Size Exclusion Chromatography Method Research Articles

Genetic and environmental variation in protein composition of Belgian soy determined with a novel size-exclusion chromatography method

To allow the determination of soy protein composition on protein subunit level in a quantitative manner, a novel method combining size-exclusion high-performance liquid chromatography (SE-HPLC) and peak deconvolution was developed. Peaks corresponding to the different soy protein (subunits) were identified based on the analysis of (i) marker proteins, (ii) 7S and 11S globulin-enriched fractions, and (iii) cross-validation with SDS-PAGE analysis. Given the recent introduction of soybean cultivation in Belgium, the optimized SE-HPLC method was here applied to study the impact of genotype, growing location, harvest year and inoculation strategy on the protein composition of Belgian soy. Especially genotype and harvest year were key to determining protein composition, while all factors influenced the chemical composition of soy flour. The developed SE-HPLC method can be used to further understand the impact of soybean cultivation practices in Belgium and elsewhere on protein composition, which is highly relevant for novel applications of soy. In addition, this SE-HPLC method can be used to determine how various soy processing methods may affect protein composition.

Two-Dimensional SEC-SEC-UV-MALS-dRI Workflow for Streamlined Analysis and Characterization of Biopharmaceuticals.

The emergence of complex biological modalities in the biopharmaceutical industry entails a significant expansion of the current analytical toolbox to address the need to deploy meaningful and reliable assays at an unprecedented pace. Size exclusion chromatography (SEC) is an industry standard technique for protein separation and analysis. Some constraints of traditional SEC stem from its restricted ability to resolve complex mixtures and notoriously long run times while also requiring multiple offline separation conditions on different pore size columns to cover a wider molecular size distribution. Two-dimensional liquid chromatography (2D-LC) is becoming an important tool not only to increase peak capacity but also to tune selectivity in a single online method. Herein, an online 2D-LC framework in which both dimensions utilize SEC columns with different pore sizes is introduced with a goal to increase throughput for biomolecule separation and characterization. In addition to improving the separation of closely related species, this online 2D SEC-SEC approach also facilitated the rapid analysis of protein-based mixtures of a wide molecular size range in a single online experimental run bypassing time-consuming deployment of different offline SEC methods. By coupling the second dimension with multiangle light scattering (MALS) and differential refractive index (dRI) detectors, absolute molecular weights of the separated species were obtained without the use of calibration curves. As illustrated in this report for protein mixtures and vaccine processes, this workflow can be used in scenarios where rapid development and deployment of SEC assays are warranted, enabling bioprocess monitoring, purity assessment, and characterization.

Quantification of Pluronic F127 used on PLGA nanoparticle preparation and comparing purification techniques by centrifugation, tangential flow filtration, and ultrafiltration

Emerging research in polymeric nanoparticles (PNP) in the field of drug delivery systems focuses on the preferable use of biocompatible materials like poly(lactic-co-glycolic) acid (PLGA). However, the substantial use of surfactant in achieving colloidal stability requires its subsequent elimination to avoid side effects. This study aimed to establish suitable conditions for detecting and quantifying Pluronic® F127 (F127), a non-ionic surfactant widely used for this specific purpose, through a precise Size Exclusion Chromatography (SEC) method. Then, comparing the F127 removal efficacy via three purification techniques: Centrifugation (Cf), Ultrafiltration (UF), and tangential flow filtration (TFF), determining the minor influence of surfactant removal on the colloidal and morphological properties of surfactant-free PLGA suspensions. Thus, to determine the most suitable strategy for purifying PNP. The initial results of the SEC method revealed two distinct molar mass distributions of F127, associated with a blend of triblock alongside a minor fraction of homopolymer or diblock. The method exhibited a high linear correlation (R2 0.99991) for surfactant concentrations ranging from 0.3 to 14.7 mg/mL. While assessing the three purification techniques, UF demonstrated proficiency in concentrating nanoparticle suspensions, albeit tending to retain a significant portion of the surfactant. In contrast, Cf displayed a remarkable capability in removing more than 90 % of the incorporated F127. However, Cf recovered only 60 % of the initial nanoparticles, predominantly eliminating smaller diameter particles. Conversely, TFF demonstrated high efficacy in surfactant removal, requiring an extended washing process and leading to minimal nanoparticle loss while leaving the colloidal variables unaffected.

Characterization of antibody surface properties and selection of a diverse subset for development of a generic size-exclusion chromatography method

Aggregates are an important quality attribute for biotherapeutics because they can affect the safety and efficacy of the drug and they are therefore routinely monitored, mainly with size-exclusion chromatography (SEC). However, there is often a need to tailor a SEC method to a specific molecule. This study describes the development of a generic SEC method tested on 138 antibodies with the variable domains taken from clinical stage antibodies. We report on the discovery of a subset of 12 antibodies that represents the full range of physiochemical properties found in the 138 antibodies. This subset is shown to be an efficient and reliable test set when developing chromatographic methods for antibodies. An understanding of the nature of the analyte-stationary phase interactions was gained when using this set with its wide range of physiochemical properties. Highly hydrophobic antibodies interact strongly with some modern silica hybrid materials causing the elution time to increase significantly, while a hydrophilically modified hybrid surface showed highly reduced interactions for the hydrophobic antibodies. Highly hydrophilic antibodies, on the other hand, exhibited asymmetric peaks to a certain extent on all stationary phases, while the elution time was not affected. The developed SEC method was shown to have satisfactory performance in terms of linearity, repeatability, range, and accuracy and exhibit very narrow distributions of elution time and peak symmetry when testing the 138 antibodies indicating its generic performance.

Immunopotentiating Polyphosphazene Delivery Systems: Supramolecular Self-Assembly and Stability in the Presence of Plasma Proteins.

Studies on the biological performance of nanomedicines have been increasingly focused on the paradigm shifting role of the protein corona, which is imminently formed once the formulation is placed in a complex physiological environment. This phenomenon is predominantly studied in the context of protein adsorption science, while such interactions for water-soluble systems remain virtually unexplored. In particular, the importance of plasma protein binding is yet to be understood for pharmaceuticals designed on the basis of supramolecular architectures, which generally lack well-defined surfaces. Water-soluble ionic polyphosphazenes, clinically proven immunoadjuvants and vaccine delivery vehicles, represent an example of a system that requires supramolecular coassembly with antigenic proteins to attain an optimal immunopotentiating effect. Herein, the self-assembly behavior and stability of noncovalently bound complexes on the basis of a model antigen─hen egg lysozyme─and polyphosphazene adjuvant are studied in the presence of plasma proteins utilizing isothermal calorimetry, asymmetric flow field flow fractionation, dynamic light scattering, and size exclusion chromatography methods. The results demonstrate that although plasma proteins, such as human serum albumin (HSA), show detectable avidity to polyphosphazene, the strength of such interactions is significantly lower than that for the model antigen. Furthermore, thermodynamic parameters indicate different models of binding: entropy driven, which is consistent with the counterion release mechanism for albumin versus electrostatic interactions for lysozyme, which are characterized by beneficial enthalpy changes. In vitro protein release experiments conducted in Franz diffusion cells using enzyme-linked immunoassay detection suggest that the antigen-adjuvant complex stability is not adversely affected by the presence of the most physiologically abundant protein, which confirms the importance of the delivery modality for this immunoadjuvant. Moreover, HSA shows an unexpected stabilizing effect on complexes with high antigen load─an important consideration for further development of polyphosphazene adjuvanted vaccine formulations and their functional assessment.

Saliva biofilm-derived outer membrane vesicles regulate biofilm formation and immune response of oral epithelial cells on titanium surfaces.

While the significant roles of outer membrane vesicles (OMVs) from individual oral bacterial species in bacterial-host interactions are known, the involvement of saliva biofilm-derived OMVs in peri-implant disease pathogenesis remains unclear. This study aimed to investigate the effect of saliva biofilm-derived OMVs on regulating saliva biofilm formation and modulating the immune response of the epithelial cells on titanium surfaces. Saliva derivedbiofilms were cultured on tissue culture plates (TCP) for 4 days using pooled saliva from four healthy donors. OMVs secreted from the TCP bound biofilm (referred to as OMVs or healthy saliva biofilm OMVs) were enriched using the size-exclusion chromatography method. We then evaluated the effects of these OMVs on theviability, metabolic activity, and the presence oforal pathogens in saliva biofilm grownon titanium discs for 24 h and 72 h. Furthermore, the impact of OMVs on the mRNA expression and inflammatory cytokines [interleukin (IL)-6, IL-1α, and monocyte chemoattractant protein-1 (MCP-1)] in human oral epithelial cells (OKF6/TERT-2) was investigated using RT-qPCR and enzyme-linked immunosorbent assay(ELISA), respectively. Healthy saliva biofilm OMVs improved the biomass and activity of saliva biofilm cultured on the titanium surfaces, with inhibited Porphyromonas gingivalis and Fusobacterium nucleatum, and enhanced Streptococcus mutansexpression. Additionally, OMVs increased pro-inflammatory cytokine IL-6 mRNA and IL-6cytokine expression in human oral epithelial cells. However, IL-1α and MCP-1 cytokines were inhibited 24-hour post-incubation with OMVs. Healthy saliva biofilm derivedOMVs regulate the activity and pathogen composition of biofilms formed on titanium, while modulating the secretion of pro-inflammation factors of oral epithelial cells grownon titanium surfaces. Healthy saliva biofilm OMVs may regulate the early biofilm formation on abutment surfaces and modulate epithelial cell immune response, which may alter the peri-implant niche and participate in the pathogenesis of peri-implant disease.

Size matters: Functional differences of small extracellular vesicle subpopulations in cardiac repair responses.

Cardiac progenitor cell (CPC)-derived small extracellular vesicles (sEVs) exhibit great potential to stimulate cardiac repair. However, the multifaceted nature of sEV heterogeneity presents a challenge in understanding the distinct mechanisms underlying their regenerative abilities. Here, a dual-step multimodal flowthrough and size-exclusion chromatography method was applied to isolate and separate CPC-derived sEV subpopulations to study the functional differences related to cardiac repair responses. Three distinct sEV subpopulations were identified with unique protein profiles. Functional cell assays for cardiac repair-related processes demonstrated that the middle-sized and smallest-sized sEV subpopulations exhibited the highest pro-angiogenic and anti-fibrotic activities. Proteasome activity was uniquely seen in the smallest-sized subpopulation. The largest-sized subpopulation showed no effect in any of the functional assays. This research uncovers the existence of sEV subpopulations, each characterized by a distinct composition and biological function. Enhancing our understanding of sEV heterogeneity will provide valuable insights into sEV mechanisms of action, ultimately accelerating the translation of sEV therapeutics.

LC‐MS/MS‐based proteome profiling of CSF‐derived extracellular vesicles in Alzheimer’s disease

AbstractBackgroundExtracellular vesicles (EVs) participate in the spread of pathological proteins between cells, contributing to the progression of neurodegenerative disorders, including Alzheimer’s disease (AD). As cerebrospinal fluid (CSF) reflects the biochemical environment of the brain, EVs from CSF constitutes a promising source to investigate novel biomarkers and therapeutic targets. We aimed to explore the unbiased proteome profile of CSF‐derived EVs from individuals with AD by label‐free quantitative liquid chromatography mass spectrometry (LC‐MS/MS).MethodEVs were obtained from 500 µL of CSF from sex and age‐matched cognitively healthy donors (HC, n = 10, mean age = 73±1.4) and patients with AD dementia (AD, n = 10, mean age = 76.2±0.9) by a high‐throughput size exclusion chromatography method (SmartSEC). Resultant EV fractions were characterized by nanoparticle tracking analysis (NTA). EVs were lysed and proteins were trypsinized overnight via S‐Trap filters (Protifi). The peptides were separated on an Evosep nano LC system and analyzed by the TimsTOF Pro (Bruker) mass spectrometer. Peptides were identified using PEAKS Online software (1% FDR). DAVID Bioinformatics Resources 6.8 was used to determine the gene ontology (GO) enrichment. We normalized log2 peptide intensities (quantiles) and determined the differential protein expression across groups by a linear mixed effects (fixed effects = diagnosis, random effects = peptide sequence) model using the MsqRob package in R. P‐values were adjusted using Benjamini‐Hochberg.ResultNTA showed homogeneous EV size profiles (mean size = 113.4±1.41nm). We identified 2096 proteins with at least 1 proteotypic peptide. GO enrichment analyses showed “extracellular exosome” as the most statistically significant enriched term (p = 1.8×10−212), confirming the enrichment of EVs. Statistical inference analysis revealed 18 differentially expressed proteins between both diagnostic groups (adj.p<0.05), all of them increased in the AD group. (Fig 1). Some of the greatest changes include CLUS (log2FC = 0.61, adj.p = 8.38×10−4, Fig.2), SPB1 (log2FC = 1.15, adj.p = 1.23×10−2, Fig.3), LRP1 (log2FC = 3.43, adj.p = 1.23×10−2), KLK7 (log2FC = 2.59, adj.p = 1.23×10−2), SAP3 (log2FC = 2.09, adj.p = 3.1×10−2) and SHPS1 (log2FC = 1.03, adj.p = 4.25×10−3), which are proteins that have been previously associated with inflammation.ConclusionOur results reveal novel protein changes in CSF‐derived EVs from individuals with AD, which could be explored further to their applicability in distinguishing the different clinical stages across the AD continuum.

Quantitation of polyethylene glycol by size exclusion chromatography with charged aerosol, differential refractive index, and multi-angle light scattering detectors

Polyethylene glycol (PEG) has found tremendous applications in pharmaceutical products and has played a critical role in PEGylated drug modalities to improve pharmacokinetic properties and biological efficacy. The characterization and quantitation of PEGs are essential to control manufacture process and drug product quality. However, the assay value of PEG could change dramatically depending on the structures of the PEG and the detection techniques used. In this study, we developed a size exclusion chromatographic (SEC) method for quantitative PEG analysis, and we systematically evaluated the performance of three online detectors with different operating principles: a charged aerosol detector (CAD), a differential refractive index (dRI) detector, and a multi-angle light scattering detector (MALS). Fourteen PEG compounds covering a wide range of molecular weight (MW, 1 – 40 kDa) and molecular architectures (linear, branched, Y-shaped and multi-arm geometries) were evaluated by these three detection techniques. Our study revealed that the dRI showed the most universal responses among all the PEGs regardless of their molecular weight or geometries. In the contrast, CAD and MALS detector showed MW-dependent and semi-universal geometry-dependent responses. Another key finding is that the relative response factor for each multi-arm PEG in the CAD and the MALS were inversely correlated, suggesting both can be applied to qualitatively assess polymers of different architectures, including the ones with subtle differences in their core structures. The comparison of the three detectors not only provides the fundamental and comprehensive understanding of PEG quantitation but also enables the process development and control of high-quality PEGs in producing PEGylated therapeutics in the pharmaceutical industry.

A novel approach to determine residual aldehyde content in conjugated polysaccharides with 3-methyl-2-benzothiazolinonehydrazon (MBTH) by colorimetric method, automation, and HP-SEC

Polysaccharide activation by periodate oxidation to form aldehydes, followed by conjugation with proteins via reductive amination is a general procedure to make polysaccharide-protein conjugate vaccines. Controlling the level of residue aldehyde content after conjugation is critical to ensure vaccine product stability. Herein, to support conjugation process optimization, we developed a 3-methyl-2-benzothiazolone hydrazone (MBTH) chemistry-based colorimetric method, which can measure low-level residual aldehyde polysaccharide-protein conjugate in high throughput 96-well plate format. This mechanism of detection works in two steps. First, MBTH reacts with the aldehyde group to form azine. Then the excess MBTH was oxidized by ferric ammonium sulfate in sulfamic acid to form a reactive cation, which reacts further with azine to form formazan, a characteristic blue chromophore at 610 nm, for colorimetric detection. The method performance, including detection limit, linearity range, matrix effect, kinetics, and color stability was systematically studied. For samples with severe matrix interference, a supplemental size exclusion chromatography (SEC) method was also developed as an alternative method.

Urinary exosome proteins PAK6 and EGFR as noninvasive diagnostic biomarkers of diabetic nephropathy

ObjectiveThe actin cytoskeleton plays an essential role in maintaining podocyte functions. However, whether the urinary exosome proteins related to the regulation of the actin cytoskeleton are changed in diabetic nephropathy (DN) is still unknown. This study was to investigate the possibility that related proteins can be applied as diagnostic biomarkers for DN.MethodsUrinary exosomes were obtained from 144 participants (Discovery phase: n = 72; Validation phase: n = 72) by size exclusion chromatography methods. Proteomic analysis of urinary exosome by LC-MS/MS. Western blot and ELISA were applied to validate the selected urinary exosome proteins. The clinical value of selected urinary exosome proteins was evaluated using correlation and receiver operating characteristic curve analyses.ResultsFifteen urinary proteins related to the regulation of the actin cytoskeleton were identified in urinary exosomes. Three upregulated proteins were selected, including Serine/threonine-protein kinase PAK6 (PAK6), Epidermal growth factor receptor (EGFR), and SHC-transforming protein 1(SHC1). The expression level of PAK6 and EGFR was negatively correlated with estimated glomerular filtration rate and positively correlated with serum creatinine levels. For diagnosing DN in the discovery phase: the area under curve (AUC) of PAK6 was 0.903, EGFR was 0.842, and the combination of two proteins was 0.912. These better performances were also observed in the validation phase (For PAK6: AUC = 0.829; For EGFR: AUC = 0.797; For PAK6 + EGFR: AUC = 0.897).ConclusionsUrinary exosome proteins PAK6 and EGFR may be promising and noninvasive biomarkers for diagnosing DN.

Comprehensive structural characterization of water-soluble and water-insoluble homoexopolysaccharides from seven lactic acid bacteria

Several lactic acid bacteria are able to produce water-soluble and water-insoluble homoexopolysaccharides (HoEPS) from sucrose. In this study, structures of all HoEPS which were fermentatively produced by Leuconostoc mesenteroides subsp. dextranicum NRRL B-1121 and B-1144, Leuconostoc mesenteroides subsp. mesenteroides NRRL B-1149, B-1438 and B-1118, Leuconostoc suionicum DSM 20241, and Liquorilactobacillus satsumensis DSM 16230 were systematically analyzed. Monosaccharide analysis, methylation analysis, NMR spectroscopy, size-exclusion chromatography, and different enzymatic fingerprinting methods were used to obtain detailed structural information. All strains produced water-soluble dextrans and/or levans as well as water-insoluble glucans. Levans showed different degrees of branching and high molecular weights, whereas dextrans had comparable structures and broader size distributions. Fine structures of water-soluble HoEPS were analyzed after endo-dextranase and endo-levanase hydrolysis. Water-insoluble glucans were composed of different portions of 1,3-linkages (5 to 40 %). Hydrolysis with endo-dextranase and endo-mutanase yielded further information on block sizes and varying fine structures. Overall, clear differences between HoEPS yields and structures were observed.

Liquid Chromatography Methods for Analysis of mRNA Poly(A) Tail Length and Heterogeneity.

Messenger RNA (mRNA) is a new class of therapeutic compounds. The current advances in mRNA technology require the development of efficient analytical methods. In this work, we describe the development of several methods for measurement of mRNA poly(A) tail length and heterogeneity. Poly(A) tail was first cleaved from mRNA with the RNase T1 enzyme. The average length of a liberated poly(A) tail was analyzed with the size exclusion chromatography method. Size heterogeneity of the poly(A) tail was estimated with high-resolution ion-pair reversed phase liquid chromatography (IP RP LC). The IP RP LC method provides resolution of poly(A) tail oligonucleotide variants up to 150 nucleotide long. Both methods use a robust ultraviolet detection suitable for mRNA analysis in quality control laboratories. The results were confirmed by the LC-mass spectrometry (LC MS) analysis of the same mRNA sample. The poly(A) tail length and heterogeneity results were in good agreement.

Purification and fractionation of phycobiliproteins from Arthrospira platensis and Corallina officinalis with evaluating their biological activities

Phycobiliproteins (PBPs) are a class of water-soluble pigments with a variety of biological functions that are present in red macroalgae and cyanobacterial species. The crude forms of phycocyanin (C-PC) from the blue green alga Arthrospira platensis and allophycocyanin (APC) from the red macroalga Corallina officinalis were extracted and purified by ammonium sulphate precipitation, anion exchange chromatography, and size exclusion chromatography methods, respectively. The obtained C-PC and APC from A. platensis and C. officinalis were 0.31 mg/mL and 0.08 mg/mL, respectively, with molecular masses of “17.0 KDa and 19.0 KDa” and “15.0 KDa and 17.0 KDa” corresponding to α and β subunits, respectively. FT-IR was used to characterize the purified APC and C-PC in order to look into their structures. Highly purified extracts (A620/A280 > 4.0) were obtained from subtractions’ PC3 and PC4 that were tested for their biological activities. APC and C-PC crude extracts plus their fractions exhibited potent anti-oxidant in different ratios by using three techniques. PC1 showed high anti-inflammatory (75.99 and 74.55%) and anti-arthritic (78.89 and 76.92%) activities for C. officinalis and A. platensis, respectively compared with standard drugs (72.02 and 71.5%). The methanolic and water extracts of both species showed greater antibacterial efficacy against Gram +ve than Gram −ve marine bacteria. Our study shed light on the potential medical uses of C-PC and APC extracted from the tested species as natural substances in a variety of foods and drugs. Further investigations are required to explore the diverse chemical natures of distinct PBPs from different cyanobacteria and red algae because their amino acid sequences vary among different algal species.

Comprehensive characterization of human brain-derived extracellular vesicles using multiple isolation methods: Implications for diagnostic and therapeutic applications.

Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and promising biomarkers and therapeutics in the central nervous system (CNS). Human brain-derived EVs (BDEVs) provide a comprehensive snapshot of physiological changes in the brain's environment, however, the isolation of BDEVs and the comparison of different methods for this purpose have not been fully investigated. In this study, we compared the yield, morphology, subtypes and protein cargo composition of EVs isolated from the temporal cortex of aged human brains using three established separation methods: size-exclusion chromatography (SEC), phosphatidylserine affinity capture (MagE) and sucrose gradient ultracentrifugation (SG-UC). Our results showed that SG-UC method provided the highest yield and collected larger EVs compared to SEC and MagE methods as assessed by transmission electron microscopy and nanoparticle tracking analysis (NTA). Quantitative tandem mass-tag (TMT) mass spectrometry analysis of EV samples from three different isolation methods identified a total of 1158 proteins, with SG-UC showing the best enrichment of common EV proteins with less contamination of non-EV proteins. In addition, SG-UC samples were enriched in proteins associated with ATP activity and CNS maintenance, and were abundant in neuronal and oligodendrocytic molecules. In contrast, MagE samples were more enriched in molecules related to lipoproteins, cell-substrate junction and microglia, whereas SEC samples were highly enriched in molecules related to extracellular matrix, Alzheimer's disease and astrocytes. Finally, we validated the proteomic results by performing single-particle analysis using the super-resolution microscopy and flow cytometry. Overall, our findings demonstrate the differences in yield, size, enrichment of EV cargo molecules and single EV assay by different isolation methods, suggesting that the choice of isolation method will have significant impact on the downstream analysis and protein discovery.

Identification and evaluation of circulating exosomal miRNAs for the diagnosis of postmenopausal osteoporosis

BackgroundPostmenopausal osteoporosis (PMOP) is a common condition that leads to a loss of bone density and an increased risk of fractures in women. Recent evidence suggests that exosomal miRNAs are involved in regulating bone development and osteogenesis. However, exosomal miRNAs as biomarkers for PMOP diagnosis have not been systematically evaluated. In this study, we aim to identify PMOP-associated circulating exosomal miRNAs and evaluate their diagnostic performance.MethodsWe performed next-generation sequencing and bioinformatics analysis of plasma exosomal miRNAs from 12 PMOP patients and 12 non-osteoporosis controls to identify PMOP-associated exosomal miRNAs, and then validated them in an independent natural community cohort with 26 PMOP patients and 21 non-osteoporosis controls. Exosomes were isolated with the size exclusion chromatography method from the plasma of elder postmenopausal women. The plasma exosomal miRNA profiles were characterized in PMOP paired with controls with next-generation sequencing. Potential plasma exosomal miRNAs were validated by qRT-PCR in the validation cohort, and their performance in diagnosing PMOP was systematically evaluated with the receiver operating characteristic curve.ResultsTwenty-seven miRNAs were identified as differentially expressed in PMOP versus controls in sequencing data, of which six exosomal miRNAs (miR-196-5p, miR-224-5p, miR320d, miR-34a-5p, miR-9-5p, and miR-98-5p) were confirmed to be differentially expressed in PMOP patients by qRT-PCR in the validation cohort. The three miRNAs combination (miR-34a-5p + miR-9-5p + miR-98-5p) demonstrated the best diagnostic performance, with an AUC = 0.734. In addition, the number of pregnancies was found to be an independent risk factor that can improve the performance of exosomal miRNAs in diagnosing PMOP.ConclusionsThese results suggested that the plasma exosomal miRNAs had the potential to serve as noninvasive diagnostic biomarkers for PMOP.

Characterization of pneumococcal serotype 7F in vaccine conjugation

Streptococcus pneumoniae is a highly invasive bacterial pathogen that can cause a range of illnesses. Pneumococcal capsular polysaccharides (CPS) are the main virulence factors that causes invasive pneumococcal disease (IPD). Pneumococcal CPS serotype 7F along with a few other serotypes is more invasive and likely to cause IPD. Therefore, 7F is a target for pneumococcal vaccine development, and is included in the two recently approved multi-valent pneumococcal conjugated vaccines, i.e. VAXNEUVANCE and PREVNAR 20.To support process and development of our 15-valent pneumococcal conjugated vaccine (PCV15), chromatographic methods have been developed for 7F polysaccharide and conjugate characterization. A size-exclusion chromatography (SEC) method with UV, light scattering and refractive index detections was employed for concentration, size and conformation analysis. A reversed-phase ultra-performance liquid chromatography (RP-UPLC) method was used for analysis of conjugate monosaccharide composition and degree of conjugation. The collective information obtained by these chromatographic analysis provided insights into the pneumococcal conjugate and conjugation process.Graphical

Changes in urinary exosomal protein CALM1 may serve as an early noninvasive biomarker for diagnosing diabetic kidney disease

BackgroundThe risk of the development and progression of diabetic kidney disease (DKD) was increased by abnormal calcium release. However, it is still unknown whether calcium signal pathway-related proteins are changed in urinary exosomes. This study aims to explore the changes in urinary exosomal proteins, which may provide novel biomarkers for diagnosing DKD. MethodsUrinary exosomes were isolated from 132 participants by size exclusion chromatography method and 72 participants were tested by LC-MS/MS (Discovery phase). Correlation and multivariate logistics analysis were applied to evaluate selected urinary proteins. Western blot and ELISA were used to validate the selected protein (Validation phase: n = 60). The diagnostic performance of the selected biomarker was evaluated by receiver operating characteristic curve analyses between the discovery and validation phases. ResultsSixteen calcium signal pathway-related proteins were identified, however, only Calmodulin-1(CALM1) was continuously increased. Different expression of CALM1 was found in patients with different level of estimated glomerular filtration rate (eGFR) in two cohorts. The level of CALM1 was correlated with eGFR and serum creatinine levels in two cohorts. Multivariate analysis revealed that serum albumin (ALB) levels and CALM1 were independent risk factors for DKD. A diagnostic model based on CALM1 and serum ALB levels that could significantly distinguish DKD was established and validated. ConclusionsSignificant changes in calcium signal pathway-related urinary exosomal proteins were observed. The CALM1 may serve as an early noninvasive biomarker for diagnosing DKD.

TNF-αand OSX mRNA of salivary small extracellular vesicles in periodontitis: a pilot study.

This cross-sectional study explored extracellular vesicle (EV)-derived gene expression of markers for bone turnover and pro-inflammatory cytokines in periodontal disease. Whole unstimulated saliva was collected from fifty-two participants (18 healthy, 13 gingivitis and 21 stages III/IV periodontitis), from which small extracellular vesicles (sEVs) were enriched using the size-exclusion chromatography (SEC) method, and characterised by morphology, EV-protein, and size distribution, using transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), and Nanoparticle Tracking Analysis (NTA), respectively. Bone turnover markers and pro-inflammatory cytokines in salivary sEVs were evaluated using RT-qPCR. Salivary sEVs morphology, mode, size distribution and particle concentration were comparable between healthy, gingivitis, and periodontitis patients. A CD9+ subpopulation was significantly higher in periodontitis-derived salivary sEVs compared to healthy. The detection of sEVs mRNA for osterix (OSX) and tumour necrosis factor-alpha (TNFα) was significantly decreased and increased, respectively, in periodontitis compared to healthy controls, with good discriminatory power for periodontitis diagnosis [area under the curve (AUC) >0.72]. Our study demonstrated that salivary sEVs mRNAs may serve as a potential non-invasive biomarker source for periodontitis diagnosis.

Multiattribute Monitoring of Aggregates and Charge Variants of Monoclonal Antibody through Native 2D-SEC-MS-WCX-MS.

Monitoring of critical quality attributes such as size and charge-related heterogeneities is essential for biopharmaceutical manufacturers. Size-exclusion chromatography (SEC) is the preferred analytical technique for the quantification of aggregates and fragments in the product, whereas weak-cation exchange chromatography (WCX) is widely used for the characterization of charge variants of biotherapeutic products, in particular monoclonal antibodies (mAbs). Multiattribute monitoring offers the ability to monitor these attributes in a single run flow using two-dimensional liquid chromatography (2D-LC). Typically, in this approach, only the second-dimension samples are directly analyzed through mass spectrometry, as the first dimension has limitations concerning direct coupling with mass spectrometry. In the present study, a novel 2D-SEC-MS/WCX-MS workflow has been proposed, in which chromatography of both dimensions (D1 and D2) was directly coupled with mass spectrometry, through which size-related and charge-related variants of monoclonal antibody mAb A were analyzed simultaneously in their native form. In comparison to stand-alone SEC and WCX methods, this method enables simultaneous analysis of size and charge variants in a single workflow without manual intervention, allowing analysis of low abundant variants. Further, this method has 75% less sample requirement and a shorter analysis time (25 min vs 90 min) when size and charge variants were analyzed individually. The proposed native 2D-LC-MS workflow was used to analyze a stressed sample of mAb A, in which D1 analysis revealed the presence of aggregates (8-20%), which were primarily dimers, whereas D2 analysis showed an increment in acidic variants (9-21%).