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1.
J Chromatogr A ; 1619: 460943, 2020 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-32061360

RESUMO

Increasing molecular diversity and market competition requires biopharmaceutical manufacturers to intensify their processes. In this respect, frontal chromatography on cation exchange resins has shown its potential to effectively remove aggregates. However, yield losses during the wash step need to be accepted in order to ensure robust product quality. In this work, we present a novel counter-current frontal chromatography process called Flow2, which uses inline dilution during an interconnected wash phase to allow high monomer recovery without contaminating the product pool with impurities. Its model-based design spaces under purity and yield constraints are compared with those corresponding to traditional batch processes in terms of size and process attributes yield and productivity. The Flow2 process shows the largest extent of feasible operating points independent of feed conditions. Thereby, it allows the implementation of higher ionic strength wash, thus widening the range of operating conditions resulting in yields above 95% compared to batch processes. Productivities of batch and counter-current processes are the same at short regeneration times and equal residence time. However, long regeneration times, while influencing the size of the Flow2 design space, are not detrimental for its productivity resulting in twice as high values as obtained for the batch process. Furthermore, process robustness is evaluated by the ability of the process to maintain the required product quality when subjected to process parameter perturbations. It is found that the Flow2 process is able to retain a larger design space associated also with higher yields showing its ability to improve process attributes without sacrificing robustness at the same time.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Química Farmacêutica/métodos , Cromatografia por Troca Iônica/normas , Anticorpos Monoclonais/química , Resinas de Troca de Cátion/química
2.
J Chromatogr A ; 1614: 460720, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31785895

RESUMO

The interest for a better understanding of ion-exchange mechanisms at the atomic level has strongly increased over the past decades. Indeed, molecular-level information about physico-chemical mechanisms could help optimizing chromatographic processes for protein purification, which are sub-optimized due to the lack of predictive models. A promising approach is based on the use of Molecular Dynamics (MD) simulations to study local phenomena inside adsorbents which can then be challenged against experimental results. In this work, macroscopic experimental data, consisting in the ion-exchange uptake of α-chymotrypsin onto SP Sepharose FF, have been compared to the adsorption behavior predicted by MD simulations. The chromatographic surface, represented as a uniform distribution of ligands with a counterion layer, in the presence of the protein was modeled using all-atom representation. The SMA formalism was used to describe single adsorption isotherms and to relate macroscopic observations with molecular simulations. Two SMA parameters based on physical principles, the characteristic charge n and the steric factor σ, have been estimated by both experiments and MD simulations. At pH 5 and NaCl concentration of 100 mM, our study shows a fairly good agreement between both results, especially for the characteristic charge. It is shown that the steric factor calculation is strongly dependent on the ligand density on the adsorbent surface, whose value must be carefully determined in order to obtain reliable predictions. In addition, four binding patches were identified as being involved in the adsorption, which have been confirmed through binding free energy calculations.


Assuntos
Resinas de Troca de Cátion/química , Quimotripsina/química , Simulação de Dinâmica Molecular , Adsorção , Cromatografia por Troca Iônica , Ligantes , Sefarose/química
3.
Artigo em Inglês | MEDLINE | ID: mdl-31812005

RESUMO

Pingyangmycin (PYM) and boanmycin (BAM), two individual components of bleomycin (bleomycin A5 and bleomycin A6), are glycopeptide antitumor antibiotics. An efficient procedure for the preparation of PYM and BAM from Streptomyces verticillus var. pingyangensis fermentation broth using macroporous cation-exchange (MCE) resin followed by medium-pressure preparative liquid chromatography (MPLC) based on monodisperse poly(styrene-co-divinylbenzene) (p(st-dvb)) microspheres was investigated in this paper. Nine frequently used MCE resins were screened by static adsorption and desorption to enrich PYM and BAM fromthe fermentation broth, and D157 resin was found to be the most effective. After one run of column-based dynamic adsorption and desorption, the contents of PYM and BAM were increased by factors of 13.8 and 12.1 with recovery yields of 84.21% and 81.47%, respectively. The enriched samples were subjected to MPLC with columns prepacked with the PolyRP 10-300 microspheres. The operational parameters of the MPLC, including the stationary phase and mobile phase compositions, sample/stationary phase ratio, sample loading scale and flow rate, were screened and optimized. The results showed that the separation and purification for PYM and BAM by MPLC were dramatically improved with a mobile phase modifier of 0.15 mol/L ammonium chloride aqueoussolution, a flow rate of 10 mL/min and a sample/stationary phase ratio of 1.0:100 (m/v, g/mL), and PYM and BAM with purities of more than 98.65% and 99.12% were obtained, respectively. The total recoveries of PYM and BAM reached 75.38% and 70.31%. The separation and purification method is simple, efficient, energy-saving, environmentally friendly and suitable for the large-scale preparation of high-purity PYM and BAM from Streptomyces verticillus var. pingyangensis fermentation broth.


Assuntos
Bleomicina/análogos & derivados , Resinas de Troca de Cátion/química , Cromatografia de Fase Reversa/métodos , Streptomyces/química , Bleomicina/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Fermentação , Reprodutibilidade dos Testes , Streptomyces/metabolismo
4.
J Chromatogr A ; 1610: 460565, 2020 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-31615624

RESUMO

One of the main steps in designing preparative chromatographic separation units is the selection of a well-performing adsorbent. This is often based on expert knowledge or based on case studies of preselected adsorbents. Therefore, the selection is usually limited in terms of an optimised choice. In this contribution, a model-based optimisation of the selection of an adsorbent on the basis of correlations between structural adsorbent properties with model parameters of a transport dispersive model is proposed. Model parameters of glucose and xylose for five cation exchanger resins with varying degree of cross-linking are experimentally determined in a sequential approach. Void fractions and particle porosities were determined by pulse experiments with different tracers. Single-component isotherms were determined threefold via breakthrough curves with concentrations of up to 250 g l-1 at 60 °C. Mass transfer coefficients were determined by batch experiments. Correlations between the degree of cross-linking of the resins and the Henry coefficients as well as the mass transfer coefficients were derived and applied in an optimisation case study. Based on the derived mathematical formula, the process performance of experimentally not investigated resins were predicted. Further, the selection of a resin for a preparative monosaccharide separation was included into optimisation algorithms.


Assuntos
Resinas de Troca de Cátion/química , Reagentes para Ligações Cruzadas/química , Modelos Teóricos , Monossacarídeos/isolamento & purificação , Adsorção , Porosidade , Temperatura
5.
J Chromatogr A ; 1611: 460586, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31606157

RESUMO

It has been known that anion exchangers prepared by grafting poly(ethyleneimine) (PEI) onto Sepharose FF (PEI-Sepharose) at ionic capacities (IC) over 600 mmol/L show both high protein adsorption capacity and uptake kinetics, and charge reversal of PEI-Sepharose by modification with succinic anhydride can produce protein cation exchangers of high capacity and uptake rate. Previously, a Charge Reversal-then-Reduction procedure (route A) was studied for preparation of cation exchangers of different IC values from PEI-Sepharose. In this work, we proposed a new route, that is, Charge Reduction-then-Reversal route (route B), to develop cation exchangers of different IC values from PEI-Sepharose FF with an IC of 700 mmol/L (FF-PEI-L700) as the starting resin. The two kinds of cation exchangers (route A, PEI-L700-CRn; route B, PEI-Rm-Cn) are compared for lysozyme (Lys) adsorption and chromatography. The two modification routes result in the difference in the ligand structures that significantly affect protein adsorption equilibrium and kinetics. Route A introduces long electroneutral groups that hinder protein adsorption and reduce equilibrium capacity. Moreover, charge reversal by reaction with succinic anhydride could cause diamide formation, which reduces remaining carboxyl groups or the IC. In the charge-reduced FF-PEI-Rm resins of the lowest IC (394 mmol/L) prepared in route B, the diamide formation was little due to the lack of primary and secondary amine groups, so its charge reversal makes a higher-IC cation exchanger. This makes PEI-Rm-Cn show a higher IC (589 mmol/L) than PEI-L700-CRn (463 mmol/L) in which De/D0 jumps about four times. The differences in the adsorption equilibrium and kinetics make the two kinds of resins behave distinctly in dynamic adsorption and chromatography. Namely, PEI-Rm-Cn resins display obviously higher dynamic binding capacities than PEI-L700-CRn resins in the IC range studied. For instance, the DBC (at 10% breakthrough) of PEI-R590-C680 (192 mg/mL) is 33% higher than that of PEI-L700-CR680 (144 mg/mL). This is proved by the purification of Lys from chicken egg white solution, in which the PEI-R590-C680 column purified Lys with a recovery yield 14% higher than the PEI-L700-CR680 column. This research thus demonstrated that Charge Reduction-then-Reversal route is superior over Charge Reversal-then-Reduction route in fabricating a high-capacity cation exchanger from PEI-Sepharose.


Assuntos
Resinas de Troca de Cátion/química , Cromatografia por Troca Iônica/métodos , Muramidase/isolamento & purificação , Polietilenoimina/química , Sefarose/química , Adsorção , Cromatografia por Troca Iônica/instrumentação , Cinética , Ligantes , Muramidase/química
6.
Molecules ; 24(24)2019 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-31861157

RESUMO

5-Acetoxymethyl-2-furfural (AMF) was prepared from D-fructose via 1,6-diacetylfructose (DAF) through a simple two-step reaction pathway. Immobilized enzyme (Novozym 435) was found to be the best enzymatic catalyst for the trans-esterification step (yielding 94.6% DAF). In the dehydration step, while soluble H2SO4 was found to be the best acidic catalyst (yielding 86.6% AMF), we opted to utilize heterogeneous cation exchange resin (Amberlyst 15) together with recyclable industrial solvents (1,4-dioxane) for a more sustainable AMF synthesis procedure. Although the total yield of AMF was a little lower, both the enzyme and the solid acid catalyst could be recycled for five cycles without a significant loss of activity, which has a major contribution to the cost-efficient aspect of the entire process.


Assuntos
Resinas de Troca de Cátion/química , Enzimas Imobilizadas , Frutose/química , Furaldeído/química , Lipase/química , Catálise , Desidratação , Esterificação , Solventes/química
7.
Artigo em Inglês | MEDLINE | ID: mdl-31378137

RESUMO

This study evaluates the application of the vegetal activated carbon (AC), vegetable AC impregnated with Ag and Cu (0.08% m/m) and cationic SupergelTM SGC650H resin for adsorption of Fe3+ and Pb2+ ions in closed and batch system. The best adsorption capacities were obtained by using the cationic resin SGC650H, pH 3, temperature of 30 °C and stirring speed of 100 rpm. Thus, the kinetic and equilibrium experiments, in mono- and bicomponent, were performed using SGC650H resin, wherein the kinetic models of pseudo-first and pseudo-second order presented a good fit to the kinetic data, for mono- and bicomponent, respectively. The Langmuir isotherm adequately represented the monocomponent equilibrium data, showing maximum adsorption capacities values of 7.18 and 4.00 meq g-1 for Fe3+ and Pb2+, respectively. An inhibitory effect between the metal species was verified by fitting the modified extended Langmuir isotherm model to the binary equilibrium data, which allowed to predict changes in the surface affinity to the adsorbent by the metal ions. Based on the observed results, the use of SGC650H resin presents great potential for water treatment systems contaminated with heavy metals.


Assuntos
Água Subterrânea , Metais Pesados/química , Poluentes Químicos da Água/química , Purificação da Água/métodos , Adsorção , Resinas de Troca de Cátion/química , Carvão Vegetal/química , Água Subterrânea/química , Concentração de Íons de Hidrogênio , Cinética , Metais Pesados/análise , Temperatura , Poluentes Químicos da Água/análise
8.
Phytochem Anal ; 30(6): 727-734, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31317605

RESUMO

INTRODUCTION: Quaternary alkaloids (QAs) are the major alkaloids in several traditional Chinese medicines, especially in Zanthoxylum simulans. However, few studies on enrichment of QAs from Z. simulans were conducted due to their high polarity and low content. OBJECTIVE: To develop a weak cation exchange (WCX) solid-phase extraction (SPE) coupled with liquid chromatography mass spectrometry (LC-MS) method to enrich and identify QAs from Z. simulans. Meanwhile, the qualitative and quantitative analyses of QAs were carried out based on the optimum conditions of the method. METHODS: Fresh stem bark of Z. simulans was extracted with 70% aqueous methanol and enriched by WCX-SPE. A high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) with an electrospray ionisation (ESI) source was used for the qualitative and quantitative analyses of QAs. RESULTS: Significant improvements were observed in resolution and abundance of the peaks with WCX-SPE. The linearity, limit of detection (LOD) and limit of quantification (LOQ) were determined for this analytical method. The linear relationship (A = 338.85C - 187.72, R2  = 0.99) was explored in the range of 0.5 to 312.5 µg/mL for chelerythrine. The LOD and LOQ for chelerythrine standard solutions were 0.0539 µg/mL and 0.1798 µg/mL, respectively. In addition, 22 peaks were detected successfully with WCX-SPE and nine of them are undetectable without the processing of WCX-SPE. CONCLUSION: A highly selective and efficient method for simultaneous enrichment and identification of QAs from crude extract of Z. simulans was developed for the first time by combining WCX-SPE with LC-MS.


Assuntos
Alcaloides/análise , Resinas de Troca de Cátion/química , Cromatografia por Troca Iônica/métodos , Extração em Fase Sólida/métodos , Zanthoxylum/química , Cromatografia Líquida de Alta Pressão/métodos , Limite de Detecção , Reprodutibilidade dos Testes
9.
J Chromatogr A ; 1602: 317-325, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31248584

RESUMO

This study demonstrates how multimodal cation exchange chromatographic systems can be successfully employed to purify an IgG4 monomer from three distinct aggregate species. In addition, the steric mass action model is shown to be capable of facilitating the development of effective bind/elute processes for aggregate removal. A variety of multimodal anion and cation exchange resin materials and conditions were initially screened for both selectivity and recovery of the product. While the multimodal anion exchangers exhibited significant recovery issues, the Capto MMC and MMC ImpRes resin systems were observed to have good recoveries and some selectivity for these challenging separations under linear gradient conditions. Mechanistic modeling was then explored as a means to expedite the development of a bind/elute process for decreasing the aggregate content of this challenging monoclonal antibody mixture. The retention behavior of the monomer and the higher molecular weight species under different linear gradient conditions were used to estimate the SMA parameters of the proteins on both the Capto MMC and MMC ImpRes systems. A range of simulations were then carried out to determine an efficient bind/elute process for the removal of higher molecular weight species while also obtaining a good yield of the monomer in both resin systems. Finally, bind/elute experiments were carried out under the suggested simulation conditions for each resin system and were shown to be in good agreement with the theoretical predictions, with purities and yields of 99% and 78.6% for Capto MMC and 99.3% and 87.9% for Capto MMC ImpRes, respectively. The simple approach described in this paper presents a rapid and useful method for model-based process development of antibody monomer-aggregate separations with multimodal cation exchange chromatography.


Assuntos
Anticorpos Monoclonais/química , Resinas de Troca de Cátion/química , Cromatografia por Troca Iônica/métodos , Modelos Moleculares , Simulação por Computador , Peso Molecular , Temperatura
10.
J Chromatogr A ; 1601: 133-144, 2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31054833

RESUMO

In Part I of this work we determined the experimental cation exchange behavior of bivalent bispecific antibodies (BiSAb) comprising a pair of single chain variable fragment (scFv) domains flexibly linked to a framework immunoglobulin G (IgG), which exhibit a complex, three-peak elution pattern dependent on the residence time. A phenomenological model was developed assuming that the BiSAb molecules exist in multiple configurations that interact differently with the resin surface and interconvert at finite rates. In Part II of this work we provide relevant biomolecular perspectives that shed light on the underlying mechanisms. Firstly, we show that crosslinking the BiSAb molecules with a bifunctional reagent, which limits conformational flexibility, suppresses multiple peak elution. Secondly, we show that of the fragments obtained by enzymatic digestion of the BiSAb molecules only those that exhibit a pair of scFv domains show three-peak elution, while only two peaks are observed if a single scFv is present. Thirdly, we analyze the roles of electrostatic and hydrophobic surface properties of the BiSAb domains, identifying regions that are likely responsible for inter-domain and protein-surface interactions. The results demonstrate that the complex elution behavior catalyzed by the combination of surface charge and hydrophobicity of the stationary phase is associated with outstretched and collapsed configurations of the scFv domains relative to the framework IgG.


Assuntos
Anticorpos Biespecíficos/química , Resinas de Troca de Cátion/química , Cromatografia por Troca Iônica , Interações Hidrofóbicas e Hidrofílicas , Imunoglobulina G/química , Anticorpos de Cadeia Única/química , Eletricidade Estática
11.
J Chromatogr A ; 1601: 121-132, 2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31056270

RESUMO

The cation exchange chromatographic behavior of three homologous bivalent bispecific antibodies (BiSAb) is characterized for two different resins, Source 15S and ProPac WCX-10, having different base matrix, particle size, pore structure, and ligand chemistry. For both resins, elution with a salt gradient results in multiple peaks for each of the three BiSAb molecules at short residence times. The peaks gradually merge into two peaks and then into one peak eluting at intermediate salt concentrations when the residence time is gradually increased. Re-injecting fractions of each the individual peaks obtained at short residence time results in nearly the same multiple peak elution pattern. This behavior, which is contrary to the behavior normally encountered in ion exchange chromatography, appears to be related to the reversible, surface -catalyzed interconversion between different conformational states of each BiSAb that interact with different strength with the chromatographic surface. This behavior is qualitatively independent of pH in the range 5-8.5, protein load in the range 0.06-5.0 mg/ml, and gradient slope, and is not associated with the formation of aggregates. Gradually increasing temperature, however, reduces the multiple peak behavior eventually resulting into a single peak at 55 °C A phenomenological model is developed that predicts the experimental behavior over a broad range of conditions using fitted rate and equilibrium constants.


Assuntos
Anticorpos Biespecíficos/química , Resinas de Troca de Cátion/química , Cromatografia por Troca Iônica , Modelos Químicos , Tamanho da Partícula
12.
J Chromatogr A ; 1599: 152-160, 2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31084900

RESUMO

A low ligand density cation exchange (CEX) chromatography resin, Eshmuno® CP-FT resin, was investigated for the removal of aggregates from monoclonal antibody (mAb) feeds using a continuous loading process. Removing mAb aggregates with a CEX resin using continuous loading is advantageous relative to a bind/elute loading process, because the resin can use nearly its full capacity to bind the aggregates enabling much higher loadings. The removal of mAb aggregates with Eshmuno® CP-FT resin using a continuous loading process was found to be consistent with a frontal chromatography mechanism where the mAb monomer initially binds to the column and is subsequently displaced by dimers and higher molecular weight aggregates. The removal of mAb aggregates with Eshmuno® CP-FT resin using a continuous loading process was compared with six other commercially available strong CEX chromatography resins and found to correlate with their ionic densities, but not their mAb static binding capacities. The influence of pH, conductivity, residence time, and mAb concentration on the removal of aggregates with Eshmuno® CP-FT resin using a continuous loading process was also investigated. Finally, the percentage of aggregates in a mAb feed was varied to examine the effect on the removal of aggregates with Eshmuno® CP-FT resin using a continuous loading process.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Resinas de Troca de Cátion/química , Cromatografia por Troca Iônica , Anticorpos Monoclonais/química , Cátions/química , Concentração de Íons de Hidrogênio
13.
J Chromatogr A ; 1595: 97-107, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-30833023

RESUMO

Salt solutions are widely used as eluents for ion-exchange chromatography. In general, salts reduce the retention of applied solutes on ion-exchange columns via electrostatic screening effects. The reverse phenomenon, namely, salt-enhanced retention, has not been reported. Here, we report that cations, including arginine, guanidine and sodium ions, enhance the retention of uncharged aromatic solutes on a cation-exchange resin, i.e., a negatively charged resin, with carboxyl groups, where we used alkyl gallates as model uncharged aromatic solutes and a carboxymethyl agarose gel (CM Sepharose) as a model negatively charged resin. Enhancement of retention was observed at concentrations of tens of millimolar of the salts, in which arginine hydrochloride was more effective than guanidinium salts and NaCl. Similar trends were observed for other phenolic compounds, including phenol and 4-hydroxybenzyl alcohol. Molecular dynamics simulations showed that the binding free energy between the alkyl gallate molecule and the CM Sepharose resin ligand molecule increased with increasing salt concentration. The increase in binding free energy caused by the salts was accounted for by the binding of the salt cations to the aromatic moiety of the alkyl gallate via cation-π interactions, leading to attenuation of intrinsic repulsive interactions between the ligand carboxyl group and the alkyl gallate aromatic moiety. Therefore, the salt-enhanced retention of the uncharged aromatic solutes on the negatively charged resins was ascribable to the increase in binding free energy induced by the cation-π interactions. This unique reverse phenomenon of the effect of salts on solute retention indicates the importance of cation-π interactions in ion-exchange chromatography. This phenomenon can be used for selective chromatographic separation of aromatic solutes, including organic solutes, proteins and nucleic acids.


Assuntos
Arginina/química , Resinas de Troca de Cátion/química , Cátions/química , Cromatografia por Troca Iônica/métodos , Sódio/química , Guanidina/química , Ligantes , Simulação de Dinâmica Molecular , Proteínas/química , Cloreto de Sódio/química , Soluções
14.
Talanta ; 196: 117-123, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30683340

RESUMO

A novel approach for green and simple solid-phase extraction of ionizable analytes using cation-exchange resin particles packed in a rotating cotton-based disk has been developed and utilized for the chemiluminescence determination of ofloxacin in biological fluids (serum and urine). The use of highly-available cation-exchange resin provided effective separation of analyte from complex sample matrixes and its elution by aqueous electrolyte solution without any organic solvents. Ofloxacin ionization in acidic sample solution allowed to provide effective separation of the analyte for subsequent chemiluminescence detection. The cotton-based disk was characterized by effective wettability. This feature promoted high mass transfer of the analyte to the cation-exchange resin surface at separation step and into elution solution at elution step. The sorption time was decreased to 10 min while the elution time was 5 min. Under the optimal conditions, the detector response for ofloxacin was linear in the concentration range from 9 × 10-8 to 3 × 10-5 mol L-1. The limit of detection, calculated from a blank test based on 3σ, was 3 × 10-8 mol L-1.


Assuntos
Antibacterianos/química , Resinas de Troca de Cátion/química , Ofloxacino/química , Adsorção , Antibacterianos/sangue , Antibacterianos/urina , Fibra de Algodão , Humanos , Luminescência , Ofloxacino/sangue , Ofloxacino/urina
15.
Biotechnol J ; 14(3): e1800132, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29809318

RESUMO

A previously developed empirical interpolation (EI) method is extended to predict highly overloaded multicomponent elution behavior on a cation exchange (CEX) column based on batch isotherm data. Instead of a fully mechanistic model, the EI method employs an empirically modified multicomponent Langmuir equation to correlate two-component adsorption isotherm data at different salt concentrations. Piecewise cubic interpolating polynomials are then used to predict competitive binding at intermediate salt concentrations. The approach is tested for the separation of monoclonal antibody monomer and dimer mixtures by gradient elution on the cation exchange resin Nuvia HR-S. Adsorption isotherms are obtained over a range of salt concentrations with varying monomer and dimer concentrations. Coupled with a lumped kinetic model, the interpolated isotherms predict the column behavior for highly overloaded conditions. Predictions based on the EI method shows good agreement with experimental elution curves for protein loads up to 40 mg mL-1 column or about 50% of the column binding capacity. The approach can be extended to other chromatographic modalities and to more than two components.


Assuntos
Anticorpos Monoclonais/química , Resinas de Troca de Cátion/química , Adsorção , Cromatografia por Troca Iônica/métodos , Cinética , Ligação Proteica , Cloreto de Sódio/química
16.
Anal Chim Acta ; 1043: 45-51, 2018 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-30392668

RESUMO

The study of low abundant proteins contributes to increasing our knowledge about (patho)physiological processes and may lead to the identification and clinical application of disease markers. However, studying these proteins is challenging as high-abundant proteins complicate their analysis. Antibodies are often used to enrich proteins from biological matrices prior to their analysis, though antibody-free approaches have been described for some proteins as well. Here we report an antibody-free workflow on the basis of strong cation exchange (SCX) enrichment and liquid chromatography-mass spectrometry (LC-MS) for quantification of the soluble Receptor of Advanced Glycation End-products (sRAGE), a promising biomarker in chronic obstructive pulmonary disease (COPD). sRAGE was quantified in serum at clinically relevant low to sub ng mL-1 levels. The method was validated according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and was compared to an antibody-based LC-MS sRAGE method. The SCX-based method builds upon the bipolar charge distribution of sRAGE, which has a highly basic N-terminal part and an acidic C-terminal part resulting in an overall neutral isoelectric point (pI). The highly basic N-terminal part (pIcalculated = 10.3) allowed for sRAGE to be enriched by SCX at pH 10, a pH at which most serum proteins do not bind. This study shows that ion exchange-based enrichment is a viable approach for the LC-MS analysis of several low abundant proteins following a thorough analysis of their physical-chemical properties.


Assuntos
Receptor para Produtos Finais de Glicação Avançada/análise , Extração em Fase Sólida/métodos , Espectrometria de Massas por Ionização por Electrospray , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Biomarcadores/sangue , Resinas de Troca de Cátion/química , Cromatografia Líquida de Alta Pressão , Humanos , Ponto Isoelétrico , Limite de Detecção , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Receptor para Produtos Finais de Glicação Avançada/imunologia , Receptor para Produtos Finais de Glicação Avançada/isolamento & purificação
17.
J Chromatogr A ; 1574: 60-70, 2018 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-30220427

RESUMO

We are currently examining the potential of amphipathic cationic α-helical peptides as a new generation of peptide standards for both cation-exchange high-performance liquid chromatography and reversed-phase chromatography. Thus, amphipathic peptides are particularly suitable for high-performance liquid chromatography standards due to the preferred binding of the non-polar face to the hydrophobic stationary phase of reversed-phase packings or the preferred binding of the polar face to the charged/hydrophilic stationary phase of cation-exchange packings. The ability of different reversed-phase or cation-exchange matrices to separate mixtures of peptide standards with only subtle hydrophilicity/hydrophobicity variations in both the non-polar and polar face of the peptides can then be assessed. Currently, we have designed de novo a mixture of six 26-residue all D-conformation amphipathic cationic α-helical peptides with a single, positively charged lysine residue in the center of the non-polar face and an increasing number of lysine residues (4-9 residues) replacing neutral residues in the polar face, resulting in an overall net positive charge of +5 to +10. Thus, the non-polar, preferred reversed-phase chromatography binding face remains constant, with only the polar face varying in hydrophilicity/hydrophobicity. Interestingly, even with the non-polar face remaining constant, reversed-phase columns of varying functional group properties (e.g., C8, C18, phenyl, polar endcapped, polar embedded) and porosity (porous versus superficially porous) were able to separate the six peptides in aq. TFA/acetonitrile gradients, albeit with different selectivities. The value of the standards in cation-exchange chromatography was expressed by monitoring the requirement of acetonitrile (0-40% in the mobile phase) to overcome hydrophobic interactions of the peptides with the cation-exchange matrix matrix when eluting with sodium perchlorate gradients at pH 6.5. Interestingly, the resolution of the higher charged peptides (+8,+9,+10) was particularly sensitive to acetonitrile levels. Our results clearly demonstrate the excellent potential of these novel peptide standards to enable optimal column choice and mobile phase conditions for reversed-phase chromatography and cation-exchange chromatography for peptide separations.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Peptídeos/isolamento & purificação , Padrões de Referência , Acetonitrilos/química , Resinas de Troca de Cátion/química , Técnicas de Química Analítica/instrumentação , Interações Hidrofóbicas e Hidrofílicas , Peptídeos/química
18.
Chemosphere ; 211: 1091-1097, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30223324

RESUMO

Cation exchange resins have proved to be efficient in removing precursors of N-nitrosodimethylamine (NDMA). NDMA is a probable human carcinogen with a calculated lifetime cancer risk of 10-6 at 0.7 ng/L in drinking water. This paper investigated the effect of pH and calcium levels on the removal of NDMA precursors using a cation exchange resin. At pH 5 and 7, 30-50% of NDMA precursors, measured by formation potentials (FPs) changes before and after the treatment, were removed by Plus resin. However, increases in NDMA FPs were observed after the treatment at pH 10 indicating that NDMA precursors were released from the resin. NDMA FPs removals in samples containing 15 and 115 mg/L Ca2+ were 40% and -10% after the ion exchange treatments at pH 7, respectively. It was found that in the presence of high concentration of calcium only one out of four cation exchange resins released NDMA precursors (probably due to manufacturing impurities). Also, the release of NDMA precursors depended on the calcium concentration and the contact time of the resin with the solution containing calcium. Nonetheless, NDMA precursors release from the resin subsided significantly with increasing the number of regeneration cycles of the resin.


Assuntos
Resinas de Troca de Cátion/química , Dimetilnitrosamina/isolamento & purificação , Poluentes Químicos da Água/isolamento & purificação , Cálcio/farmacologia , Dimetilnitrosamina/análise , Água Potável/química , Concentração de Íons de Hidrogênio , Poluentes Químicos da Água/análise , Purificação da Água/métodos
19.
Water Sci Technol ; 2017(3): 770-781, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30016295

RESUMO

A magnetic cation-exchange resin (MCER) was prepared by copolymerization of oleic acid-grafted magnetite with styrene, divinylbenzene (DVB), and triallylisocyanurate (TAIC) for removing Cd(II) from wastewater. A non-magnetic cation-exchange polystyrene resin (CEPR) was also prepared as a reference. Structural and morphological analyses revealed that the MCER and CEPR were mesoporous microspheres; the MCER contained about 25% Fe3O4. The influence of temperature, pH, contact time, and the initial concentration of Cd(II) on the adsorption of Cd(II) was investigated. The maximum adsorption capacity of the MCER reached 88.56 mg/g, which was achieved at 343 K using a Cd(II) initial concentration of 200 mg/L. The adsorption processes attained equilibrium within 120 min for the MCER and 300 min for the CEPR, and were well described by a pseudo-second-order kinetic model. Furthermore, the equilibrium adsorption data fitted the Freundlich isotherm model better than the Langmuir model. The superior magnetic response and regeneration of the MCER make it a good candidate as an adsorbent for removing Cd(II) from wastewater.


Assuntos
Cádmio/química , Resinas de Troca de Cátion/química , Poliestirenos/química , Águas Residuárias/química , Poluentes Químicos da Água/química , Adsorção , Cátions/análise , Óxido Ferroso-Férrico , Concentração de Íons de Hidrogênio , Cinética , Temperatura , Purificação da Água/métodos
20.
Anal Chem ; 90(14): 8478-8486, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-29847097

RESUMO

A design of electromembrane extraction (EME) as a lab on-a-chip device was proposed for the extraction and determination of phenazopyridine as the model analyte. The extraction procedure was accomplished by coupling EME and packing a sorbent. The analyte was extracted under the applied electrical field across a membrane sheet impregnated by nitrophenyl octylether (NPOE) into an acceptor phase. It was followed by the absorption of the analyte on strong cation exchanger as a sorbent. The designed chip contained separate spiral channels for donor and acceptor phases featuring embedded platinum electrodes to enhance extraction efficiency. The selected donor and acceptor phases were 0 mM HCl and 100 mM HCl, respectively. The on-chip electromembrane extraction was carried out under the voltage level of 70 V for 50 min. The analysis was carried out by two modes of a simple red-green-blue (RGB) image analysis tool and a conventional HPLC-UV system. After the absorption of the analyte on the solid phase, its color changed and a digital picture of the sorbent was taken for the RGB analysis. The effective parameters on the performance of the chip device, comprising the EME and solid phase microextraction steps, were distinguished and optimized. The accumulation of the analyte on the solid phase showed excellent sensitivity and a limit of detection (LOD) lower than 1.0 µg L-1 achieved by an image analysis using a smartphone. This device also offered acceptable intra- and interassay RSD% (<10%). The calibration curves were linear within the range of 10-1000 µg L-1 and 30-1000 µg L-1 ( r2 > 0.9969) for HPLC-UV and RGB analysis, respectively. To investigate the applicability of the method in complicated matrixes, urine samples of patients being treated with phenazopyridine were analyzed.


Assuntos
Dispositivos Lab-On-A-Chip , Membranas Artificiais , Fenazopiridina/isolamento & purificação , Fenazopiridina/urina , Microextração em Fase Sólida/instrumentação , Adulto , Resinas de Troca de Cátion/química , Cromatografia Líquida de Alta Pressão/instrumentação , Eletricidade , Técnicas Eletroquímicas/instrumentação , Eletrodos , Desenho de Equipamento , Feminino , Humanos , Fenazopiridina/análise , Espectrofotometria Ultravioleta/instrumentação , Adulto Jovem
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