Concentrations Of Phosphate Salts Research Articles

Purification of secretory IgA monoclonal antibodies enriched fraction directly from cell culture medium using aqueous two-phase systems

Secretory immunoglobulin A [sIgA] is a promising candidate for enteric therapeutics applications, and several sIgA-based constructs are currently being developed by groups utilizing clarified Chinese hamster ovary [CHO] cell culture supernatants. To the monoclonal antibody downstream processing typically entails chromatography-based purification processes beginning with Protein A chromatography. In this paper, aqueous two-phase systems [ATPS] were employed for the preliminary purification of secretory immunoglobulin A [sIgA] monoclonal antibody [mAb] from clarified CHO-cell culture supernatants. A 24 full factorial design was utilized. The influence of various process parameters such as pH, PEG molecular weight [MPEG], PEG concentration [CPEG], and phosphate salt concentration [CPHO], on the sIgA partition coefficient [K sIgA] and the recovery index [Y] in the PEG phase were evaluated. The Elisa assay revealed that, in the ATPS conditions tested, sIgA mAb was mostly detected in PEG upper phase. Run 14 with the highest sIgA activity exhibited the following conditions: MPEG 8.000 g/mol, CPEG 12,5 %, pH 7,0 and CPHO 10 %, and a sIgA K of 94.50 and a recovery index [Y] of 33.52 %. The proposed platform provides straightforward implementation, yields comparable results, and offers significantly improved economics for manufacturing sIgA mAb biotherapeutics.

Optimize purification of a cellulase from Bacillus velezensis A4 by aqueous two-phase system (ATPS) using response surface methodology

Abstract This work aimed to study and optimize the parameters affecting partition of a cellulase from the strain Bacillus velezensis A4 by aqueous two-phase system (ATPS). Four factors, molecular weight of polyethylene glycol (PEG), PEG concentration, phosphate salt concentration and pH, were investigated. A L9(34) orthogonal experiment was initially carried out to screen important factors affecting partitioning of the enzyme in ATPS. The detailed study of the parameters on enzyme partitioning in ATPS was carried out using Box-Behnken (Box-B) design of response surface methodology (RSM). The statistical analysis indicated the molecular weight of PEG, PEG concentration, and pH affect the recovery rate and purification factor of cellulase partitioning significantly. The model was built and analyzed through RSM analysis and validated by our experiment. The maximum recovery rate of enzyme (67.8%) and purification factor (1.14) could be achieved under the conditions of PEG 4000 (20.75%, w/w), K2HPO4 (8.5%, w/w), pH 8.5 in ATPS. The cellulase molecular weight was 35 kDa exhibited in zymogram and has an optimum temperature and pH at 60 °C and 5.0.

Effect of Phosphate Salt Concentration and Solution pH on the Aqueous‐Phase Homo and Copolymerization of N‐Vinyl Pyrrolidone

Abstract N‐vinyl pyrrolidone (NVP) aqueous‐phase polymerization is known to depend on the degree of hydration of the NVP molecules, resulting in an increase in the rate of propagation with decreasing monomer concentration. When potassium monophosphate and sodium hexametaphosphate are added to the monomer solution the reaction kinetics slow down, with the degree of retardation being dependent on salt‐to‐monomer concentration ratio. Using polymerization reaction models in combination with ion coordination theory, this effect can be associated with an active interaction of the ions in solution with the NVP molecules. When varying the solution pH, an optimum operating zone ranging approximately from pH 4 to pH 7 for the NVP–V‐50 2,2′‐Azobis(2‐methylpropionamidine) dihydrochloride (AAPH) system is found. Potassium persulfate is unable to initiate NVP homopolymerization. The addition of poly(ethylene glycol) diacrylate as a comonomer reduces the kinetic slow‐down caused by the addition of salts or by extreme pH and significantly increases the overall conversion rate.

Elution profile of di-peptides on a sulfonated ethylstyrene-divinylbenzene copolymer resin column by high-performance liquid chromatography.

This study investigates the characteristics of a partially sulfonated ethylstyrene-divinylbenzene copolymer for the separation of di-peptides by high-performance liquid chromatography. Di-peptides (VE, VA, VH, VK, and VR) with different isoelectric points (pI, 4.0 to 9.7) and log P values (-1.63 to -0.72) were used to optimize the separation conditions of the columns packed with sulfonated copolymer resin. The retention factor (k) of the di-peptides on the column with a 0.81 wt% sulfo group content decreased with increasing concentrations of phosphate salts (2.5 - 20 mmol L(-1)) in the mobile phase. The complete separation of the five di-peptides was obtained with a gradient of 10% methanol containing 5 mmol L(-1) NaH2PO4 (pH 4.8) to 50% methanol containing 5 mmol L(-1) Na2HPO4 (pH 8.9) for 60 min at 0.5 mL min(-1) at 50°C. Under the optimal conditions, a good relationship between the k and pI values of the di-peptides, with the exception of VE (pI 4.0), was observed, suggesting that the retention of the di-peptides on the column packed with sulfonated copolymer resin was dependent on the pI value, when it was greater than 5. The log P value also influenced the separation characteristics of the column; peptides possessing the same pI value (6.4 for GH, VH, IH, and FH) showed a higher retention on the column with increasing log P values. In conclusion, the prototype sulfonated ethylstyrene-divinylbenzene copolymer column was applicable for the separation of basic di-peptides, and the separation depended on the pI and hydrophobicity of the di-peptides.

Aqueous two phase extraction of alkaline fungal xylanase in PEG/phosphate system: Optimization by Box–Behnken design approach

Alkaline xylanase produced under submerged fermentation by Aspergillus candidus was extracted in aqueous two phase system (ATPS) composed of PEG 4000/NaH2PO4 system. A Box–Behnken design was adopted to optimize critical factors like PEG molecular weight, PEG concentration and phosphate salt concentration and to enhance purification fold of xylanase. Enzyme purification factor was found maximum in presence of low molecular weight of PEG (4000), intermediate concentration of PEG (8.66% w/w) and high salt concentration (22.4% w/w). Under optimized condition threefold increase of purification factor with partition coefficient 8.41% and 88.10% enzyme yield at top phase was attained. The extracted enzyme was found to be stable at high alkaline pH and activity was stimulated in presence of Mn2+ ions. The ATPS was emerged as an effective alternative for primary purification of xylanase.

Unexpected phosphate salt-catalyzed hydrolysis of glycosidic bonds in model disaccharides: Cellobiose and maltose

Monobasic sodium phosphate salt is shown to unexpectedly catalyze cellobiose and maltose hydrolysis to glucose at temperatures between 90 °C and 120 °C and pH 4 in aqueous solution. A selectivity of up to 80% glucose is achieved, which increases with increasing disaccharide dilution. The catalytic role of the phosphate salt is evident in a comparison of the measured activation energy of the catalyzed process for cellobiose hydrolysis of 59 kJ mol −1 versus that reported for the background reaction of 136 kJ mol −1. The normalized rate of catalysis over background significantly increases further at higher pH. Zero-order dependence of hydrolysis rate on disaccharide concentration as well as a logarithmic dependence of hydrolysis rate on phosphate salt concentration is observed. A heterogeneous catalysis mechanism is proposed, which is supported by the presence of condensed phosphate in reaction solution via 31P NMR spectroscopy. This mechanism involves disaccharide chemisorption and subsequent activation via interactions with surface acid/base sites.

An investigation of phosphate based ECMP electrolyte performance on feature scale planarization

Conventional copper chemical mechanical planarization (CMP) techniques are being pushed to their limits by increasing industrial standards caused by device miniaturization and the use of new materials. There is a need to investigate alternative methods of polishing to maintain and/or improve planarization standards while operating at low downforce. In this study, electrochemical mechanical planarization (ECMP) is considered as an alternative and/or an extension to current CMP processes. ECMP is unique due to the combination of an applied voltage to oxidize Cu and an abrasion from a polishing pad, which potentially allows the system to achieve high levels of planarization through the use of an appropriately tailored electrolyte. An electrolyte containing 1.0 M potassium phosphate salt concentration with a pH value of 2 and a benzotriazole (BTA) concentration of 0.001 M was tested for its planarization capability on patterned Cu structures using a custom built ECMP tool. Feature sizes of the Cu structures were varied from 1 to 6 μm. Similar planarization results were achieved using three pad types. All experiments were performed at 0.5 V versus Ag/AgCl reference. The average step height reduction (SHR) was ~840 nm while the decrease in the average metal thickness removed (λavg) was on the order of ~430 nm. Because features were approximately 50% of the substrate area, the total average metal thickness removed was approximately half of the SHR for all three pad types.

Aqueous two-phase extraction (ATPE): An attractive and economically viable technology for downstream processing of Aspergillus oryzae α-galactosidase

Aqueous two-phase systems (ATPSs) have many applications for protein purification in biochemistry and biotechnology. One of the major challenges in the biotechnology industry is the large-scale purification of a desired protein from a fermentation broth containing a wide variety of biomolecules. One possible strategy for addressing this challenge is to use an aqueous two-phase system as the initial primary downstream processing (DSP) for partial purification of industrial enzymes. The aim of this work is to extract and purify Aspergillus oryzae α-galactosidase using an aqueous two-phase system. The α-galactosidase was extracted by partitioning in ATPS composed of polyethylene glycol (PEG) and phosphate. The effects of phase composition, molecular weight of the PEG, PEG concentration, phosphate salt concentration, pH, temperature and neutral salt (sodium chloride) concentration on enzyme partition and purification were studied. The optimal system was found at pH 5.0, containing 12% (w/w) PEG 4000 and 11.9% (w/w) phosphate with a K α-galactosidase of 0.156, purification factor of 3.6 and 87.71% of yield of enzyme activity in the bottom phase. After purification by ATPS, the α-galactosidase optimum pH and temperature were not altered.

An iron-based T 1 contrast agent made of iron-phosphate complexes: In vitro and in vivo studies

A new iron-based T1 contrast agent consisting of a complex of iron ions coordinated to phosphate and amine ligands (Fe(phos) in short) has been characterized by spectroscopic and magnetic measurements. NMR relaxation studies showed r1 values to be dependent on the phosphate salt concentration, K2HPO4, present in the medium. r1 reaches a maximum value of 2.5 mM(-1) s(-1) for measurements carried out at 7 T and 298 K. 31P MRS, Mössbauer spectroscopy and magnetic measurements of Fe(phos) solutions suggest paramagnetic Fe3+ ions present in the studied iron-phosphate complex. In vitro and in vivo toxicity experiments with C6 cells and CD1 mice, respectively, demonstrated lack of toxicity for Fe(phos) at the highest dose tested in the MRI experiments (12 mM iron for C6 cells and 0.32 mmol iron/kg for mice). Finally, T1 weighted images of brain tumours in mice have shown positive contrast enhancement of Fe(phos) for tumour afflicted regions in the brain.

Optimum conditions for transformed Panax ginseng hairy roots in flask culture.

Panax ginseng hairy roots were transformed by Agrobacterium rhizogenes KTCT 2744. They showed an active branching pattern and fast growth in hormone-free medium, and good growth at 23 degrees C, pH 5.8, 1/2 MS medium, and 3% sucrose. Sucrose provided the highest growth among seven carbon sources tested. Six complex media were also tested. In the combined sugar study, hairy roots grew better on sucrose without glucose or fructose than with glucose or fructose. In the 1/2 MS basal medium, 30 mM in nitrogen and 0.62 mM phosphate salt concentration was the optimum. The growth ratio was maximal at an inoculum size of 0.4% (w/v). Crude saponin and polysaccharide levels were also measured.

Induced Natural Transformation of Acinetobacter calcoaceticus in Soil Microcosms

Factors affecting natural transformation of Acinetobacter calcoaceticus BD413 with homologous chromosomal DNA in a silt loam soil microcosm were investigated. Inducible transformation of declining populations of noncompetent A. calcoaceticus cells was detectable for up to 6 days when a simple carbon source, salts, and freshly added DNA were used. In two different experimental setups, the residence time in soil of induced cells could be increased to either 11 or 24 h before DNA addition without reduced transformation frequency; 200-to 1,000-fold fewer transformants were observed following the addition of water. These observations suggest that A. calcoaceticus remains transformable for several hours after its activation by nutrients in soil. Increasing the levels of phosphate salts significantly enhanced the numbers of transformants without increasing the recipient counts correspondingly. Variable levels of ammonium or divalent cations (Mg(sup2+) and Ca(sup2+)) did not have a similar major influence. Soil moisture content significantly affected the transformation frequency of A. calcoaceticus cells, with a general tendency of higher frequencies in drier soil. A minimal frequency was observed at around 35% soil moisture. The data indicate that A. calcoaceticus cells in soil which cannot be detectably transformed are easily induced by nutrients to undergo natural transformation with chromosomal DNA. Access to nutrients seems to be critical for the development and maintenance of competence in soil, which is also affected by abiotic factors like moisture level and phosphate salt concentration.

Effects of salts and poly(ethylene glycol)-palmitate on the partitioning of proteins and Bacillus subtilis neutral protease in aqueous two-phase systems

Polyethylene glycol (PEG) and dextran, and PEG and potassium phosphate buffer (PPB) create aqueous two-phase systems. In the PEG/dextran aqueous two-phase systems, the partition coefficient of bovine serum albumin (BSA), of which surface charged negatively in the neutral pH region, decreased by adding phosphate salt. However, the partition coefficient of lysozyme, which is charged positively at neutral pH, increased as phosphate salt was added. Bacillus subtilis neutral protease, produced by fermentation, showed increasing partition coefficient in PEG/PPB aqueous two-phase systems with increasing phosphate salt concentration. When PEG-palmitate was substituted for PEG, Bacillus subtilis neutral protease moved to the upper PEG-palmitate phase and was purified to 10.2-fold compared to the crude enzyme. Palmitic acid in PEG-palmitate showed not only high affinity to Bacillus subtilis neutral protease but also an activation effect (factor of 2.5) on the proteolytic activity. This affinity partitioning with palmitic acid is due to the high affinity of palmitic acid in PEG-palmitate for the enzyme via hydrophobic interactions giving increased proteolytic activity after binding. The results of this study show that aqueous two-phase systems are suitable for purifying proteins and enzymes by bulk liquid partitioning, and can also give useful information on the surface properties of proteins.

Liver uptake of 99mTc-pyrophosphate

A 56-yr-old chronic schizophrenic with a prior history of anterior myocardial infarction was hospitalized with a 2-day history of progressive abdominal pain and shortness of breath. Physical examination revealed an agitated male with regular pulse of 100/min, blood pressure of 112/70, respiratory rate of 30/rain, and slightly elevated temperature of 100~ The jugular veins were distended to the level of the jaw and bibasilar rates were ausculted in the lungs. The liver was enlarged, firm, and mildly tender. The chest roentgenogram showed pulmonary interstitial edema and pulmonary venous congestion. On admission, creatine phosphokinase (CPK) was 2464, lactate dehydrogenase (LDH) 1110, glutamic oxalactic transaminase (SGOT) 1210, bilirubin 0.8, and alkaline phosphatase 177. The prothrombin time and partial thromboplastin time were elevated at 22.5 and 45.4 sec, respectively. Serum urea nitrogen (BUN) was 66 with creatinine level 1.9. The EKG showed right bundle branch block, old Q waves in the precordiaI leads, and new Q waves in leads I1, lI1, and AVF. Diagnosis on admission was recent inferior MI, rightand left-sided congestive heart failure, congestive hepatopathy, and acute renal failure. Subsequent CPK isoenzyme fractionation failed to support the diagnosis of acute myocardial infarction, and a 99mTc-pyrophosphate myocardial scintigram was performed on the third hospital day that showed intense labeling of the liver without demonstrable myocardial activity. (Fig. 1). It was postulated that the patient's myocardial infarction had occurred 3-5 days prior to admission, and that the persistent elevation of CPK was due to rhabdomyolysis. Right heart catheterization performed after correction of coagulation parameters with fresh frozen plasma revealed markedly elevated right-sided pressures, with right atrial pressure 23 mm Hg, RV 47/20, and pulmonary artery wedge 28. The cardiac output was 2.8 liter/min. On nitroprusside and dopamine, the hemodynamic parameters improved somewhat with diuresis and concomitant improvement in hepatic function. On the seventh hospital day, the patient developed supraventricular tachycardia, which progressed to ventricular fibrillation. Resuscitation was performed; however, a downhi|l course ensued and the patient expired 3 days later. Permission for autopsy was not granted. This case demonstrates diffuse hepatic labeling by 99mTc-pyrophosphate in a clinical setting with high likelihood of central hepatic necrosis secondary to severe congestive heart failure. Liver uptake of bone imaging agents is unusual; in a study of 250 consecutive bone scans, liver uptake occurred only in 5 cases, 2 of which were associated with metastases and the remaining 3 due to poor radiopharmaceutical quality. ''~ Diffuse hepatic uptake of 99roTe-phosphate compounds has since been shown to occur in massive liver necrosis, 2 in the presence of high serum AI +§ levels, 3 and in amyloidosis. 4 Animal studies have shown elevated liver uptake with increased phosphate salt concentration: and with combination of tin and 99mTc-reagents prior to the addition of the phosphate-chelating agent during radiopharmaceutical preparation. 6 In liver necrosis and amyloidosis, labeling occurs presumably due to phosphate localization at sites of cellular damage by mechanisms to be discussed, while in the other cases, reticuloendothelial uptake of microcolloidal particles is the postulated mechanism: ~ Focal uptake of 99roTe-phosphate compounds by hepatic metastases from adenocarcinoma of the colon, breast carcinoma, oat cell carcinoma of the lung, malignant melanoma, and esophageal squamous cell carcinoma has been reported. 7-~~ A single case of uptake by cholangiocarcinoma has also been described. 8 Tumor localization of phosphate compounds probably