes dominate the titania MALDI mass spectra. Surfactants, peptides, tryptic digest products, and small proteins with molecular weights below ca. 24 000 Da, are observed in the titania MALDI mass spectra. Detection limits for insulin are as low as ca. 2 fmol with mass resolution of ca. 660.

Increase of local hydrogen ion gradient near bilayer lipid membrane under the conditions of catalysis of proton transfer across the interface

The measurements of pH profiles in the unstirred layers (USLs) near planar bilayer lipid membranes (BLM) were applied for the evaluation of the hydrogen ion fluxes which were induced by nigericin in the presence of potassium ion gradients. It was shown that at high concentrations of KCl the increase in the concentration of citrate buffer caused an anomalous effect, namely, an increase in the local pH shifts in the USLs. The hydrogen ion flux rose 50 times upon the increase in the citrate concentration from 1 mM to 20 mM. Phosphate stimulated the flux 7 times under these conditions. In agreement with our previous results, at low KCl concentrations, when the process is limited by the K +-nigericin interaction, an increase in the buffer concentration led to a reduction of the local pH shifts, under these conditions the usual concentration dependence was observed. The data obtained favor the model implying the existence of the kinetic barrier for proton transfer at the membrane-water interface.

Capillary zone electrophoretic separation of β-blockers using citrate buffer at low pH

Ten β-blockers were simultaneously separated by capillary zone electrophoresis using citrate buffer as a background electrolyte at low pH. The effects of buffer concentration and buffer pH on the migration behavior and separation of β-blockers were systematically investigated. The results indicate that the resolution of co-migrating analytes improves with increasing buffer concentration at a low pH and that the optimum pH of the buffer decreases with increasing buffer concentration. Complete separation of the β-blockers tested was achieved within a relatively short time with an appropriate concentration of citrate buffer in the range 160–400 m M and a proper pH in the range 3.0-1.8 using a 43 cm×50 μm I.D. fused-silica capillary at 15 kV.

Capillary zone electrophoresis/electrospray ionization mass spectrométry for the characterization of drugs of forensic interest

AbstractSolutions containing between six and thirteen drug substances of forensic interest have been examined by capillary zone electrophoresis/electrospray mass spectrometry (CZE/ES‐MS). The effects of citrate buffer concentration and its content of organic modifiers on the migration time, resolution and migration order of the various components were assessed. Performing CZE/ES‐MS in full‐scan mode at elevated cone (skimmer) voltage allowed differentiation between nalorphine and thebaine which have isomeric structures.

Displacement chromatography on cyclodextrin silicas: V. Separation of the enantiomers of 5, 10-dideazatetrahydrofolic acid

A displacement chromatographic method has been developed for the preparative separation of the enantiomers of 5,10-dideazatetrahydrofolic acid on a β-cyclodextrin silica column. The retention behavior of the chiral solute was studied in detail to find the separation conditions which provide the largest chiral selectivity values: the κ′ vs. acetonitrile modifier concentration, the κ′ vs. pH, the κ′ vs. citrate buffer concentration and the κ′ vs. 1/ T relationships; also, the α vs. acetonitrile concentration, the α vs. pH, the α vs. citrate buffer concentration and the log α vs. 1/ T relationships have been determined. Cetyltrimethylammonium bromide, more retained and more strongly adsorbed than the chiral solute, was selected as displacer for the separation. With an α value of 1.14, good preparative chiral separations were observed both in the displacement mode and in the overloaded elution mode.

Environment and Spray Additive Effects on Picloram Absorption and Translocation in Leafy Spurge (Euphorbia esula)

Relative humidity after application, spray additives, and solution pH affected both foliar absorption and translocation of14C-picloram to leafy spurge roots.14C-picloram absorption increased from 11 to 34% and translocation increased from 5 to 21% as time at posttreatment humidity increased from 0 to 48 h. Absorption and translocation were not different when pre- or posttreatment temperatures were 30/18 or 18/10 C (day/night).14C-picloram absorption and translocation to the roots were 18 and 6%, respectively, when applied alone, and increased to 46 and 12%, respectively, when applied with ammonium sulfate at 2.5 kg/ha. Absorption and translocation were unaffected by ammonium nitrate. Foliar absorption and translocation of14C-picloram in leafy spurge were unaffected by pH of unbuffered spray solution but increased at least 50% when applied in a solution buffered at pH 4.8 with trisodium citrate. Foliar absorption in detached leafy spurge leaves increased from 26 to 51% of applied14C as the citrate buffer concentration increased from 0.01 to 0.1 mM, respectively.