Nano Differential Scanning Fluorimetry-Based Thermal Stability Screening and Optimal Buffer Selection for Immunoglobulin G

Nano differential scanning fluorimetry (nanoDSF) is a high-throughput protein stability screening technique that simultaneously monitors protein unfolding and aggregation properties. The thermal stability of immunoglobulin G (IgG) was investigated in three different buffers (sodium acetate, sodium citrate, and sodium phosphate) ranging from pH 4 to 8. In all three buffers, the midpoint temperature of thermal unfolding (Tm) showed a tendency to increase as the pH increased, but the aggregation propensity was different depending on the buffer species. The best stability against aggregation was obtained in the sodium acetate buffers below pH 4.6. On the other hand, IgG in the sodium citrate buffer had higher aggregation and viscosity than in the sodium acetate buffer at the same pH. Difference of aggregation between acetate and citrate buffers at the same pH could be explained by a protein–protein interaction study, performed with dynamic light scattering, which suggested that intermolecular interaction is attractive in citrate buffer but repulsive in acetate buffer. In conclusion, this study indicates that the sodium acetate buffer at pH 4.6 is suitable for IgG formulation, and the nanoDSF method is a powerful tool for thermal stability screening and optimal buffer selection in antibody formulations.

DEGRADATION OF REACTIVE DYES USING IMMOBILIZED PEROXIDASE PURIFIED FROM NIGELLA SATIVA

The present study was aimed to exploit the free and immobilized peroxidase from Nigella sativa seeds to degradation of textile dyes polluting the environment and water. The optimum conditions for extracting the enzyme from the Nigella seeds were determined and the highest specific activity of the enzyme was obtained 1750 units / mg protein when extracting the enzyme from the ground seeds at a ratio of 1:20 (w: v) with sodium acetate buffer at 0.2 M and pH 5.0 for 15 minutes. The enzyme was purified using two steps including the concentration by sucrose and gel filtration by using Sephadex G-150. The results shown an increase in final purification folds 2.8 time with an enzyme yield of 35%. The immobilization of peroxidase were done by entrapment method using Ca- alginate and the immobilization ratio was reached to 49%. The removal efficiency of dyes by crude enzyme (free, immobilized) and partial purified peroxidase were studied with textile dyes such as yellow, red, black and blue dyes at optimum conditions pH 5, temperature 37oC after 3 hr. Maximum removal efficiency of dyes observed with crude peroxidase and reached (76.9, 88.7, 91 and 88) % respectively. These results were close to the efficiency of the purified enzyme in removing the four dyes, while the efficiency of the crude immobilized enzyme in removing the dyes was about (70, 81, 72 and 56.4)%, respectively.

DETERMINATION OF THE OPTIMUM CONDITIONS FOR UREASE INHIBITTION EXTRACTED FROM SOME LOCAL PLANTS

In the current study, four types of plants commonly used namely Soybean, chickpea, bean, pea were obtained and screened for urease activity, among this plants, chickpea was chosen with maximum enzymatic activity, and it had the highest productivity of urease enzyme (1243 U/mg protein). Also sodium acetate buffer (0.2 M, pH 5.0) was chosen as a best extraction buffer with specific activity 1460 U/mg protein. The optimum extraction ratio represented by 1:8 (w:v) after 15 min, it was given 1988 U/mg protein. As well as four types of plants include garlic, red onion, green onion and cabbage were used to select the optimum plant material that inhibited urease enzyme. Cabbage was chosen, it had the highest inhibition activity of the enzyme (41%). Also tris buffer (0.2 M, pH 9) was selected as a best extraction buffer of plants inhibitor with inhibition activity 80%. The optimum extraction ratio represented by 1:8 (w:v) after 60 min, it was given 86% enzyme inhibition activity.

Investigate Retention Behavior of 2-deoxycytidine in Hydrophilic Interaction Liquid Chromatography

A hydrophilic interaction chromatography has been investigated to separate 2-deoxycytidine chosen for nucleoside. A small molecule with specific features for human serum samples was 2-deoxycytidine tested. 2-deoxycytidine has been applied to self-made stationary hydrophilic phases (ZIC1 and ZIC5). The deoxycytidine (dCD) retention was investigated with varying concentrations of sodium acetate buffer, acetonitrile%, and pH. The results confirmed the hydrophilicity of 2-deoxycytidine. The exchanger retention mechanism was studied taking into account 2-deoxycytidine used for describing the interaction of hydrophilic and cation. For both ZIC1 and ZIC5 exchangers, we described and discussed the influence of chromatographic conditions (concentration of sodium acetate buffer, a percent of CAN, and pH). The two methods developed are a useful alternative to current 2-deoxycytidine separation methods.

DEVELOPMENT AND VALIDATION OF A RP-HPLC METHOD FOR DETERMINATION OF ERDAFITINIB IN MARKETED FORMULATIONS

A simple and precise RP-HPLC method for the estimation of Erdafitinib in tablet dosage form was developed and validated. The chromatographic separation of the drug was done with a Hypersil™ ODS C18 Column (150 mm × 4.6 mm i.d., particle size 5 μ) using 20mM sodium acetate buffer (pH 4. ±0.02), methanol and acetonitrile (60:10:30 v/v/v) as a mobile phase. The instrument was set at flow rate of 1.0 mLmin-1 at ambient temperature and the wavelength of UV-visible detector at 310nm. The method showed excellent linearity over a range of 5-35 μgmL-1 for the drug. The correlation coefficient for Erdafitinib was noted to be 0.9999. The mean recovery values were found to be 99.77% and 100.88%. The results suggest that the proposed method could be suitable for quantitative determination of Erdafitinib in pharmaceutical preparations and also for quality control in bulk manufacturing. The F-test and t-test at 95% confidence level were applied on data for statistical analysis.

XRD Identification of Ore Minerals during Cruises: Refinement of Extraction Procedure with Sodium Acetate Buffer

The on-board identification of ore minerals during a cruise is often postponed until long after the cruise is over. During the M127 cruise, 21 cores with deep-seafloor sediments were recovered in the Trans-Atlantic Geotraverse (TAG) field along the Mid Atlantic Ridge (MAR). Sediments were analyzed on-board for physicochemical properties such as lightness (L*), pH and Eh. Selected samples were studied for mineral composition by X-ray powder diffraction (XRD). Based on XRD data, sediment samples were separated into high-, low- and non-carbonated. Removal of carbonates is a common technique in mineralogical studies in which HCl is used as the extraction agent. In the present study, sequential extraction was performed with sodium acetate buffer (pH 5.0) to remove carbonates. The ratio between the highest calcite XRD reflection in the original samples (Iorig) vs its XRD-reflection in samples after their treatment with the buffer (Itreat) was used as a quantitative parameter of calcite removal, as well as to identify minor minerals in carbonated samples (when Iorig/Itreat > 24). It was found that the lightness parameter (L*) showed a positive correlation with calcite XRD reflection in selected TAG samples, and this could be applied to the preliminary on-board determination of extraction steps with acetate buffer (pH 5.0) in carbonated sediment samples. The most abundant minerals detected in carbonated samples were quartz and Al- and Fe-rich clays. Other silicates were also detected (e.g., calcic plagioclase, montmorillonite, nontronite). In non-carbonated samples, Fe oxides and hydroxides (goethite and hematite, respectively) were detected. Pyrite was the dominant hydrothermal mineral and Cu sulfides (chalcopyrite, covellite) and hydrothermal Mn oxides (birnessite and todorokite) were mineral phases identified in few samples, whereas paratacamite was detected in the top 20 cm of the core. The present study demonstrates that portable XRD analysis makes it possible to characterize mineralogy at cored sites, in particular in both low- and high-carbonated samples, before the end of most cruises, thus enabling the quick modification of exploration strategies in light of new information as it becomes available in near-real time.

Fabrication of novel bone haemostasis sheet by using sugar-containing hydroxyapatite and plant-derived polymer

The fabrication conditions of bone-haemostasis sheet were examined by using (i) phosphoryl oligosaccharides of calcium (POs-Ca), sugar-containing hydroxyapatite ( s -Ca 10 (PO 4 ) 6 (OH) 2 : s -HAp) derived from POs-Ca and (ii) natural plant-derived polymers (locust bean gum (LBG), guar gum (GG) and alginate (AG)). The sol, which had been prepared by dissolving 2 mass% LBG/GG and 2 mass% AG into 200 cm 3 deionized water and then by agitating at the speed of 20 000 r.p.m., was immersed into 3 mass% POs-Ca solution at room temperature for 24 h; it was hydrothermally treated at 100°C for 5 h, and then freeze-dried at −50°C for 24 h to form porous composite sheet. The microscopic observation showed that the pore sizes were controlled in the range of 5–100 µm by the optimization of LBG/GG ratio. The composite sheet showed the noted uptake of simulated body fluid (1426%) at 37.0°C and also the human blood. Thus, the porous composite sheet was found to be a promising candidate of the bone haemostasis, on the basis of the data of haemostasis, uptake ability of SBF and solubility in acetic acid–sodium acetate buffer solution.

Formation of Polyaniline and Polypyrrole Nanocomposites with Embedded Glucose Oxidase and Gold Nanoparticles

Several types of polyaniline (PANI) and polypyrrole (Ppy) nanocomposites with embedded glucose oxidase (GOx) and gold nanoparticles (AuNPs) were formed by enzymatic polymerization of corresponding monomers (aniline and pyrrole) in the presence of 6 and 13 nm diameter colloidal gold nanoparticles (AuNPs(6nm) or AuNPs(13nm), respectively) or chloroaurate ions (AuCl4−). Glucose oxidase in the presence of glucose generated H2O2, which acted as initiator of polymerization reaction. The influence of polymerization bulk composition and pH on the formation of PANI- and Ppy-based nanocomposites was investigated spectrophotometrically. The highest formation rate of PANI- and Ppy-based nanocomposites with embedded glucose oxidase and gold nanoparticles (PANI/AuNPs-GOx and Ppy/AuNPs-GOx, respectively) was observed in the solution of sodium acetate buffer, pH 6.0. It was determined that the presence of AuNPs or AuCl4− ions facilitate enzymatic polymerization of aniline and pyrrole.

RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF ASPIRIN AND OMEPRAZOLE IN BINARY COMBINATION

A simple, accurate, rapid and precise RP-HPLC method for the simultaneous estimation of aspirin and Omeprazole in binary mixture has been developed and validated. The drugs were resolved using HPLC column (ACE 250 x 4.6 mm C18 column) with mobile phase of HPLC grade methanol: Sodium acetate buffer (70:30 v/v) at a flow rate of 1 mL/min. The retention times of aspirin and omeprazole were 3.10 ± 0.3min and 5.01 ± 0.02min with UV detection at 230 nm. The method was validated with respect to linearity, sensitivity, accuracy, precision and robustness as per the International Conference on Harmonization (ICH) guidelines. The method was specific and it was observed that no interference with diluents. The linearity was established over the concentration range of 40-140μg/ml and 20-120μg/mL with correlation coefficients (r2) 0.9998 and 0.9986 for aspirin andomeprazole magnesium respectively. The mean recoveries were found to be in the range of 97.05%-99.75% and 97.21% -98.76% for aspirin and omeprazole respectively. The % R.S.D. values for intra-day precision study and inter-day study were <1.0%, confirming that the method is precise. The method can be successfully employed for the simultaneous determination of aspirin and omeprazole.