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Due to its versatility in formulation and manufacturing, self-emulsifying drug delivery systems (SEDDS) can be used to design parenteral formulations. Therefore, it is necessary to understand the effects of excipients on the behavior of SEDDS formulations upon parenteral administration, particularly their interactions with blood plasma and cell membranes. In this study, we prepared three neutrally charged SEDDS formulations composed of medium-chain triglycerides as the oil phase, polyoxyl-35 castor oil (EL35) and polyethylene glycol (15)-hydroxystearate (HS15) as the nonionic surfactants, medium-chain mono- and diglycerides as the co-surfactant, and propylene glycol as the co-solvent. The cationic surfactant, didodecyldimethylammonium bromide (DDA), and the anionic surfactant, sodium deoxycholate (DEO), were added to the neutral SEDDS preconcentrates to obtain cationic and anionic SEDDS, respectively. SEDDS were incubated with human blood plasma and recovered by size exclusion chromatography. Data showed that SEDDS emulsion droplets can bind plasma protein to different extents depending on their surface charge and surfactant used. At pH 7.4, the least protein binding was observed with anionic SEDDS. Positive charges increased protein binding. SEDDS stabilized by HS15 can adsorb more plasma protein and induce more plasma membrane disruption activity than SEDDS stabilized by EL35. These effects were more pronounced with the HS15 + DDA combination. The addition of DDA and DEO to SEDDS increased plasma membrane disruption (PMD) activities, and DDA (1% w/w) was more active than DEO (2% w/w). PMD activities of SEDDS were concentration-dependent and vanished at appropriate dilution ratios. [ABSTRACT FROM AUTHOR]

During the last years, there has been an increasing research interest in the analysis of biological fluids requiring non-invasive sampling for biomedical and clinical applications. In this work, we have focused on the nasal exudate with the aim of investigating the potential use of this fluid to know the role of iron in stroke and also for diagnosis. Potential differences in the nasal exudate, collected in swabs, from diagnosed hemorrhagic stroke, ischemic stroke, and control groups were investigated with regard to total iron by inductively coupled plasma-mass spectrometry, iron fractionation studies by size exclusion chromatography together with post-column isotope dilution analysis, and four proteins containing iron (ferritin, transferrin, lactoferrin, and ferroportin) with ELISA kits. All these analyses represent an analytical challenge, considering the rather limited amount of sample (10–40 mg) available, being the nasal exudate extracted from the swab with 300 µL 10 mM Tris/HCl, pH = 7.4. Studies to obtain reliable analytical information, such as the blank contribution of the sampling step, evaluation of the extraction efficiency of the nasal exudate from the swab, and normalization strategies for data treatment, have been carried out. Results showed that despite the limited number of investigated samples, fractionation studies as well as the concentrations of ferritin and ferroportin obtained with ELISA kits showed a differential behavior between the different cohorts. [ABSTRACT FROM AUTHOR]

Purposes: Size exclusion chromatography (SEC) is widely used to characterize molecular size variants of antibody drugs. However, SEC analysis is hindered by secondary interactions (or nonspecific interactions) between proteins and stationary phase packing, which result in poor column efficiency. Previous studies have reported that chaotropic salt can inhibit these interactions, but the corresponding applications of this aspect are relatively rare. Therefore, this study introduces a novel approach using sodium iodide (NaI) as a mobile-phase component in SEC and investigates the influence of the mobile-phase composition on secondary interactions. Methods: SEC analysis was performed on one antibody–drug conjugate and four monoclonal antibodies (mAbs) using three different mobile-phase systems (i.e., sodium chloride/L-arginine hydrochloride/NaI mobile phases system) to compare the column efficiency. Subsequently, mAb-1 was used as a model to investigate the effects of these factors on secondary interactions by adjusting the ionic strength (salt concentration) and pH of the NaI mobile-phase system. Results: NaI exhibits superior column efficiency performance in the SEC analysis of most products. The ionic strength will affect nonideal electrostatic and hydrophobic interaction. An appropriate ionic strength can inhibit electrostatic interactions, while an excessive ionic strength increases hydrophobic interactions. pH primarily influences electrostatic interactions. Determining the appropriate pH necessitates consideration of the isoelectric point of the protein and the pH tolerance of the column. Conclusions: In SEC analysis, using NaI as the salt component in the mobile phase reduces secondary interactions and improves column efficiency. This approach is advantageous for samples with intense secondary interactions and is a suitable alternative. [ABSTRACT FROM AUTHOR]

This work deals with the influence of different heat treatment temperatures (140, 150, 160, 170, 180, 190, 200, and 210 °C) on changes in sapwood and red heartwood of European beech (Fagus sylvatica L.). According to the results of wet chemistry methods, HPLC (high-performance liquid chromatography), FTIR (Fourier transform infrared spectroscopy), SEC (size exclusion chromatography), the wood constituents in sapwood and red heartwood behaved similarly to heat treatment, but the individual proportions were different. The loss of hemicelluloses and the increase in extractives with increasing temperature were more pronounced in sapwood. The amount of cellulose in sapwood and red heartwood showed similar behaviour with increasing temperature. Thermal treatment causes changes in cellulose crystallinity, and the formation of aromatic structures, mainly in beech sapwood. However, the increase in the lignin content of red heartwood was significantly lower than that of sapwood due to its auto condensation, and formation of pseudo-lignin. Among the carbohydrates, the most significant changes were observed in xylose content, which was almost twice as high in red heartwood as in sapwood. Other carbohydrates (glucose, mannose, galactose, and arabinose) reached similar values in sapwood and red heartwood. [ABSTRACT FROM AUTHOR]

Extracellular Vesicles (EV) have become an interesting focus as novel biomarkers of disease and are increasingly reported upon in humans and other species. The Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018) guidelines were published to improve rigor and standardisation within the EV field and provide a framework for the reliable isolation and characterisation of EV populations. However, this rigor and standardisation has been challenging in the area of comparative medicine. Herein we present the successful isolation of EVs from human and canine plasma using Size Exclusion Chromatography and characterise these EVs according to best international practice. This study provides evidence for the reliable comparison of human and canine EVs isolated by this approach, and a baseline description of the EVs from healthy dogs to inform future biomarker studies. This work also demonstrates that the MISEV2018 guidelines can be successfully applied to EVs isolated from canine plasma. [ABSTRACT FROM AUTHOR]

Owing to a supposed quantitative transformation, oximation of dialdehyde cellulose (DAC) with hydroxylamine hydrochloride is commonly employed in chemical DAC analysis, e.g., for the determination of the degree of oxidation (DO) by titration or elemental analysis. In this study, this modification was utilized for the indirect determination of molecular weight distributions (MWD) by gel permeation chromatography (GPC). The presumably quantitative conversion of aldehyde groups in DAC to the corresponding oxime also breaks up the intermolecular and intramolecular hemiacetal crosslinks, which were associated with solubility issues in the DMAc/LiCl solvent system in previous studies. The limits of the procedure and the material's stability during oximation were investigated. For samples with a DO up to approximately 9% a good applicability was observed, before at higher DO values residual crosslinks led to solubility problems. The oximation/GPC protocol was used to examine the development of the MWD in the early stages of DAC formation under different reaction conditions. The time-dependent partial depolymerization of the polymer backbone was observed. Furthermore, the stability of DAC towards different pH conditions ranging from strongly acidic to strongly alkaline was tested. The depolymerization of DAC in alkaline media occurred with concomitant degradation of aldehyde moieties. In turn, DAC proved to be remarkably stable in acidic and neutral solutions up to a pH of 7. [ABSTRACT FROM AUTHOR]

Blood extracellular vesicles (EVs) play essential roles in cell–cell communication and their molecular cargo is a promising source of disease biomarkers. However, proteomic characterization of plasma-derived EVs is challenged by the presence of highly abundant plasma proteins, which limits the detection of less abundant proteins, and by the low number of EVs in biological fluids. The aim of this study was to investigate if the removal of abundant plasma proteins prior to EV isolation could improve plasma-derived EV characterization by LC–MS/MS and expand the proteome coverage. Plasma depletion was performed using a single-use spin column and EVs were isolated from only 100 µL of non-depleted and depleted plasma by size exclusion chromatography. Afterwards, EVs were characterized by nanoparticle tracking analysis and mass spectrometry–based proteomics using a data-independent acquisition approach. Depleted plasma-derived EVs had higher particle concentrations and particle-to-protein ratios. Depletion did increase the protein coverage with a higher number of identifications in EVs from depleted plasma (474 proteins) than from non-depleted (386 proteins). However, EVs derived from non-depleted plasma carried a slightly higher number of common EV markers. Overall, our findings suggest that plasma depletion prior to EV isolation by size exclusion chromatography provides higher yield and protein coverage, but slightly lower identification of EV markers. This study also showed the possibility to characterize the proteome of EVs derived from small plasma volumes, encouraging the clinical feasibility of the discovery of EV biomarkers. [ABSTRACT FROM AUTHOR]

Biopolymers, polymers derived from renewable biomass sources, have gained increasing attention in recent years due to their potential to replace traditional petroleum-based polymers in a range of applications. Among the many advantages of biopolymers can be included their biocompatibility, excellent mechanical properties, and availability from renewable feedstock. However, the development of biopolymers has been limited by a lack of understanding of their properties and processing behaviours. Continuous analysis techniques have the potential to hasten progress in this area by providing real-time insights into the properties and processing of biopolymers. Significant research in polymer chemistry has focused on petroleum-derived polymers and has thus provided a wealth of synthetic and analytical methodologies which may be applied to the biopolymer field. Of particular note is the application of flow technology in polymer science and its implications for accelerating progress towards more sustainable and environmentally friendly alternatives to traditional petroleum-based polymers. In this mini review we have outlined several of the most prominent use cases for biopolymers along with the current state-of-the art in continuous analysis of polymers in flow, including defining and differentiating atline, inline, online and offline analysis. We have found several examples for continuous flow analysis which have direct application to the biopolymer field, and we demonstrate an atline continuous polymer analysis method using size exclusion chromatography. [ABSTRACT FROM AUTHOR]

Biopharmaceuticals are large, complex and labile therapeutic molecules prone to instability due to various factors during manufacturing. To ensure their safety, quality and efficacy, a wide range of critical quality attributes (CQAs) such as product concentration, aggregation, particle size, purity and turbidity have to be met. Size exclusion chromatography (SEC) is the gold standard to measure protein aggregation and degradation. However, other techniques such as dynamic light scattering (DLS) are employed in tandem to measure the particle size distribution (PSD) and polydispersity of biopharmaceutical formulations. In this study, the application of multi-angle dynamic light scattering (MADLS) was evaluated for the determination of particle size, particle concentration and aggregation in 3 different protein modalities, namely bovine serum albumin (BSA) and two biopharmaceuticals including a monoclonal antibody (mAb) and an enzyme. The obtained calibration curve (R2 > 0.95) for the particle number concentration of the 3 proteins and the observed correlation between MADLS and SEC (R2 = 0.9938) for the analysis of aggregation in the enzyme can be employed as a 3-in-1 approach to assessing particle size, concentration and aggregation for the screening and development of products while also reducing the number of samples and experiments required for analysis prior to other orthogonal tests. [ABSTRACT FROM AUTHOR]

This study focused on the bio-characterization of a GH38 α-mannosidase from the hyperthermophile Pseudothermotoga thermarum DSM 5069. We aimed to successfully express and characterize this thermophilic α-mannosidase and to assess its functional properties. Subsequently, recombinant α-mannosidase PtαMan was expressed in Escherichia coli BL21(DE3) and purified via affinity chromatography, and native protein was verified as a tetramer by size exclusion chromatography. In addition, the activity of α-mannosidase PtαMan was relatively stable at pH 5.0–6.5 and temperatures up to 75 ℃. α-Mannosidase PtαMan was active toward Co2+ and had a good catalytic efficiency deduced from the kinetic parameters. However, its activity was strongly inhibited by Cu2+, Zn2+, SDS, and swainsonine. In summary, this cobalt-required α-mannosidase is putatively involved in the direct modification of glycoproteins. [ABSTRACT FROM AUTHOR]

The small-angle X-ray scattering method allows studying the structure of solutions of proteins, polymers, and metal nanoparticles in the range of 1–200 nm. The development of new and improvement of the available algorithms for the analysis of experimental small-angle X-ray scattering data is an important task. This study presents a number of algorithms that make it possible to find the particle size distribution functions, restore the intensity profiles of individual components in protein mixtures, and estimate the size of the region of crystallinity and spacing distances in partially ordered systems. A number of algorithms are implemented as programs using the Qt cross-platform graphics library, which greatly expands the number of their potential users. The efficiency of the algorithms has been demonstrated on a number of theoretical and experimental small-angle X-ray scattering data. [ABSTRACT FROM AUTHOR]

Synthetic polymers used in various technical applications were analyzed by asymmetric flow field flow fractionation (AF4) and size exclusion chromatography size exclusion without dash is more frequent (SEC) followed by a multi-angle light scattering (MALS) photometer and the results were compared from the view point of the resolution of the two methods and their ability to determine the molar mass distribution. The analyzed polymers covered broad molar mass range and included linear, randomly branched and star-shaped molecular topologies. Narrow polystyrene standards were used to determine the molar mass dependence of the efficiency. The number-average molar masses obtained by SEC-MALS were systematically lower compared to those from AF4-MALS with the exception of highly branched polymers, whereas the weight-averages and z-averages were well consistent except for polymers containing ultra-high molar mass fractions undergoing shear degradation in SEC columns. The number of theoretical plates in AF4 was molar mass-dependent as predicted by the theory. Using linear cross flow decay, the molar mass dependence of the AF4 selectivity showed an arc-shaped pattern. The AF4 mass recovery was close to 100% for all samples not containing molecules below the cut-off of the channel membrane. [ABSTRACT FROM AUTHOR]

Purpose: Host cell proteins (HCPs) are impurities derived from expression systems during the manufacturing of biotherapeutics. Even trace amounts of certain HCPs can potentially compromise product safety and quality. Therefore, comprehensive analytical characterization is necessary. In particular, understanding how each HCP co-purifies with the biotherapeutics throughout the purification process would help guide process development to avoid further contamination. Methods: We developed a new strategy based on size exclusion chromatography (SEC) fractionation followed by mass spectrometry (MS) analysis to study HCPs. Results: Through an optimized experimental procedure, HCPs were effectively separated from monoclonal antibody (mAb) drug substances via SEC fractionation and sensitively detected with MS. Many HCPs were enriched in the high molecular weight fraction, thus indicating the formation of HCP-mAb complexes. SEC separation under mild denaturing conditions was demonstrated to disrupt weak interactions between certain HCPs and mAbs. The binding profiles of HCPs to mAbs were further characterized through comparison of the relative abundance of HCPs in each fraction under either native or mild denaturing SEC conditions. Conclusions: This new method not only achieves improved identification of HCPs in biotherapeutic drug substances but also offers an effective means to evaluate the binding properties between biotherapeutics and a wide range of HCPs. [ABSTRACT FROM AUTHOR]

Carbonic anhydrases (CA, EC 4.2.1.1) catalyze the hydration of carbon dioxide and take part in many essential physiological processes. In humans, 15 CAs are characterized, including the only secreted isoenzyme CA VI. CA VI has been linked to specific processes in the mouth, namely bitter taste perception, dental caries, and maintenance of enamel pellicle, and implicated in several immunity-related phenomena. However, little is known of the mechanisms of the above. In this study, we characterized human CA VI purified from saliva and milk with biophysical methods and measured their enzyme activities and acetazolamide inhibition. Size-exclusion chromatography showed peaks of salivary and milk CA VI corresponding to hexameric state or larger at pH 7.5. At pH 5.0 the hexamer peaks dominated. SDS- PAGE of milk CA VI protein treated with a bifunctional crosslinker further confirmed that a majority of CA VI is oligomers of similar sizes in solution. Mass spectrometry experiments confirmed that both of the two putative N-glycosylation sites, Asn67 and Asn256, are heterogeneously glycosylated. The attached glycans in milk CA VI were di- and triantennary complex-type glycans, carrying both a core fucose and 1 to 2 additional fucose units, whereas the glycans in salivary CA VI were smaller, seemingly degraded forms of core fucosylated complex- or hybrid-type glycans. Mass spectrometry also verified the predicted signal peptide cleavage site and the terminal residue, Gln 18, being in pyroglutamate form. Thorough characterization of CA VI paves way to better understanding of the biological function of the protein. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) represent a major category of biopharmaceutical products which due to their success as therapeutics have recently experienced the emergence of mAbs originating from different types of trafficking. We report the development of an analytical strategy which enables the structural identification of mAbs in addition to comprehensive characterization and quantification in samples in potentially counterfeit samples. The strategy is based on the concomitant use of capillary zone electrophoresis analysis (CZE-UV), size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and liquid chromatography hyphenated to tandem mass spectrometry (LC–MS/MS). This analytical strategy was applied to the investigation of different samples having unknown origins seized by the authorities, and potentially incorporating an IgG 4 or an IgG 1. The results achieved from the different techniques demonstrated to provide orthogonal and complementary information regarding the nature and the structure of the different mAbs. Therefore, they allowed to conclude unequivocally on the identification of the mAbs in the potentially counterfeit samples. Finally, a LC–MS/MS quantification method was developed which specificity was to incorporate a different mAbs labeled with stable isotopes as internal standard. The LC–MS/MS quantification method was validated and thus demonstrated the possibility to use common peptides with the considered IgG in order to achieve limit of quantification as low as 41.4 nM. The quantification method was used to estimate the concentration in the investigated samples using a single type of internal standard and experimental conditions, even in the case of mAbs with no stable isotope labeled homologues available. [ABSTRACT FROM AUTHOR]

Oil-immersed paper insulation and paper pressboards for structural support are widely used in electrical power transformers. Cellulose thus fulfills an essential task for the smooth power supply of our societies. However, the prevailing temperatures in such equipment, combined with a targeted service life of several decades, pose a serious challenge to the long-term integrity of cellulosic paper insulation. Therefore, numerous studies have been conducted to obtain kinetic data on the degradation processes that contribute to the thermally induced decomposition of cellulose. These studies usually rely on the assessment of the average degree of polymerization by viscosity measurements. In this work, we applied and optimized more advanced methods for the characterization of cellulosic materials based on gel permeation chromatography for the special case of thermally stressed unbleached Kraft paper samples. This allowed studying the molar mass distributions of paper polymers upon exposure to heat, as well as the investigation of changes in their conformation in solution and the observation of thermally induced cross-linking. In combination with group-selective fluorescence labeling, it was possible to track over time the changes in molar mass-dependent profiles of carbonyl and carboxyl groups of authentic Kraft insulator paper samples under thermal stress. In addition, changes of the hemicellulose composition were quantified. We hope that this analytical approach to the in-depth characterization of thermally stressed insulator paper will prove useful for future studies of this important cellulose product, and that our findings will contribute to a better understanding of the thermal decomposition of paper in general. [ABSTRACT FROM AUTHOR]

The iconic Tasmanian devil (Sarcophilus harrisii) is endangered due to the transmissible cancer Devil Facial Tumour Disease (DFTD), of which there are two genetically independent subtypes (DFT1 and DFT2). While DFT1 and DFT2 can be differentially diagnosed using tumour biopsies, there is an urgent need to develop less-invasive biomarkers that can detect DFTD and distinguish between subtypes. Extracellular vesicles (EVs), the nano-sized membrane-enclosed vesicles present in most biofluids, represent a valuable resource for biomarker discovery. Here, we characterized the proteome of EVs from cultured DFTD cells using data-independent acquisition–mass spectrometry and an in-house spectral library of > 1500 proteins. EVs from both DFT1 and DFT2 cell lines expressed higher levels of proteins associated with focal adhesion functions. Furthermore, hallmark proteins of epithelial–mesenchymal transition were enriched in DFT2 EVs relative to DFT1 EVs. These findings were validated in EVs derived from serum samples, revealing that the mesenchymal marker tenascin-C was also enriched in EVs derived from the serum of devils infected with DFT2 relative to those infected with DFT1 and healthy controls. This first EV-based investigation of DFTD increases our understanding of the cancers' EVs and their possible involvement in DFTD progression, such as metastasis. Finally, we demonstrated the potential of EVs to differentiate between DFT1 and DFT2, highlighting their potential use as less-invasive liquid biopsies for the Tasmanian devil. [ABSTRACT FROM AUTHOR]

A method for determining residual protein in commercial human milk oligosaccharides is described. Size exclusion chromatography is used to separate any protein from the oligosaccharides, and UV absorbance is used to estimate its concentration. The analysis is calibrated with casein-spiked oligosaccharide solutions, with protein mass fractions at 5 mg/kg–100 mg/kg of oligosaccharide (or when expressed as mass/volume of calibration solution, 0.75 mg/L–15 mg/L). Method performance was defined by assessments of linearity (correlation coefficient ≥ 0.998; residuals from -2.9 % to -5.6 %); reproducibility (analyses of multiple lots of oligosaccharides, with and without a quantifiable protein presence); accuracy (verification by three alternate methods); selectivity (reagent blanks and peak area ratio); and limits of quantitation and detection (~ 5 mg/kg and ~ 2 mg/kg, respectively, as mass fraction protein/oligosaccharide). The method provides a simple means for determining residual protein in oligosaccharides, capable of quantifying low protein levels (e.g., 5 mg/kg oligosaccharide) in the presence of a high oligosaccharide concentration (150 g/L, the HMO mass/volume in the prepared sample) by conventional LC/UV. [ABSTRACT FROM AUTHOR]

The development and applications of asymmetric flow field-flow fractionation (AF4) are outlined in comparison with the older and better-known size exclusion chromatography. Recent advances in AF4 instrumentation and predictions for further progress of the technique are given. [ABSTRACT FROM AUTHOR]

Abstract Proteins are major effectors and regulators of biological processes that can elicit multiple functions depending on their interaction with other proteins. The organization of proteins into macromolecular complexes and their quantitative distribution across these complexes is, therefore, of great biological and clinical significance. In this paper, we describe an integrated experimental and computational technique to quantify hundreds of protein complexes in a single operation. The method consists of size exclusion chromatography (SEC) to fractionate native protein complexes, SWATH/DIA mass spectrometry to precisely quantify the proteins in each SEC fraction, and the computational framework CCprofiler to detect and quantify protein complexes by error‐controlled, complex‐centric analysis using prior information from generic protein interaction maps. Our analysis of the HEK293 cell line proteome delineates 462 complexes composed of 2,127 protein subunits. The technique identifies novel sub‐complexes and assembly intermediates of central regulatory complexes while assessing the quantitative subunit distribution across them. We make the toolset CCprofiler freely accessible and provide a web platform, SECexplorer, for custom exploration of the HEK293 proteome modularity.

Both angiotensin converting enzyme (ACE) and carbonic anhydrase III (CAIII) are zinc-containing enzymes. Interestingly, blocking of the both enzymes is attributed with clinically significant outcomes against hyperlipidemia and obesity. An optimized in-vitro screening approach based on HPLC–size exclusion chromatography was adopted to study the angiotensin converting enzyme inhibitors (ACEIs) affinity against CAIII enzyme. Series of concentrations of the enzyme were injected in the column with a mobile phase containing one of ACEIs in each time. The affinity of the ACEIs toward CAIII was characterized by vacancy (negative) peak whose intensity representing the fraction of the drug bound with CAIII. To explore whether the binding is within the binding site or just a promiscuous binding, exact procedure was repeated with apo-protein part of CAIII. Furthermore, an esterase activity of CAIII was performed to determine if the binding with ACEIs is excitatory or inhibitory. It has been found that ACEIs have real in-vitro inhibitory effects against CAIII at a micro-molar level. The chromatographic study revealed that the ionized carboxylate groups are essential for binding with Zn+2 ion in the enzyme active site. Among the four tested ACEIs, captopril, ramipril, enalapril, and lisinopril, captopril was found to be the most potent inhibitor with Ki = 12.1 μM, while lisinopril is the least potent one with Ki = 25.3 μM. This finding might open the door for further investigation to optimize ACEIs as lead compounds for the discovery and development of selective and potent CAIII inhibitors. [ABSTRACT FROM AUTHOR]

Glycogen is conventionally considered as a transient energy reserve that can be rapidly synthesized for glucose accumulation and mobilized for ATP production. However, this conception is not completely applicable to prokaryotes due to glycogen structural heterogeneity. A number of studies noticed that glycogen with small average chain length gc in bacteria has the potential to degrade slowly, which might prolong bacterial environment survival. This phenomenon was previously examined and later formulated as the durable energy storage mechanism hypothesis. Although recent research has been warming to the hypothesis, experimental validation is still missing at current stage. In this review, we summarized recent progress of the hypothesis, provided a supporting mathematical model, and explored the technical pitfalls that shall be avoided in glycogen study. [ABSTRACT FROM AUTHOR]

The comprehensive branching analysis of complex polymers is still a challenge in advanced polymer analysis. Average branching information (average number and length of branches) can be obtained by spectroscopic methods, mainly NMR spectroscopy. The determination of the branching distribution, i.e., the concentration of macromolecules with a given number of branches, however, requires fractionation. Typically, size exclusion chromatography is used that separates the complex mixture with regard to molecular size in solution and not strictly with regard to the number of branches. In the present approach, model star-shaped polystyrenes were synthesized with a pre-determined architecture to give theoretical three-arm, four-arm, and six-arm structures. The branched samples were compared with a linear analogue of comparable molar mass known not to contain branching. Triple detector size exclusion chromatography with refractive index, multiangle light scattering, and online viscometer detection was used to determine absolute molar masses, radii of gyration, and branching distributions of the star-shaped polymers. 1H-NMR was used to calculate the average functionality and a reasonable agreement between the results of the two methods was obtained. Thermal gradient interaction chromatography and solvent gradient interaction chromatography were employed to separate the complex reaction products according to chemical composition (number of branches) and to resolve by-products. The separation capabilities of the two chromatographic techniques were compared and evaluated. Comprehensive two-dimensional liquid chromatography was used to separate the polydisperse star-shaped polystyrenes with regard to both branching and molar mass. [ABSTRACT FROM AUTHOR]

Dynamic light scattering and size exclusion chromatography were used to investigate the size of sodium lignosulfonate particles in water—ethanol media and the changes occurring at temperatures of 115–440 °C (13 MPa). It was found that the characteristic size (diameter) of particles of the starting sodium lignosulfonate in solutions with a concentration of 1.5 and 13.3 g L−1 was 2.2−5.2 nm. Treatment in a flow reactor at temperature of 235–325 °C led to a disintegration of larger particles with an increase in the fraction of particles with size of 2.2 nm and a simultaneous formation of particles with a size of ~100 nm. Increasing the treatment temperature (>415 °C) led to a complete disappearance of particles with size of 2.2–5.2 nm and the formation of particles 100–130 nm in diameter, as well as the precipitation of large soot-like particles. IR spectroscopy showed that increasing the treatment temperature above 275 °C led to a decrease in the specific content of oxygen-containing and sulfoxyl groups in the sodium lignosulfonate structure, with the exception of carboxyl groups, whose fraction remained almost unchanged, as well as CH3 groups (methoxy and aliphatic), whose content increased slightly. The temperature range of 235–275 °C was optimal for hydrolysis of sodium lignosulfonate and preparation of aromatic monomers. [ABSTRACT FROM AUTHOR]

In the present analytical workflow, chromatographic methods have been developed and hyphenated to mass spectrometry (MS) for the characterization of protein size, charge, hydrophobic, and hydrophilic variants of daratumumab. Multiple critical quality attributes (CQAs) were characterized in forced degraded daratumumab sample, using size exclusion, ion exchange (IEX), and hydrophobic interaction (HIC) chromatography coupled to fluorescence detection for relative quantification and fractionation. Mass assignment was performed by using a fast, non-denaturing and universal size exclusion chromatography (SEC) method prior to native MS analysis of the collected fractions (off-line approach). This allowed the identification of N-terminal lysine clipping, and the extent of glycation and oxidation at intact protein level. Finally, middle-up analysis of daratumumab was performed using reversed phase (RPLC) and hydrophilic interaction (HILIC) chromatography coupled to MS to obtain a comprehensive overview of all PTMs after the forced stressed conditions and a fine characterization of the glycosylation profile. Conveniently, the presented workflow maintains the established golden standard non-denaturing chromatography techniques and additionally introduces a straightforward and automated desalting procedure prior to MS analysis. Therefore, it is expected that the off-line coupling of SEC, IEX, and HIC to SEC-MS has great potential to be implemented in routine characterization of mAbs. [ABSTRACT FROM AUTHOR]

Exosomes are cell-derived functional microparticles which exist in most body fluids. They carry abundant signaling molecules to transfer information between cells and microenvironment. Research on exosomes' heterogeneity and constitute variations has been a heated topic in recent years. In this work, size-dependent sub-proteome analysis of urinary exosomes was investigated by size exclusion chromatography (SEC) firstly. The particle size of urinary exosomes is distributed in four main ranges naturally. We found out that these fractions contained sub-proteomes with great difference in constitution. In each fraction, 206, 134, 157, and 276 unique proteins were identified by LC-MS/MS. Differential expression of exosomal markers such as TSG101, CD9, CD63, and caveolin-1 was observed in these fractions by western blots. Biological function annotation indicated that the proteins identified in each fraction were involved in different molecular and cellular processes. It is proven that SEC can serve as an efficient analytical tool for exosomes isolation and fractionation. This work provides a new strategy to classify exosomes into sub-populations for comprehensive study of heterogeneous functionalities. [ABSTRACT FROM AUTHOR]

Erythropoietin (EPO) is a glycoprotein hormone which promotes red cell replenishment and is also a global biotherapeutic medicine widely used to treat anaemia resulting, for example, from chemotherapy. Requirements of the European Pharmacopoeia stipulate that the level of dimer must be quantified in clinical EPO products (with a limit of 2%). Quantification is hampered by the lack of reference preparations containing stable measurable levels of EPO dimer, but the reproducible generation of a stable dimerised EPO preparation is challenging. We describe here the development of a lyophilised, chemically cross-linked EPO preparation, which has good stability and may be used for calibration and system suitability assurance for the size exclusion chromatographic separation of EPO preparations. [ABSTRACT FROM AUTHOR]

The aim of this research was to develop a method for simultaneous quantification of proteins and main polyphenolic compounds extracted from oleaginous meal by aqueous media. Size exclusion chromatography with a Biosep column (exclusion range from 1 to 300 kDa) and acetonitrile/water/formic acid (10:89.9:0.1 v/v) eluent at 0.6 mL min−1 yielded the most efficient separation of sunflower proteins and chlorogenic acid monoisomers (3-caffeoylquinic acid, 5-caffeoylquinic acid, and 4-caffeoylquinic acid). After a study of the stability of the extract components, the incorporation of a stabilization buffer (0.5 mol L−1 tris(hydroxymethyl)aminomethane-hydrochloric acid/1.0 mol L−1 sodium chloride at pH 7) was proposed to avoid polyphenol-protein interactions and/or isomeric transformation. The use of 214 nm as the wavelength for protein quantification was also included to minimize the effect of interference from polyphenol-protein interactions on the quantification. Under the used experimental conditions, the protein and chlorogenic acid monoisomer signals remained stable during 300 min at 20 °C (95–125% of the starting value). The developed method was validated and parameters such as specificity, sensitivity, precision, and accuracy were determined. The results from size exclusion chromatography correlated well with the results of protein determination by the reference Kjeldahl method. The proposed method was successfully applied for rapeseed extract analysis making simultaneous quantification of proteins and major rapeseed polyphenols (sinapine and sinapic acid) possible. Graphical abstract [ABSTRACT FROM AUTHOR]

A simple and sensitive stability-indicating size exclusion chromatography method was developed and validated for the quantitative analysis of cetuximab. The effect of variety of parameters including mobile phase composition, pH, flow rate and injection volume was investigated to achieve acceptable peak resolution and the optimum condition was selected. The proposed method was validated in accordance with the International Conference on Harmonization guidelines. Method validation showed good linearity over the concentration range of 1.56–250 µg mL−1 (r2 = 0.9997), acceptable precision (relative standard deviations < 2.8%) and accuracy (recovery of 97.6–99.5%). The limits of detection and quantitation were 0.34 µg mL−1 and 1.03 µg mL−1, respectively. The robustness of the method was evaluated by small variation in buffer composition, buffer pH and flow rate and was determined to be acceptable. Assessment of the specificity and stability-indicating capability of the method using thermally stressed, photo degraded, acidic and oxidative stressed samples revealed no interference between cetuximab and excipients or force degradation products. Furthermore, evaluation of bioactivity of stressed samples showed significant differences (p < 0.05). The proposed method could be utilized as a precise and robust stability-indicating method which can be reproduced in any labs for high-throughput quantitative analysis, stability monitoring and quality control of cetuximab in pharmaceutical formulation. [ABSTRACT FROM AUTHOR]

Abstract: Two commercial carboxymethyl celluloses (CMC) with degree of substitution (DS) 0.75 and 0.77, respectively, have been submitted to exhaustive methanolysis until the amount of the insoluble residue did not further decrease. Dissolved and insoluble material were determined gravimetrically and further analyzed with respect to their substituent distribution in the glucosyl units by capillary electrophoresis, and by size exclusion chromatography. While the substitution of the soluble fraction was slightly above the average DS and in accordance with a random distribution (Spurlin), unsubstituted glucose was steadily enriched in the retained solid material (10 and 20 mol%), resulting in a final DS of 0.46 and 0.42, respectively. An "excess" of unsubstituted glucose is mainly responsible for the heterogeneity of this portion, since deviation from the Spurlin model could be minimized by reducing the molar fraction of glucosyl units. Estimation of the DS/DP-distribution profile of the insoluble residue from molar mass and substituent distribution data, and comparison with the profile of the original CMC (DS 0.75) showed that roughly only about half of the residue could be covered by sequences of the original CMC if random substitution is assumed. This indicates that there is a certain portion of unsubstituted domains in a low substituted heterogeneous part of the CMC. By this procedure, low or unsubstituted areas as a minor part of the CMC material can be concentrated and heterogeneity detected with higher sensitivity.Graphical abstract: [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are highly potent biopharmaceuticals designed for targeted cancer therapies. mAbs and ADCs can undergo modifications during production and storage which may affect binding to target receptors, potentially altering drug efficacy. In this work, liquid chromatography was coupled online to surface plasmon resonance (LC-SPR) to allow label-free affinity evaluation of mAb and ADC sample constituents (size and charge variants), under near-native conditions. Trastuzumab and its ADC trastuzumab emtansine (T-DM1) were used as a test sample and were analyzed by aqueous size-exclusion chromatography (SEC)-SPR before and after exposure to aggregate-inducing conditions. SEC-SPR allowed separation of the formed aggregates and measurement of their affinity towards the ligand-binding domain of the human epidermal growth factor receptor 2 (HER2) receptor immobilized on the surface of the SPR sensor chip. The monomer and aggregates of the mAb and ADC were shown to have similar antigen affinity. Conjugation of drugs to trastuzumab appeared to accelerate the aggregate formation. In addition, cation-exchange chromatography (CEX) was coupled to SPR enabling monitoring the maximum ligand-analyte binding capacity (Rmax) of individual charge variants present in mAbs. Deamidated species and lysine variants in trastuzumab sample were separated but did not show different binding affinities to the immobilized HER2-binding domain. In order to allow protein variant assignment, parallel MS detection was added to the LC-SPR setup using a column effluent split. The feasibility of the LC-MS/SPR system was demonstrated by analysis of trastuzumab and T-DM1 providing information on antibody glycoforms and/or determination of the drug-to-antibody ratio (DAR), while simultaneously monitoring binding of eluting species to HER2.ᅟ [ABSTRACT FROM AUTHOR]

Key message: Euglena gracilis is a unicellular microalga showing characteristics of both plants and animals, and extensively used as a model organism in the research works of biochemistry and molecular biology. Biotechnological applications of E. gracilis have been conducted for production of numerous important compounds. However, chitin-mediated defense system intensively studied in higher plants remains to be investigated in this microalga. Recently, Taira et al. (Biosci Biotechnol Biochem 82:1090-1100, 2018) isolated a unique chitinase gene, comprising two catalytic domains almost homologous to each other (Cat1 and Cat2) and two chitin-binding domains (CBD1 and CBD2), from E. gracilis. We herein examined the mode of action and the specificity of the recombinant Cat2 by size exclusion chromatography and NMR spectroscopy. Both Cat1 and Cat2 appeared to act toward chitin substrate with non-processive/endo-splitting mode, recognizing two contiguous N-acetylglucosamine units at subsites − 2 and − 1. This is the first report on a chitinase having two endo-splitting catalytic domains. A cooperative action of two different endo-splitting domains may be advantageous for defensive action of the E. gracilis chitinase.Abstract: The unicellular alga, E. gracilis, produces a chitinase consisting of two GH18 catalytic domains (Cat1 and Cat2) and two CBM18 chitin-binding domains (CBD1 and CBD2). Here, we produced a recombinant protein of the Cat2 domain to examine its mode of action as well as specificity. Cat2 hydrolyzed N-acetylglucosamine (A) oligomers (An, n = 4, 5, and 6) and partially N-acetylated chitosans with a non-processive/endo-splitting mode of action. NMR analysis of the product mixture from the enzymatic digestion of chitosan revealed that the reducing ends were exclusively A-unit, and the nearest neighbors of the reducing ends were mostly A-unit but not exclusively. Both A-unit and D-unit were found at the non-reducing ends and the nearest neighbors. These results indicated strong and absolute specificities for subsites − 2 and − 1, respectively, and no preference for A-unit at subsites + 1 and + 2. The same results were obtained from sugar sequence analysis of the individual enzymatic products from the chitosans. The subsite specificities of Cat2 are similar to those of GH18 human chitotriosidase, but differ from those of plant GH18 chitinases. Since the structures of Cat1 and Cat2 resemble to each other (99% similarity in amino acid sequences), Cat1 may hydrolyze the substrate with the same mode of action. Thus, the E. gracilis chitinase appears to act toward chitin polysaccharide chain through a cooperative action of the two endo-splitting catalytic domains, recognizing two contiguous A-units at subsites − 2 and − 1. [ABSTRACT FROM AUTHOR]

The aim of the present work was to experimentally evaluate an alternative advanced wastewater treatment system, which combines the action of photocatalytic oxidation with ceramic membrane filtration. Experiments were carried out using laboratory scale TiO2/UV photocatalytic reactor and tubular ceramic microfiltration (CMF) system to treat the secondary effluent (SE). A 100-nm pore size CMF membrane was investigated in cross flow mode under constant transmembrane pressure of 20 kPa. The results show that specific flux decline of CMF membrane with and without TiO2/UV photocatalytic treatment was 30 and 50%, respectively, after 60 min of filtration. Data evaluation revealed that the adsorption of organic compounds onto the TiO2 particles was dependent on the pH of the suspension and was considerably higher at low pH. The liquid chromatography-organic carbon detector (LC-OCD) technique was used to characterise the dissolved organic matter (DOM) present in the SE and was monitored following photocatalysis and CMF. The results showed that there was no removal of biopolymers and slight removal of humics, building blocks and the other oxidation by-products after TiO2/UV photocatalytic treatment. This result suggested that the various ions present in the SE act as scavengers, which considerably decrease the efficiency of the photocatalytic oxidation reactions. On the other hand, the CMF was effective for removing 50% of biopolymers with no further removal of other organic components after photocatalytic treatment. Thus, the quantity of biopolymers in SE has an apparent correlation with the filterability of water samples in CMF. [ABSTRACT FROM AUTHOR]

Biopharmaceutical drugs are large, complex protein molecules derived from living cells. The characterization of the protein-based products is a complicated and challenging task necessary to guarantee their efficacy and safety. A major concern in manufacturing protein biopharmaceuticals is their propensity to form aggregates. The separation of proteins by size-exclusion chromatography (SEC) is a well-established method for the characterization of macromolecules and their aggregates. The demand for increased resolution and faster analysis in SEC has led to the development of small, more rigid fully porous particles and superficially porous particles with different pore sizes. The coupling of SEC with three or four detectors has enhanced protein characterization by generating accurate molecular weights (absolute determination) and by giving insight into the degradation pathways and products associated with aggregation. The intent of this review is to report the continuous developments of SEC stationary phases and their applications on the characterization of intact proteins, their variants or aggregates and protein complexes. Thus, the most recent applications of SEC with the focus on proteins and protein aggregates are reported and discussed. [ABSTRACT FROM AUTHOR]

Zirconium-89 (Zr) is an interesting immuno-PET imaging agent due to its favorable physical characteristics of 78.4 h half-life and a relatively low positron energy of 0.9 MeV. Radiochemical separation of Zr from irradiated yttrium target matrix has been achieved by different separation methodologies like anion-exchange chromatography, cation-exchange chromatography and size exclusion chromatography (Sephadex G-25 resin). A novel and simple method based on size exclusion chromatography (Sephadex G-25) for radiochemical separation of Zr with no harmful substances has been explored. The experimental yield of Zr from natural yttrium target was found to be 27 MBq/μAh using 12 MeV proton. The radiochemical separation yield, radionuclidic purity and chemical purity of Zr using an anion exchanger, Dowex-1 resin/2 M HCl column system and Sephadex G-25 resin column was found to be high enough. [ABSTRACT FROM AUTHOR]

Rapid estimation of the degree of polymerization (DP) of pure tannins or mean degree of polymerization (mDP) of tannin samples is an analytical challenge and a necessity to better understand their bioactivity. In the present paper, four spectrophotometric analyses and normal phase HPLC/MS were performed to estimate mDP of tannin fractions from pecan nut ( Carya illinoinesis) kernels and shells. Tannins were obtained by acetone extraction of phenolic compounds followed by size exclusion chromatography fractionation. Four final fractions were obtained, and their mDP was estimated by spectrophotometric and HPLC/MS analysis and results compared among the different methods. All assays showed good reproducibility. The combination of butanol-HCl depolymerization reaction with vanillin-acetic acid assay yielded mDP values equal to those estimated by HPLC/MS (alpha = 5%), proving this method both suitable and reliable for rapid mDP estimation of tannins in samples. [ABSTRACT FROM AUTHOR]

Four new green deep eutectic solvents (DESs) based on betaine were prepared. In addition, one conventional (i.e., not synthesized using DESs) and four betaine-based mesoporous materials were generated by hydrothermal synthesis. All of the mesoporous materials had spherical shapes. The proposed mesoporous materials were characterized by scanning electron microscopy, nitrogen sorption analysis, and thermogravimetric analysis, and were used as sorbents in high-performance size-exclusion chromatography to separate polysaccharides. Six dextrans with different molecular weights (5, 25, 50, 270, 670, and 1100 kDa) were then applied as target compounds for separation. The DES-based mesoporous materials provided better separation of the dextrans than the conventional mesoporous materials, and the betaine-urea-based column was found to give the best results, especially for dextrans <270 kDa in molecular weight. These results indicate that the presence of a betaine-based DES during mesoporous material synthesis had a positive influence on the size-exclusion separation capabilities of the resulting material. Graphical abstract: [ABSTRACT FROM AUTHOR]

Elements derived from lentiviral particles such as viral vectors or virus-like particles are commonly used for biotechnological and biomedical applications, for example in mammalian protein expression, gene delivery or therapy, and vaccine development. Preparations of high purity are necessary in most cases, especially for clinical applications. For purification, a wide range of methods are available, from density gradient centrifugation to affinity chromatography. In this study we have employed size exclusion columns specifically designed for the easy purification of extracellular vesicles including exosomes. In addition to viral marker protein and total protein analysis, a well-established single-particle characterization technology, termed tunable resistive pulse sensing, was employed to analyze fractions of highest particle load and purity and characterize the preparations by size and surface charge/electrophoretic mobility. With this study, we propose an integrated platform combining size exclusion chromatography and tunable resistive pulse sensing for monitoring production and purification of viral particles. [ABSTRACT FROM AUTHOR]

The effects of amino acid composition and peptide molecular mass on ACE-inhibitory and antioxidant activities of protein fragments obtained from tomato waste fermented using Bacillus subtilis were evaluated. The addition of B. subtilis increased the relative amounts of aromatic and positively-charged amino acids which have been described to influence the biological activities of peptide fragments. IC values of hydrolysates for ACE-inhibitory and 2, 2′-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities were found to be 1.5 and 8.2 mg/mL, respectively. Size-exclusion chromatography (SEC) pattern of the hydrolysate indicated the breakdown of parent proteins to smaller peptides with molecular weights mainly below 1400 Da. MALDI-TOF mass spectrometry analysis revealed that the highest ACE-inhibitory activity was due to peptides showing molecular mass range 500-800 Da, while the most active antioxidant peptides were found to be mainly at the two different peptide weight ranges 500-800 Da and 1200-1500 Da. [ABSTRACT FROM AUTHOR]

Recent findings indicate there is only a small window of sulfuric acid concentration (60-65 %) and temperature (45-65 °C) which allows efficient extraction of cellulose nanocrystals in significant quantities from bleached chemical pulp. In the present report, we develop a systematic explanation for how hydrolysis temperature, at a specific acid concentration, governs CNC surface properties. We demonstrate that CNCs with different suspension viscosity, stability in electrolyte-containing solutions, and optical properties can be produced, based on the presence or not of a precipitated oligosaccharide layer (OSL) on the surface of the nanocrystals. At hydrolysis temperatures below 65 °C, the degree of polymerization (DP) distribution of cellulose chains in CNC samples exhibits a bimodal distribution, indicating an accumulation of oligosaccharides on the CNC surface which increases as the hydrolysis temperature is decreased. At low hydrolysis temperature (45 °C), the oligosaccharides dissolved in the strong acid phase have a DP between 7 and 20 and precipitate onto CNCs when the reaction is quenched by diluting with water. As the temperature of hydrolysis is increased (50-60 °C), the dissolved oligosaccharides are hydrolyzed faster and their DP decreases such that they remain soluble after quenching. At 65 °C, no precipitated oligosaccharides can be detected on the CNC surface. Based on these results, we propose possible explanations to account for the effects of the OSL on the CNC suspension viscosity and stability and on optical properties of CNC films. [ABSTRACT FROM AUTHOR]

The molecular weights of poly( N-isopropylacrylamide) (PNIPA), calculated according to polystyrene calibration standards upon the elution of THF on styragel columns, appear to be much lower than their actual values determined using independent approaches. This is likely due to interactions between the nitrogen-containing units of PNIPA polymer chains and the sorbent, so the polymer is eluted in the mode intermediate between exclusion and critical. An effective exclusion mode during the elution of PNIPA on a styragel column can be achieved by using an eluent more polar than tetrahydrofuran (particularly, 1-methylpyrrolidone). [ABSTRACT FROM AUTHOR]

Unreduced gluten proteins of Indian wheat varieties viz.C306, DBW16, HI977 and HW2004 were separated using size-exclusion chromatography (SEC). Statistical correlation of area % of eluted peaks with properties of flour, dough and noodles was elucidated. Chromatograms of gluten proteins were classified primarily into five peaks in decreasing molecular size range and relative proportion were expressed in terms of area % of individual peaks which depicts the quantitative variation in protein eluted at different retention times. Cooking time and cooked weight of noodles depicted positive correlation with peak I and negative correlation with peak II which predominantly composed of glutenins and gliadins, respectively. Oil uptake and cooking loss were negatively association with peak I and positively with peak II. Noodle hardness, springiness, cohesiveness and chewiness were positively correlated with peak I and negatively to peak II, though adhesiveness was unaffected by SEC eluted peaks statistically. [ABSTRACT FROM AUTHOR]

Hydrocolloids were extracted from the alcohol insoluble solids of defatted, finely crushed and freeze-dried winery waste of the Greek variety Xinomavro, using temperature-controlled aqueous solutions buffered at pH 5, pH 7 and pH 9. The extracts were characterized in terms of their macromolecular populations distribution using size exclusion chromatography; of their generic chemical composition and homogeneity using Fourier-transform infra-red (FTIR) spectroscopy and micro-FTIR mapping; of their polymeric particle distribution using dynamic light scattering; and of their charge using zeta potential measurements. These were compared with extracts obtained from fresh grape. The capacity of the extracts toward emulsification and emulsion stabilization was studied at neutral and acidic conditions (pH 7 and pH 3) using light diffraction for droplet distribution measurement and confocal microscopy. The extracts show potential for use as emulsifiers for acidic and neutral model emulsions. [ABSTRACT FROM AUTHOR]

The article describes sensitive and selective homogeneous immunoassays for the liver cancer biomarker alpha-fetoprotein (AFP) in human serum by using single wavelength excitation fluorescence cross-correlation spectroscopy (SW-FCCS). Both competitive and sandwich immunoassay modes were applied, and AFP served as a model analyte. Fluorescent CdSe/ZnS quantum dots (with a 655 nm emission peak) and the fluorophore Alexa Fluor 488 (520 nm emission) were chosen to label the antibodies in the sandwich mode, and the antibody and the antigen in the competitive mode. Under optimized conditions, the sandwich assay has a linear dynamic range that covers the 20 pM to 5.0 nM concentration range. The competitive assay, in turn, extends from 180 pM to 15.0 nM. The respective detection limits are 20 pM and 180 pM. The method was successfully applied to directly determine AFP in (spiked) clinical samples, and results were in good agreement with data obtained via ELISAs. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Recombinant proteins are frequently hampered by aggregation during the refolding and purification process. A simple and rapid method for in vitro refolding and purification of recombinant human parathyroid hormone (rhPTH 1-34) expressed in Escherichia coli with protein folding size exclusion chromatography (PF-SEC) was developed in the present work. Discrete effects of potential solution additives such as urea, polypolyethylene glycol, proline, and maltose on the refolding with simultaneous purification of rhPTH were investigated. The results of individual additives indicated that both maltose and proline had remarkable influences on the efficiency of refolding with a recovery yield of 65 and 66 % respectively. Further, the synergistic effect of these additives on refolding was also explored. These results demonstrate that the additive combinations are more effective for inhibiting protein aggregation during purification of rhPTH in terms of recovery yield, purity, and specific activity. The maltose and proline combination system achieved the highest renatured rhPTH having a recovery yield of 78 %, a purity of ≥99 %, and a specific activity of 3.31 × 10 cAMP pM/cell respectively, when compared to the classical dilution method yield (41 %) and purity (97 %). In addition, the role of maltose and proline in a combined system on protein aggregation and refolding has been explained. The molecular docking (in silico) scores of maltose (−10.91) and proline (−9.0) support the in vitro results. [ABSTRACT FROM AUTHOR]

An in vitro digestion-size exclusion chromatography set-up has been used as to study transfer of macromolecules from bread to simulated gastrointestinal (GI) juices and their subsequent breakdown along the GI tract. Bread samples underwent mechanical processing in a sequence of simulated GI environments, namely in simulated oral, gastric, then intestinal fluids. In the absence of GI enzymes, transfer of starch from the bolus to the GI fluids has been monitored to occur without any apparent hydrolysis. α-Amylase incorporation in the simulated oral fluids resulted in hydrolysis further down the GI tract, namely in the duodenum, suggesting a pH-dependent enzyme deactivation in the stomach and reactivation in the simulated intestinal area. Pepsin in the simulated gastric fluids influenced the release of protein in the simulated stomach environment. [ABSTRACT FROM AUTHOR]

The antioxidants in food materials have recently attracted researchers' attention because many reports have shown that the oxidative stress is closely related to the aging process of the cells and acts as a trigger to various diseases including cancer. Since reactive oxygen species (ROS) is involved in initiating and promoting several diseases such as cancer and cardiovascular events, this study was designed to evaluate the antioxidant capacity of pectic polysaccharides extracted from the bark of Cinnamomum zeylanicum, locally known as Daruchini. An arabinogalactan ( A), one partly methyl esterified galacturonic acid ( B) and a neutral glucan ( C) were isolated. The glucan is made up of β-(1 → 3)-linked glucopyranosyl residues and has a molecular mass of 7 kDa. The arabinogalactan is highly branched and has an average molecular mass of 40 kDa. The in vitro antioxidant capacity of the fractions was studied by ferric reducing antioxidant power (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays. The arabinogalactan ( A) showed the highest potential followed by the uronic acid ( B) and glucan ( C). Taken together, these findings demonstrate that these polysaccharides could be used as natural antioxidants by the food industry. [ABSTRACT FROM AUTHOR]

In this study, five different dissolved organic matter (DOM) fractions, defined based on a size exclusion chromatography with simultaneous detection of organic carbon (OCD) and ultraviolet (UVD), were quantitatively tracked with a treatment train of coagulation/flocculation-sand filtration-ozonation-granular activated carbon (GAC) filtration in a full-scale advanced drinking water treatment plant (DWTP). Five DOM samples including raw water were taken after each treatment process in the DWTP every month over the period of three years. A higher abundance of biopolymer (BP) fraction was found in the raw water during spring and winter than in the other seasons, suggesting an influence of algal bloom and/or meltwater on DOM composition. The greater extent of removal was observed upon the coagulation/flocculation for high-molecular-weight fractions including BP and humic substances (HS) and aromatic moieties, while lower sized fractions were preferentially removed by the GAC filtration. Ozone treatment produced the fraction of low-molecular-weight neutrals probably resulting from the breakdown of double-bonded carbon structures by ozone oxidation. Coagulation/flocculation was the only process that revealed significant effects of influent DOM composition on the treatment efficiency, as revealed by a high correlation between the DOM removal rate and the relative abundance of HS for the raw water. Our study demonstrated that SEC-OCD-UVD was successful in monitoring size-based DOM composition for the advanced DWTP, providing an insight into optimizing the treatment options and the operational conditions for the removal of particular fractions within the bulk DOM. [ABSTRACT FROM AUTHOR]

Background: It has been shown from the chain length distributions (CLDs) that amylose chains can be divided into at least two groups: long and short amylose chains. These molecular structures influence some functional properties of starch, such as digestibility and mouth-feel. GBSSI is the key enzyme for the elongation of amylose chains; however, the effect of other starch biosynthesis enzymes in amylose synthesis is still not fully understood. Two advanced starch characterization techniques, size exclusion chromatography (SEC) and fluorophore-assissted carbohydrate electrophoresis (FACE), together with a newly developed starch biosynthesis model, are used to improve understanding of amylose biosynthesis. Results: SEC and FACE were used to determine the CLD of amylose and amylopectin in various native and mutant rice starches. The types of starch branching enzymes (SBEs) involved in the synthesis of the distinct features seen for shorter degrees of polymerization, DP, < 2000, and longer (DP > 2000) amylose chains are identified by combining these data with a mathematical model of amylopectin biosynthesis. The model enables each feature in the amylopectin CLD to be parameterized in terms of relative SBE activities, which are used to explain differences in the genotypes. Conclusions: The results suggest that while GBSSI is the predominant enzyme controlling the synthesis of longer amylose chains, some branching enzymes (such as BEI and BEIIb) also play important roles in the synthesis of shorter amylose chains. [ABSTRACT FROM AUTHOR]

Purpose: To identify High Molecular Weight Products (HMWP) formed in human insulin formulation during storage. Methods: Commercial formulation of human insulin was stored at 37°C for 1 year and HMWP was isolated using preparative size exclusion chromatography (SEC) and reverse phase (RP) chromatography. The primary structure of the isolated species was analysed using liquid chromatography mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS). To test the hypothesis that amino groups of insulin are involved in HMWP formation, the HMWP content of various formulations spiked with amine compounds or formulations of insulin with modified amino groups was measured. Results: More than 20 species of HMWP were observed and 16 species were identified using LC-MS. All identified species were covalent dimers of human insulin linked via A21Asn and B29Lys, formed via the formation of an anhydride intermediate at A21Asn. Two types of HMWP were identified, with the covalent link in the open or closed (succinimidyl) form. Some species also contained single deamidation at B3 or the desPhe(B1)-N-oxalyl-Val(B2) modification. Reduced rate of HMWP formation was observed after addition of L-lysine, L-arginine or piperazine or when insulin analogues with methylated N-terminals and side chain amines and A21Gly mutation were used. Formulations of human insulin without zinc and m-cresol were found to contain a different pool of HMWP. Conclusions: HMWP formed in formulation of human insulin at pH 7.4 with zinc and m-cresol consists primarily of covalent dimers linked via A21Asn and B29Lys. Insulin formulation properties determine the amount and identity of formed HMWP. [ABSTRACT FROM AUTHOR]