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1.
Food Chem ; 340: 127876, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32871354

RESUMO

Jackfruit is a sweet tropical fruit with very pleasant aroma, and the ripe seeds are edible. In this study, jackfruit seed proteins were isolated and subjected to trypsin digestion. The resultant protein hydrolysate was then subjected to antioxidant assay-guided purification, using centrifugal filtration, C18 reverse-phase and strong cation exchange (SCX) fractionations. The purified SCX fraction was further analyzed by de novo peptide sequencing, and two peptide sequences were identified and synthesized. Peptide JFS-2 (VGPWQK) was detected with antioxidant potential, with EC50 value comparable to that of commercial GSH antioxidant peptide. Additionally, the identified peptides were tested with protein protection potential, in an albumin protein denaturation inhibitory assay. Concurrently, we also investigated the pH, temperature, and gastrointestinal-digestion stability profiles for the identified peptide. With further research efforts, the identified peptides could potentially be developed into preservative agent for protein-rich food systems or as health-promoting diet supplements.


Assuntos
Antioxidantes/análise , Artocarpus/química , Peptídeos/análise , Peptídeos/química , Hidrolisados de Proteína/química , Sementes/química , Antioxidantes/química , Cromatografia por Troca Iônica , Digestão , Conservantes de Alimentos , Concentração de Íons de Hidrogênio , Peptídeos/metabolismo , Temperatura , Tripsina
2.
Food Chem ; 343: 128543, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33187742

RESUMO

Lysozyme from crude chicken egg white (CEW) feedstock was successfully purified using a stirred fluidized bed adsorption system ion exchange chromatography where STREAMLINE SP and SP-XL high density adsorbents were selected as the adsorption carrier. The thermodynamic and kinetic studies were carried out to understand the characteristics of lysozyme adsorption by adsorbents under various conditions, including adsorption pH, temperature, lysozyme concentration and salt concentrations. Results showed that SP and SP-XL adsorbents achieved optimum lysozyme adsorption at pH 9 with capacity of ~139.77 and ~251.26 mg/mL, respectively. The optimal conditions obtained from batch studies were directly employed to operate in SFBA process. For SP-XL adsorbent, the recovery yield and purification factor of lysozyme were 93.78% and ~40 folds, respectively. For SP adsorbent, lysozyme can be eluted ~100% with purification factor of ~26 folds. These two adsorbents are highly suitable for use in direct recovery of lysozyme from crude CEW.


Assuntos
Cromatografia por Troca Iônica/métodos , Clara de Ovo/química , Muramidase/isolamento & purificação , Adsorção , Animais , Galinhas , Cinética , Muramidase/química , Temperatura
3.
Food Chem ; 343: 128549, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33189480

RESUMO

Whole grains and pulses are rich in nutrients but often avoided by individuals with gastrointestinal disorders, due to high levels of fermentable oligo-, di-, monosaccharides and polyols (FODMAPs). This study investigated the impact of malting as delivery-system for endogenous enzymes. Malts from barley and wheat (naturally high in fructans), lentils and chickpeas (high in galactooligosaccharides), oat and buckwheat (low in FODMAPs) were produced. While barley and wheat malts had slightly elevated fructan-levels, in oat malt 0.8 g/100 g DM fructans were de novo synthesized. In lentils and chickpeas galactooligosaccharides diminished by 80-90%. Buckwheat did not contain any FODMAPs commonly investigated, but fagopyritols which may have a similar physiological effect. Also fagopyritols were degraded. While malted pulses and buckwheat are directly suitable for low FODMAP applications, using the combined approach of malting and fermentation, malted cereals could contribute to high nutritional values of such products.


Assuntos
Dissacarídeos/metabolismo , Grão Comestível/metabolismo , Manipulação de Alimentos/métodos , Frutanos/metabolismo , Monossacarídeos/metabolismo , Oligossacarídeos/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Cicer/crescimento & desenvolvimento , Cicer/metabolismo , Dissacarídeos/análise , Fagopyrum/crescimento & desenvolvimento , Fagopyrum/metabolismo , Frutanos/análise , Germinação , Lens (Planta)/crescimento & desenvolvimento , Lens (Planta)/metabolismo , Monossacarídeos/análise , Oligossacarídeos/análise , Triticum/crescimento & desenvolvimento , Triticum/metabolismo
4.
J Chromatogr A ; 1634: 461633, 2020 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-33189959

RESUMO

The current study investigates a method for purification of the G-quadruplex secondary structure, naturally formed by a guanine-rich 21-mer oligonucleotide strand using a monolithic convective interaction media-quaternary amine (CIM-QA) column under ion-exchange conditions. The monolithic support was initially evaluated on a preparative scale against a highly efficient TSKgel SuperQ-5PW ion-exchange support designed for oligonucleotide purification. The CIM analogue demonstrated clear advantages over the particle-based support on the basis of rapid separation times, while also affording high purity of the G-quadruplex. Various parameters were investigated including the type of mobile phase anion, cation, pH and injection load to induce and control quadruplex formation, as well as enhance chromatographic separation and final purity. Potassium afforded the most prominent quadruplex formation, yet sodium allowed for the highest resolution and purity to be achieved with a 30 mg injection on an 8 ml CIM-QA monolithic column. This method was applied to purify in excess of 300 mg of the quadruplex, with excellent retention time precision of under 1% RSD. Native mass spectrometry was utilized to confirm the identity of the intact G-quadruplex under non-denaturing conditions, while ion-pairing reversed-phase methods confirmed the presence of the single-stranded oligonucleotide in high purity (92%) under denaturing conditions. The key advantage of the purification method enables isolation of the G-quadruplex in its native state on a milli-gram scale, allowing structural characterization to further our knowledge of its role and function. The G-quadruplex can also be subsequently denaturated at elevated temperature causing single strand formation if additional reactions are to be pursued, such as annealing to form a duplex, and evaluation in in vitro or in vivo studies.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia por Troca Iônica , Quadruplex G , Espectrometria de Massas , Oligonucleotídeos/análise
5.
J Chromatogr A ; 1634: 461648, 2020 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-33207292

RESUMO

Novel anion exchangers with mono- and dialkanolamines in the external part of covalently-bonded hyperbranched functional layer were synthesized. Comparison of the chromatographic properties of the prepared stationary phases in suppressed ion chromatography (IC) mode using hydroxide eluent allowed us to evaluate the effect of the number of substituents at nitrogen atom in alkanolamine on selectivity of anion exchangers toward organic acids. Obtained anion exchangers were also examined together with previously described hyperbranched stationary phases with different mono- and diamines in the external part of the layer for evaluating the influence of various parameters on their selectivity. Effects of hydrophilicity, functionality of amine, and the number of substituents at nitrogen atom of amine used in the last modification cycle were established independently from each other, which provided the possibility to tailor selectivity toward organic acids when preparing anion exchanger for solving particular analytical task. Predominance of hydrophilicity as a key factor affecting the separation of weakly retained organic acids over other studied parameters was demonstrated.


Assuntos
Ácidos/química , Algoritmos , Cromatografia por Troca Iônica/métodos , Aminas/química , Diaminas/química , Interações Hidrofóbicas e Hidrofílicas , Hidróxidos/química
6.
J Chromatogr A ; 1634: 461669, 2020 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-33166892

RESUMO

Polymer-grafted media have been a focus of recent development for ion exchange chromatography (IEC) because of their capacity and uptake kinetics that can lead to high dynamic capacity in protein purification. This work is devoted to developing novel cation exchangers of high adsorption performance by grafting polymerization of sodium methacrylate (MA) onto a commercial agarose gel Sepharose FF (FF). Five polyMA (pMA)-grafted FF gels were prepared with the same grafting density but different chain lengths (i.e., different ionic capacities, ICs), and named as FF-pMA-IC (IC denotes IC value in mmol/L). The effects of chain length (IC) and ionic strength (IS) on protein adsorption and chromatographic behaviors were examined using lysozyme (at pH 8.0) and γ-globulin (at pH 5.0) as model proteins. It was found that lysozyme adsorption capacity increased with increasing IC till reaching a plateau (390 mg/mL) over IC=540 mmol/L (FF-pMA-540), while there was an optimum IC (320 mmol/L, FF-pMA-320) at which γ-globulin adsorption capacity reached the highest (208 mg/mL). With increasing chain length (IC), the uptake rates of both the proteins presented decreasing trends due to the steric hindrance caused by the polymer chains. At the same IC, however, the uptake rate of lysozyme was much higher than that of γ-globulin because of the different sizes of the two proteins. Increasing salt concentration obviously promoted the uptake rates of the proteins, which led to the increase of dynamic binding capacities (DBCs) in different salt concentration ranges. The DBC value of lysozyme on FF-pMA-540 kept as high as 108-198 mg/mL in the salt concentration range of 0-150 mmol/L, and the DBC of γ-globulin on FF-pMA-320 increased to 27 mg/mL with increasing salt concentration from 100 mmol/L. This work clearly indicated the presence of optimal IC values (chain lengths) for different sized proteins, and IS was also crucial for reaching a high DBC for a specific protein. The findings provided insight into the selection of FF-pMA-n gels and operational conditions (e.g., IS) for the purification of a target protein of defined size.


Assuntos
Cromatografia por Troca Iônica/métodos , Ácidos Polimetacrílicos/química , Proteínas/análise , Proteínas/isolamento & purificação , Sefarose/química , Adsorção , Cátions , Cinética , Muramidase/análise , Muramidase/isolamento & purificação , Concentração Osmolar , Polietilenoimina/química , Proteínas/química , gama-Globulinas/análise , gama-Globulinas/isolamento & purificação
7.
J Chromatogr A ; 1634: 461688, 2020 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-33221654

RESUMO

A fast method for assessing the stability of monoclonal antibodies (mAbs) adsorbed on ion exchange resins has been developed. The method exploited a real time polymerase chain reaction equipment to determine the temperature of protein phase transition, i.e., the so called melting temperature, based on differential scanning fluorimetry. Changes to the melting temperature were screened under various adsorption conditions and correlated with the protein stability upon adsorption. The method was tested for two different mAbs bound to various types of strong cation exchangers at different pH and loading concentrations. The mAbs destabilized upon adsorption due to strong binding, which manifested itself in aggregate formation and recovery reduction. The phenomenon depended on the resin type and binding conditions. However, regardless of the process conditions and resins used, drop in the melting temperatures to a critical value of about 30° could serve as an indicator of destructive changes in the protein structure in the adsorbed phase. The measurements were simultaneously accomplished for a number of samples with very small material consumption. Therefore, the method may be applied for screening resins and operating variables for a given mAb to exclude conditions that induce structure destabilization and aggregation.


Assuntos
Anticorpos Monoclonais/química , Resinas de Troca de Cátion/química , Cromatografia por Troca Iônica/métodos , Adsorção , Anticorpos Monoclonais/metabolismo , Fluorometria , Estabilidade Proteica , Temperatura
8.
PLoS One ; 15(10): e0240580, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33045024

RESUMO

Human serum albumin presents in its primary structure only one free cysteine (Cys34) which constitutes the most abundant thiol of plasma. An antioxidant role can be attributed to this thiol, which is located in domain I of the protein. Herein we expressed domain I as a secretion protein using the yeast Pichia pastoris. In the initial step of ammonium sulfate precipitation, a brown pigment co-precipitated with domain I. Three chromatographic methods were evaluated, aiming to purify domain I from the pigment and other contaminants. Purification was achieved by cation exchange chromatography. The protein behaved as a non-covalent dimer. The primary sequence of domain I and the possibility of reducing Cys34 to the thiol state while avoiding the reduction of internal disulfides were confirmed by mass spectrometry. The reactivity of the thiol towards the disulfide 5,5´-dithiobis(2-nitrobenzoate) was studied and compared to that of full-length albumin. A ~24-fold increase in the rate constant was observed for domain I with respect to the entire protein. These results open the door to further characterization of the Cys34 thiol and its oxidized derivatives.


Assuntos
Antioxidantes/química , Cisteína/genética , Albumina Sérica Humana/genética , Compostos de Sulfidrila/química , Cromatografia por Troca Iônica , Cisteína/química , Expressão Gênica/genética , Humanos , Domínios Proteicos/genética , Multimerização Proteica , Saccharomycetales/genética , Albumina Sérica Humana/química
9.
J Chromatogr A ; 1632: 461603, 2020 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-33099238

RESUMO

The herbicide glyphosate and its main metabolite aminomethylphosphonic acid (AMPA) are generally studied in environmental samples in the investigation of contamination of soil, plants, water and food. Many analytical methods are based on liquid chromatography or high-performance liquid chromatography, with pre-column or post-column derivatization; in addition, the chromatograph can be coupled to mass spectrometers for detection and quantification. Gas chromatography and spectroscopic and electrochemical methods have also been used. In this work, a simple low-cost method is presented for the analysis of water intended for human consumption with the quantification not only of glyphosate and AMPA, but also of other ions of interest to public health (fluoride, chlorite, bromate, chloride, nitrite, nitrate, sulfate and phosphate). Based on ion chromatography with conductivity detection (chemical suppression of eluent conductivity), the key point in this method is the use of gradient elution with two eluents of different pH and ionic strength, not requiring derivatization. There is no interference from the other ions at higher concentrations. The detection limits obtained for glyphosate and AMPA were 15 µg L-1 and 80 µg L-1, respectively. As the method allows the analysis of a large number of samples, it has been successfully applied to monitoring the quality of tap water in 89 municipalities in the northeast region of the State of São Paulo, Brazil.


Assuntos
Cromatografia por Troca Iônica/métodos , Água Potável/química , Monitoramento Ambiental/métodos , Glicina/análogos & derivados , Saúde Pública , Poluentes Químicos da Água/análise , Glicina/análise , Herbicidas/análise , Humanos , Limite de Detecção , Compostos Organofosforados/química , Padrões de Referência , Soluções
10.
J Chromatogr A ; 1633: 461635, 2020 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-33128974

RESUMO

Viral clearance is an important performance metric for the downstream process of monoclonal antibodies (mAbs) due to its impact on patient safety. Anion exchange chromatography (AEX) has been well-accepted in the industry as one of the workhorse techniques for removing viruses, and is considered to be able to achieve high log clearance values under most operating conditions. However, it is not uncommon for viral clearance results on AEX to fall below the desired level despite operating under conditions that should achieve high clearance levels according to conventional wisdom of how this mode of chromatography operates. In this study, a design of experiment (DoE) approach was used to develop a more fundamental understanding of viral clearance during AEX chromatography using Minute Virus of Mice (MVM) on POROS HQ resin. Load pH, conductivity and virus concentration were evaluated as design factors for three mAbs with varying physical and chemical properties. The hydrophobicity and surface charge distributions of the molecules were found to be the most significant factors in influencing viral clearance performance, and the viral clearance trends did not seem to fit with conventional wisdom. To explain this seemingly unconventional behavior, we propose a new mechanism that suggests that interactions between the mAb and the virus have a major contribution on retention of the virus on the resin. This furthered understanding may help improve the predictability, performance and robustness of viral clearance during AEX chromatography.


Assuntos
Anticorpos Monoclonais/metabolismo , Cromatografia por Troca Iônica/normas , Vírus Miúdo do Camundongo/metabolismo , Vírus/metabolismo , Animais , Ânions/química , Anticorpos Monoclonais/química , Camundongos , Vírus/química
11.
J Chromatogr A ; 1629: 461483, 2020 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-32823015

RESUMO

Polymer-grafting to porous materials is an effective way to create protein ion-exchangers of high capacity and uptake rate because the 3D architecture of the polymeric ion exchange groups provides high binding space and facilitated transport of the bound protein. Herein, a new anion exchanger was fabricated by grafting (3-acrylamide propyl) trimethyl ammonium chloride (APTAC) onto Sepharose FF gel for protein adsorption and chromatography. The ion exchanger, denoted as FF-pAPTAC, presented high capacity but limited uptake rate in bovine serum albumin (BSA) adsorption due to the high charge density of the cationic polymer chains. To solve the problem, we proposed to copolymerize APTAC with an electroneutral monomer, acrylamide (AM), onto Sepharose FF to modulate the charge density of the grafted polymer chains. By decreasing the feeding molar ratio of APTAC to AM, the ionic capacity (IC) of the copolymerized resins, FF-p(AM-APTAC)n (n denotes IC in mmol/L), decreased, but the chain length could be remained almost unchanged due to the similar reactivity ratios of the two monomers. With decreasing IC, the static adsorption capacity (qm) of FF-p(AM-APTAC)n decreased gradually because of the decline of protein binding sites. The uptake rate, however, represented by the ratio of effective pore diffusivity to the free solution diffusivity (De/D0), exhibited a strong uptrend with decreasing IC, reaching ~2.5-fold of the maximum observed with FF-pAPTACn. It is considered that the decrease of charge density weakened protein binding strength to the chains and increased the chain flexibility, which consequently facilitated the transport of bound proteins on the chains. Moreover, at the same IC, each FF-p(AM-APTAC)n displayed similar adsorption capacity but high uptake rate as compared with its FF-pAPTACn counterpart mainly due to the longer chain length of the copolymer. Particularly, ~6.5-fold enhancement of De/D0 was observed at IC = 50 ± 2 mmol/L. Both high adsorption capacity and uptake rate made FF-p(AM-APTAC)n exhibit superior dynamic binding performance. The findings proved that reducing chain charge density by copolymerizing an electroneutral monomer was promising for fabrication of high-performance protein ion exchangers.


Assuntos
Cromatografia por Troca Iônica/métodos , Proteínas/química , Compostos de Amônio Quaternário , Adsorção , Ânions , Cinética , Ligação Proteica , Proteínas/isolamento & purificação , Sefarose , Soroalbumina Bovina/química , Soroalbumina Bovina/isolamento & purificação
12.
J Chromatogr A ; 1629: 461495, 2020 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-32846340

RESUMO

In the chromatographic separation process of oligonucleotides (ONs), mechanistic understanding of their binding and diffusion processes is of significant importance to determine operating conditions in a fast and robust way. In this work, we determined the number of binding sites and the diffusivities of ONs in a polymer grafted anion exchange chromatography through linear gradient experiments (LGE) being carried out at selected four to five gradient slopes. Synthetic poly (T)s with length ranging from 3 to 90-mer were employed as a model of an antisense oligonucleotide with typical lengths of 10 - 30 bases. Comparison of the retention was also conducted between the grafted anion exchanger with a conventional ligand and an anion monolith disk. For the ONs up to 50 bases, the number of binding sites determined can be correlated with the length of ONs, and the grafted resin showed a better diffusion and narrower peak width compared to the nongrafted one. The retention behavior became similar for porous media when the longer ONs (> 50mer) were applied. The results obtained suggest that antisense ONs can be separated with grafted ligands without sacrificing mass transfer properties.


Assuntos
Cromatografia por Troca Iônica/métodos , Oligonucleotídeos/isolamento & purificação , Ânions , Sítios de Ligação , Difusão , Oligonucleotídeos/química , Polímeros
13.
J Chromatogr A ; 1628: 461444, 2020 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-32822983

RESUMO

The effect of bead and ligand structure on protein adsorption was investigated for multimodal anion exchangers combining a quaternary ammonium ion group with hydrophobic moieties: Nuvia aPrime 1 and aPrime 2, based on a 54 µm diameter polymeric bead, and Capto Adhere ImpRes and Capto Adhere, based on agarose beads 51 and 78 µm diameter, respectively. Bovine serum albumin (BSA) monomer, BSA dimer, and thyroglobulin (Tg) were used as model proteins. Based on TEM imaging and iSEC, the Nuvia resins have a microgranular structure and large pores (110 nm radius), while the Capto resins have a fibrous structure and smaller pores (32-36 nm radius). Comparable binding capacities (80-110 mg/mL), decreasing as salt is added, are observed for all three proteins on the Nuvia resins. Higher capacities (110-130 mg/mL), also decreasing as salt is added, are observed for BSA monomer and dimer on the Capto resins. However, the Tg binding capacity is very low in this case and increases as salt is added. Confocal laser scanning microscopy show that the kinetics are controlled by pore diffusion for all four resins, but with diffusivities that decrease as the protein size increases especially for the Capto resins. For Tg at low salt, binding is restricted to a thin shell close to the bead surface for both Capto resins. The ratio of effective and free diffusivity is about 0.30, 0.18, and 0.08 for BSA monomer, BSA dimer, and Tg, respectively, on the Nuvia resin. These values decrease to about 0.11, 0.04, and 0.01, respectively, for the Capto resins as a result of diffusional hindrance. Dynamic binding capacities are consistent with the equilibrium and rate behaviors.


Assuntos
Resinas de Troca de Ânions/química , Cromatografia por Troca Iônica , Proteínas/metabolismo , Adsorção , Ânions/química , Difusão , Interações Hidrofóbicas e Hidrofílicas , Cinética , Ligantes , Polímeros/química , Sefarose/química , Soroalbumina Bovina/química
14.
J Chromatogr A ; 1627: 461394, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823099

RESUMO

The early identification of unstable glass objects in museum collections is essential for their conservation, but as yet cannot be accomplished straightforwardly. Accordingly, this paper describes the development and validation of a simple protocol for quantitative determination of ions characteristic of the chemical decay of historic glass, using surface swabbing combined with ion-exchange chromatography. The establishment of a robust protocol is an important step in the development of an early warning system for the chemical deterioration of unstable glass. Using a model system, the protocol was validated for specificity, linearity, accuracy, precision, limits of detection, and limits of quantification for 10 anionic species (fluoride, acetate, formate, chloride, nitrite, bromide, nitrate, carbonate, sulfate and phosphate) and 6 cationic species (lithium, sodium, ammonium, potassium, magnesium and calcium). Good validation parameters (R2 > 0.995; RSD < 5%; Recovery 90-100%) were obtained for acetate, formate, nitrite, nitrate, phosphate, lithium, sodium, potassium, magnesium and calcium. Chloride (R2 = 0.934; RSD = 13.6%; recovery 71.4%) and carbonate (R2 = 0.993; RSD = 10.3%; recovery 120%) had poor validation parameters. Sulfate had low recovery (78.2%), but high reproducibility (RSD = 4.32%) with R2 = 0.997. Limits of quantification were below 1 mg/L for all analytes, which is satisfactory for the study of unstable glass in museum collections. The validated sampling protocol was trialled using artificially aged unstable glass fragments, which resulted in a high relative standard deviation (between 1 and 30%). The ability to achieve improved care of historic glass by application of the validated protocol in museum collections is discussed in the context of a pilot study undertaken at the Rijksmuseum, Amsterdam.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Vidro/química , Íons/análise , Cloretos/análise , Cromatografia por Troca Iônica , Fluoretos/análise , Íons/química , Limite de Detecção , Metais/análise , Metais/química , Museus , Nitratos/análise , Reprodutibilidade dos Testes , Propriedades de Superfície
15.
Sci Rep ; 10(1): 13356, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770127

RESUMO

The present study report for the first time on the one-pot production and purification of fibrinolytic protease from Bacillus cereus by extractive fermentation using natural deep eutectic solvents (NADES). Cheese whey was chosen as a sustainable low-cost production alternative yielding a significantly high amount of protease (185.7 U/mg). Five natural deep eutectic solvents with menthol as hydrogen bond donor and sugar molecules as corresponding hydrogen bond acceptors were synthesized and their association was confirmed with H1 NMR. Thermophysical investigation of the synthetic NADES was accomplished as a function of temperature to define their extraction ability. Response surface methodology based optimization of concentration of NADES (77.5% w/w), Na2SO4 (14% w/v) and cheese whey (1% w/w) were accomplished for extractive fermentation. Further, preparative purification using size exclusion chromatography was used to quantify the amount of enzyme obtained in the extraction phase (190 U/ml). On subsequent purification with an anion exchange column, the maximum purity fold (21.2) with enzyme activity (2,607.8 U/ml) was attained. The optimal pH (8.0), temperature (50 °C) were determined and the in-vitro fibrinolytic activity has been confirmed using a fibrin plate assay.


Assuntos
Bacillus cereus/metabolismo , Fibrinolíticos/metabolismo , Peptídeo Hidrolases/metabolismo , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Fermentação , Fibrinólise , Fibrinolíticos/isolamento & purificação , Humanos , Espectroscopia de Ressonância Magnética , Peptídeo Hidrolases/isolamento & purificação , Solventes
16.
J Chromatogr A ; 1628: 461429, 2020 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-32822971

RESUMO

Recent studies have shown that by combining orthogonal, non-affinity chromatography steps, it is possible to rapidly develop efficient purification processes for molecules of interest. Here, we build upon previous work to develop a flexible framework for identifying resins that remove optimally orthogonal sets of impurities for a wide variety of products. Our approach involves screening a library of proteins with diverse properties (pI ranging from 5.0-11.4 and varying hydrophobicity measured by retention in a HIC gradient) on a library of resins and quantifying each resin's ability to separate every protein pair in the library. Orthogonality is then defined as the degree to which two resins separate mutually exclusive sets of protein pairs. We applied this approach to a library of model proteins and a series of strong, salt tolerant, and multimodal ion exchangers and evaluated which resin combinations performed well and which performed poorly. In particular, we found that strong cation and strong anion exchangers were orthogonal, while strong and salt tolerant anion exchangers were not orthogonal. Interestingly, salt tolerant and multimodal cation exchangers were found to be orthogonal and the best resin combination included a multimodal cation exchange resin and a tentacular anion exchange resin. This approach for quantifying orthogonality is valuable in that it can be used both as a criteria for resin design as well as process design. We envision that, using this framework, it will be possible to design a set of next generation chromatography ligands that are explicitly engineered to optimize separability and orthogonality.


Assuntos
Resinas de Troca de Ânions/química , Resinas de Troca de Cátion/química , Cromatografia por Troca Iônica/métodos , Animais , Bovinos , Galinhas , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Proteínas/análise , Proteínas/química , Sais/química , Suínos
17.
J Chromatogr A ; 1626: 461367, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797846

RESUMO

This paper analyzes the use of animal-component free chromatographic materials for the efficient purification of the human fibroblast growth factor 2 (hFGF-2). hFGF-2 is produced in Escherichia coli and purified via three different chromatography steps, which include a strong cation exchange chromatography as a capture step, followed by heparin affinity chromatography and an anion exchange chromatography as a polishing step. The affinity chromatography step is based on the animal-derived material heparin. Chemically produced ligands provide a viable alternative to animal-derived components in production processes, since they are characterized by a defined structure which leads to reproducible results and a broad range of applications. The alternative ligands can be assigned to adsorber of the mixed-mode chromatography (MMC) and pseudo-affinity chromatography. Eight different animal-component free materials used as adsorbers in MMC or pseudo-affinity chromatography were tested as a substitute for heparin. The MMCs were cation exchangers characterized with further functional residues. The ligands of the pseudo-affinity chromatography were heparin-like ligands which are based on heparin's molecular structure. The alternative methods were tested as a capture step and in combination with another chromatographic step in the purification procedure of hFGF-2. In each downstream step purity, recovery and yield were analysed and compared to the conventional downstream process. Two types of MMC - the column ForesightTM NuviaTM cPrimeTM from Bio-Rad Laboratories and the column HiTrapTM CaptoTM MMC from GE Healthcare Life Sciences - can be regarded as effective animal-component free alternatives to the heparin - based adsorber.


Assuntos
Fator 2 de Crescimento de Fibroblastos/isolamento & purificação , Adsorção , Animais , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Heparina/química , Humanos , Ligantes
18.
J Chromatogr A ; 1626: 461350, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797830

RESUMO

In ionexchange chromatography, the pH gradient mode becomes more and more popular today for the analysis of therapeutic proteins as this mode can provide higher or alternative selectivity to the commonly used salt gradient mode. Ideally, a linear pH response is expected when performing linear gradients. However up to now, only a very few buffer systems have been developed and are commercially available which can perform nearly linear pH responses when flowing through a given column. It is also known that a selected buffer system (mobile phase) can work well on one column but can fail on other column. The goal of this study was to practically evaluate the effects that ionexchange columns (weak and strong exchangers) might have on effluent pH, when performing linear pH gradient separations of therapeutic monoclonal antibodies. To attain this objective, the pH was monitored on-line at the column outlet using a specific setup. To make comprehensive observations of the phenomenon, four different mobile phase conditions and five cation exchange columns (weak and strong exchangers) were employed. The obtained pH responses were systematically compared to responses measured in the absence of the columns. From this work, it has become clear that both the column and mobile phase can have significant effects on pH gradient chromatography and that their combination must be considered when developing a new method. Phase systems (column + mobile phase) providing linear pH responses are indeed the most suitable for separating mAbs with different isoelectric points and, with them, it is possible to elute mAbs across wide retention time ranges and with high selectivity.


Assuntos
Cromatografia por Troca Iônica/métodos , Anticorpos Monoclonais/análise , Cátions/química , Ácido Cítrico/química , Concentração de Íons de Hidrogênio , Troca Iônica , Força Próton-Motriz , Hidróxido de Sódio/química , Taurina/análogos & derivados , Taurina/química
19.
J Chromatogr A ; 1626: 461351, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797831

RESUMO

Traditional polymer-separation methods, such as size-exclusion chromatography and (gradient) liquid adsorption chromatography, cannot provide separations exclusively based on the number of deprotonated carboxylic-acid groups along the backbone chain of polymers. A novel separation method, based on non-aqueous ion-exchange chromatography (NAIEX), was developed, which allows such a separation of acid-functional polymers that are soluble in organic solvents. The polar, aprotic N-methyl-2-pyrrolidone was found to be a suitable solvent. It features a high relative permittivity (favouring dissociation of ion pairs into free ions) and it is a good solvent for polymers and organic salts, such as triethyl-ammonium formate. A negative charge is established on these polymers by deprotonation of the carboxylic-acid groups in the presence of an organic superbase (tetramethyl guanidine). Traditional potent organic bases, such as triethylamine, do not possess the base strength to compensate for the increase in pKa of polymeric carboxylic acid groups in non-aqueous conditions. Triethyl-ammonium formate is proposed as an alternative to traditional salts used for elution in aqueous ion-exchange chromatography. Separation was performed on an industry-standard strong-anion-exchange column and (near-)universal detection of the polymers was performed by high-temperature evaporative-light-scattering detection. The NAIEX method yielded a separation based on the acid-functionality distribution of the polymer. NAIEX was compared with traditional normal- and reversed-phase liquid-chromatography approaches for the separation of acid-functional copolymers.


Assuntos
Cromatografia por Troca Iônica/métodos , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Polímeros/química , Espectroscopia de Prótons por Ressonância Magnética , Sais/química , Solventes/química
20.
J Chromatogr A ; 1628: 461479, 2020 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-32822997

RESUMO

In this study, the thermodynamics of binding of two industrial mAbs to multimodal cation exchange systems was investigated over a range of buffer and salt conditions via a van't Hoff analysis of retention data. Isocratic chromatography was first employed over a range of temperature and salt conditions on three multimodal resins and the retention data were analyzed in both the low and high salt regimes. While mAb retention decreased with salt for all resins at low salts, retention increased at high salts for two of the resins, suggesting a shift from electrostatic to more hydrophobic driven interactions. The retention data at various temperatures were then employed to generate non-linear van't Hoff plots which were fit to the quadratic form of the van't Hoff equation. At low salts, retention of both mAbs decreased with increasing temperature and the van't Hoff plots were concave downward on Capto MMC and Nuvia cPrime, while being concave upward on Capto MMC ImpRes. Different trends were observed on some of the resins with respect to both the concavity of the van't Hoff plots as well as the impact of temperature on the favorable enthalpies in the low salt regime. Interestingly, while increasingly favorable enthalpy with temperature was observed with Capto MMC and Nuvia cPrime at low salt, favorable enthalpy decreased with temperature for Capto MMC ImpRes. At high salts, binding of both mAbs on the two Capto resins were consistently entropically driven, consistent with desolvation. While the negative heat capacity data at low salts indicated that desolvation of polar/charged groups were important in Capto MMC and Nuvia cPrime, the positive data suggested that desolvation of non-polar groups were more important with Capto MMC ImpRes. Finally, the data at high salts indicated that desolvation of non-polar groups was the major driver for binding of both mAbs to the Capto resins under these conditions.


Assuntos
Anticorpos Monoclonais/química , Cromatografia por Troca Iônica/métodos , Adsorção , Cátions , Interações Hidrofóbicas e Hidrofílicas , Dinâmica não Linear , Eletricidade Estática , Termodinâmica
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