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Background: Extracellular vesicles (EVs) are amongst the most promising candidates for developing blood-based biomarkers. However, patient sample availability is a key barrier to translational research whilst most biobanks store samples of 1.5mL volume or less. To date, there is no consensus on the most suitable method of EV separation and current techniques frequently require large volumes of biofluids, complicated technology, technical expertise, or significant operating costs, which prevents their widespread adoption by less EV-focussed laboratories. Therefore, there is a need for an easy and reproducible method that separates representative EVs from clinically relevant 1mL volumes of plasma prior to subsequent biomarker identification. Methods: In this study, EVs were separated from a clinically relevant 1mL volume of human plasma using four different separation techniques: size exclusion chromatography (SEC), differential ultracentrifugation, precipitation, and immunoaffinity magnetic bead capture. The EVs were characterised using several orthogonal techniques (protein quantification, nanoparticle tracking analysis, transmission electron microscopy, Western blot, single particle interferometric reflectance imaging sensing, and mass spectrometry-based proteomics) to comprehensively compare the separated samples. Results: We provide examples of anticipated results highlighting that SEC-processed samples have greater protein quantification yield, greater particle yield of the expected size for EVs, and sufficient EV purity, which facilitates effective EV cargo assessment by proteomics. Moreover, we confirm significant overlap with known EV-related proteins within the Vesiclepedia database. Additionally, using single particle interferometric reflectance imaging sensing (Leprechaun®), we identify that SEC has the most representative surface tetraspanin distribution of the separated EV population compared to unprocessed plasma. Discussion: Given that SEC requires minimal expertise, no complicated technology and can separate EVs within 90 min, this comparison reinforces SEC as a clinically relevant EV separation method from 1mL of plasma making it suitable for widespread implementation. [ABSTRACT FROM AUTHOR]

Poly(N‐vinylcaprolactam) (PVCL), a salt responsive ligand, was grafted from electrospun cellulose nanofiber membranes by atom transfer radical polymerization (ATRP). The membranes were used for hydrophobic interaction chromatography. The modified PVCL‐cellulose membranes were used to separate BSA dimers from monomers as well as monoclonal antibody (mAb) aggregates. Fast protein liquid chromatography (FPLC) was used for protein separation and fraction collection. High performance liquid chromatography with a size exclusion column (HPLC‐SEC) analysis confirmed the separation of monomers and aggregates from both BSA and the mAb. The results suggest that use of a responsive ligand that changes its conformation with ionic strength could improve the performance of hydrophobic interaction chromatography enabling the separation of compounds based on slight differences in hydrophobicity. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are cell derived membranous nanoparticles. EVs are important mediators of cell–cell communication via the transfer of bioactive content and as such they are being investigated for disease diagnostics as biomarkers and for potential therapeutic cargo delivery to recipient cells. However, existing methods for isolating EVs from biological samples suffer from challenges related to co‐isolation of unwanted materials such as proteins, nucleic acids, and lipoproteins. In the pursuit of improved EV isolation techniques, we introduce multimodal flowthrough chromatography (MFC) as a scalable alternative to size exclusion chromatography (SEC). The use of MFC offers significant advantages for purifying EVs, resulting in enhanced yields and increased purity with respect to protein and nucleic acid co‐isolates from conditioned 3D cell culture media. Compared to SEC, significantly higher EV yields with similar purity and preserved functionality were also obtained with MFC in 2D cell cultures. Additionally, MFC yielded EVs from serum with comparable purity to SEC and similar apolipoprotein B content. Overall, MFC presents an advancement in EV purification yielding EVs with high recovery, purity, and functionality, and offers an accessible improvement to researchers currently employing SEC. [ABSTRACT FROM AUTHOR]

Polymerized impurities in β -lactam antibiotics can induce allergic reactions, which seriously threaten the health of patients. In order to study the polymerized impurities in cefoxitin sodium for injection, a novel approach based on the use of two-dimensional liquid chromatography coupled with time-of-flight mass spectrometry (2D-LC-TOF MS) was applied. In the 1st dimension, high performance size exclusion chromatography (HPSEC) with a TSK-G2000SWxl column was employed. Column switching was applied for the desalination of the mobile phase used to separate polymerized impurities in the 1st dimension before they were transferred to the 2nd dimension which utilized reversed phase liquid chromatography (RP-LC) and TOF MS for further structural characterization. The structures of four polymerized impurities (which were all previously unknown) in cefoxitin sodium for injection were deduced based on the MS2 data. One novel polymerized impurity (PI–I), with 2H less than the molecular weight of two molecules of cefoxitin (M r. 852.09), was found to be the most abundant (>50 %) in almost all the samples examined and could be regarded as the marker polymer of cefoxitin sodium for injection. This work also showed the great potential of the 2D-LC-TOF MS approach in structural characterization of unknown impurities separated with a mobile phase containing non-volatile phosphate in the 1st dimension. [Display omitted] • Novel approach to characterize unstable impurities separated by a non-volatile mobile phase. • Four novel PIs found in cefoxitin based on MSn data. [ABSTRACT FROM AUTHOR]

• SEC-MALS was firstly applied to measure the MW of ULC and SC. • The relationship between relative contents of ULC and PHR were investigated. • The relationship between sizes of ULC and PHR were investigated. • The relationship between heparin MW and the ULC size were investigated. Heparin-induced thrombocytopenia (HIT) is an immune complication of heparin therapy. Antibodies binding to complexes of platelet factor 4 (PF4) and heparin is the trigger of HIT. A method using size exclusion chromatography with multi-angle laser light scattering detector (SEC-MALS) was developed in this work. The soluble ultra-large complex (ULC) was separated from the small complex (SC) and their molecular weights (MWs) were firstly measured. The complexes of PF4 and three heparins with different MW, including unfractionated heparin (UFH), dalteparin (Daltep) and enoxaparin (Eno) were characterized using this method. The contents and the sizes of ULC increased gradually when heparins were added to PF4 to certain amounts. While, they reduced after more heparins were added. It is the first time to measure the MWs of the biggest ULC of PF4-heparins as millions of Dalton. at the proper ratios of PF4 to heparin (PHR). Meanwhile, those mixtures at those certain PHRs induced the higher expression of CD83 and CD14 markers on dendritic cells (DCs) suggesting that they had stronger immunogenicity and is critical for HIT. [ABSTRACT FROM AUTHOR]

The accurate characterization of post-consumer recycled (PCR) polyethylene (PE) and polypropylene (PP) blends is essential for repurposing them into higher-demand applications. This study improves the accuracy of the Temperature Rising Elution Fractionation coupled with Gel Permeation Chromatography (TREFxGPC) approach for both virgin and post-consumer recycled PP/PE blends. Model blends and virgin polymers were used to estimate polymer-specific IR-detector response factors. Moreover, a correction for the molecular weight-dependent amount of PP eluting in the TREF temperature range characteristic of PE was found. Accounting for these factors improved the results of the method, reducing the error in composition determination from 8 % to 2 %. Therefore, our approach provides better quantitative assessment capabilities for recycled blends. Furthermore, the refined TREFxGPC not only reliably determines the PCR PP/PE blend composition but also reveals their bivariate molecular weight and chemical composition distribution (MWD-CCD). Understanding the full microstructure of the blend can lead to superior recycled products, enabling the use of PCR in more demanding applications. • Assessing TREFxGPC's accuracy for PE & PP composition in blends & recyclates. • Adjusting for PP eluting in PE range and polymer-specific detector response. • Method validation on model and recycled blends matches true composition. [ABSTRACT FROM AUTHOR]

Purpose: This study focuses on the effect of length and structure of attached polyethylene glycol (PEG) chain on hydrodynamic radius (Rh) and chromatographic retention of PEGylated protein. To this aim human serum albumin (HSA) as a standard protein was PEGylated site specifically with mPEG-maleimide. Methods: Separated PEG_HSA fractions were analyzed by size exclusion and anion exchange chromatography (AExC). The purity of fractions and the relative mobility of PEGylated and native proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Hydrodynamic radius was determined based on the retention time of fractions on size exclusion chromatography (SEC), and also according to the previously reported equations. Results: A linear relation was shown between the molecular weight of attached PEG and Rh of PEGylated HSA. No significant difference between Rh of proteins modified with linear and branched PEG was shown. In SDS-PAGE, the delaying effect of branched PEG on movement of PEGylated protein was higher than that of linear PEG. Conclusion: As PEGylated HSA and dimer HSA have almost the same size and in SEC they elute at very close retention times, so in this case ion exchange chromatography (IExC) is more effective than SEC in separation of PEGylated HSA. Branched PEG- HSA showed earlier elution on anion exchange chromatography compared to linear PEG-HSA, that this can explain the different shielding effect of various structures of attached PEGs. The smaller size of PEGylated HSA in compare to the sum of the hydrodynamic radiuses of native HSA and attached PEG could be as a result of shielded attachment of polymer around protein. [ABSTRACT FROM AUTHOR]

Aflatoxins (AFs) are one of the most harmful carcinogenic natural toxins that affect food. Crops containing reasonably high oil content may be affected by Aspergillus species and consequently by AF contamination. In this study, a proposed testing method for AF detection in oilseed was developed, validated, and used for a market survey to assess the probabilistic risk exposure caused by consuming contaminated oilseeds including corn, sunflower seed, and soybean. The test method was optimized for selective extraction and then validated for fitness of purpose; the limits of quantification (LOQs) were 0.2, 0.4, 0.2, and 0.2 μg kg−1 for aflatoxin G1 (AFG1), aflatoxin B1 (AFB1), aflatoxin G2 (AFG2), and aflatoxin B2 (AFB2), respectively. The method was linear from the LOQs up to 20 μg kg−1, and its budget of measurement uncertainties were estimated at 25, 24, 26, and 30 for AFG1, AFB1, AFG2, and AFB2, respectively. The contamination levels were from 10,000 body weight (BW) day−1, respectively. The main finding of the present study highlights the possibility of some risk of AF exposure from corn consumption, which may represent a health concern. • Size exclusion chromatography has an excellent and selective extraction of aflatoxins from heavy matrix oilseeds. • According to the consumption rate of corn, the estimated margin of exposure to aflatoxin B1 was 721 body weight (BW) day−1. • There is a possibility of some risk of aflatoxins exposure from corn consumption, which may represent a health concern. [ABSTRACT FROM AUTHOR]

• Explain the separation mechanism of ultrasound-assisted chromatography (UAC). • Comparatively analyzed ultrasound effects on C18 PEEK and SS columns. • Distinguished the differences between acoustic and temperature effects in UAC. • Ultrasound can be utilized as an external factor in chromatography. Based on the previous ultrasound-assisted chromatography (UAC) studies on plastic and stainless steel (SS) columns, this study explores the UAC mechanism by comparatively analyzing ultrasound effects on plastic and SS columns with C18 stationary phase when separating a mixture of polycyclic aromatic hydrocarbons (PAHs) under various ultrasound intensities. The results showed a substantial difference in H values between the PEEK and SS columns under the influence of ultrasound agitation. Specifically, for the pyrene peak, as the ultrasonic intensity increased from 0% to 100% of 900 W, the H values of the SS column slightly rose from 8.82 μm to 9.86 μm. Conversely, the corresponding values for the PEEK column exhibited a significant 12-fold increase from 11.5 μm to 134 μm. The findings demonstrated poor penetration of ultrasound energy through the SS column, and the temperature rise of the medium induced by the ultrasound was the primary contributing factor to PAH separation. However, ultrasound easily penetrated through the plastic column, resulting in acoustic cavitation within the C18 polyether ether ketone (PEEK) column. Cavitation induced heat generation and contributed to a decrease in retention time and the magnitude of peak broadening or distortion, depending on the specific ultrasonic energy. Based on the estimated change in inlet temperature of the PEEK column due to an acoustic effect, the comparison with temperature effects under non-sonic conditions consistently demonstrated a stronger acoustic effect in reducing the retention time, by 2–9%, depending on specific peaks and pairs. We revisited the previously described separation mechanism of ultrasound-assisted ion chromatography and conjoined with our findings to infer and establish a thorough explanation for the previously unexplained separation mechanism of chiral separation and size exclusion chromatography by UAC using SS columns. [ABSTRACT FROM AUTHOR]

• Capillary-channeled polymer fibers provide high throughput as 2D in 2DLC. • Fiber columns in 2D eliminates the need for UPLC hardware in the second dimension. • SEC x RP with fiber columns provides for potential implementation for ESI sampling. • PET and PP fibers exhibit different levels of selectivity, under RP conditions. • RP x RP with fiber columns provides orthogonality in chemical information. Polymeric materials are readily available, durable materials that have piqued the interest of many diverse fields, ranging from biomedical engineering to construction. The physiochemical properties of a polymer dictate the behavior and function, where large polydispersity among polymer properties can lead to problems; however, current polymer analysis methods often only report results for one particular property. Two-dimensional liquid chromatography (2DLC) applications have become increasingly popular due to the ability to implement two chromatographic modalities in one platform, meaning the ability to simultaneously address multiple physiochemical aspects of a polymer sample, such as functional group content and molar mass. The work presented employs size exclusion chromatography (SEC) and reversed-phase (RP) chromatography, through two coupling strategies: SEC x RP and RP x RP separations of the water-soluble polymers poly(methacrylic acid) (PMA) and polystyrene sulfonic acid (PSSA). Capillary-channeled polymer (C-CP) fiber (polyester and polypropylene) stationary phases were used for the RP separations. Particularly attractive is the fact that they are easily implemented as the second dimension in 2DLC workflows due to their low backpressure (<1000 psi at ∼70 mm sec−1) and fast separation times. In-line multi-angle light scattering (MALS) was also implemented for molecular weight determinations of the polymer samples, with the molecular weight of PMA ranging from 5 × 104 to 2 × 105 g mol−1, while PSSA ranges from 105 to 108 g mol−1. While the orthogonal pairing of SEC x RP addresses polymer sizing and chemistry, this approach is limited by long separation times (80 min), the need for high solute concentrations (PMA = 1.79 mg mL−1 and PSSA = 0.175 mg mL−1 to yield comparable absorbance responses) due to on-column dilution and subsequently limited resolution in the RP separation space. With RP x RP couplings, separation times were significantly reduced (40 min), with lower sample concentrations (0.595 mg mL−1 of PMA and 0.05 mg mL−1 of PSSA) required. The combined RP strategy provided better overall distinction in the chemical distribution of the polymers, yielding 7 distict species versus 3 for the SEC x RP coupling. [ABSTRACT FROM AUTHOR]

• Simulated moving bed chromatography can be performed at a microliter scale. • Custom-made 3D printed valve reduces overall dead volume to 358 µL. • Pressure-driven closed-loop flow control is a suitable flow source. • Desalting level of up to 99% for continuous protein desalting • Feed flow rate as low as 15 µL/min. In the 1960s, chromatography processes were revolutionized by the invention of simulated moving bed chromatography. This method not only enhances the separation performance and resin utilization in comparison to batch-chromatography, it has also a much lower buffer consumption. While simulated moving bed chromatography nowadays is applied for a wide range of industrial applications, it was never transferred to the micro-scale (in regards to column and system volume). In our opinion a micro simulated moving bed chromatography system (µSMB) would be a useful tool for many applications, ranging from early process development and long term studies to downstream processing of speciality products. We implemented such a µSMB with a 3D printed central rotary valve and a microfluidic flow controller as flow source. We tested the system with a four zone open loop setup for the separation of bovine serum albumin and ammonium sulfate with size exclusion chromatography. We used four process points and could achieve desalting levels of BSA ranging from 94% to 99%, with yields ranging form 65% to 88%. Thus, we were able to achieve comparable results to common lab scale processes. With a total dead volume of 358 µL, including all sensors, connections and the valve, this is, to the best of our knowledge, the smallest SMB system that was ever built and we were able to perform experiments with feed flow rates reaching as low as 15 µL/min. [ABSTRACT FROM AUTHOR]

• SEC packing materials with three different bondings were prepared. • The three materials were characterized and compared. • Non-specific interactions were systematically studied. • The new HO-PEO phase was clearly the most favourable one. • A fast platform method (3 min analysis time) has been developed for protein SEC. Size exclusion chromatography has become an essential tool for the protein therapeutics industry. Conceptually, it is a simple form of chromatography that is driven by entropy and sieving effects. An ideal size exclusion column would exhibit no adsorptive interactions between its internal surfaces and the solutes being analysed, but that is not easily achieved. To this end, we have studied the utility of three unique packing materials in pursuit of additional column chemistries that might be less prone to interacting with proteins. These packing materials were each prepared from bridged ethylene hybrid organic/inorganic particles but uniquely derivatized into either hydroxy terminated PEO bonded, methoxy terminated PEO bonded, or diol bonded packing materials. All three materials were packed into column hardware modified with hydrophilic hybrid surface technology (h-HST) so that packing material effects could be more clearly observed without any influence from the secondary interactions that can originate from metal hardware. Non-specific interactions were compared for various challenging protein samples in the presence of ammonium acetate (volatile) and phosphate buffered saline (non-volatile) buffers. It was reconfirmed that the h-HST column hardware mitigates a majority of non-desired secondary interactions. However, during studies on hydrophobic interactions, the new hydroxy terminated PEO packing material showed clear benefit to obtaining higher apparent recoveries to better ensure accurate aggregate quantitation. Further experiments were explored to show that a hydroxy terminated PEO column could be effectively paired with a mobile phase comprised of standard strength phosphate buffered saline to make a fast platform method capable of baseline resolving monoclonal antibody monomer and aggregate peaks within a 3 min analysis time. [ABSTRACT FROM AUTHOR]

Herein, a striking anion-tunnel transfer effect was demonstrated in 1-butyl-3-methylimidazolium benzoate (BmimPhCOO) ionic liquid, so a rapid, mild and efficient benzoylation of cellulose is accomplished under catalyst-free condition in BmimPhCOO. In a wide temperature range of 20–80 °C, the equilibrium of reaction is reached within only 15 min, which is much faster than the reported acylation of cellulose. Furthermore, the resultant cellulose tribenzoates have excellent solubility in conventional organic solvents, thus they can be used to accurately reflect the molecular weight and dispersity index of cellulose raw materials by gel permeation chromatography. This method is suitable for various cellulose. Therefore, we found a new principle to realize the extremely-rapid acylation of cellulose, and proposed an effective approach to accurately determine the molecular weight parameters of cellulose. A new principle to realize the rapid acylation of cellulose was found to establish an effective strategy for accurately characterizing the molecular weight parameters of cellulose. [Display omitted] [ABSTRACT FROM AUTHOR]

• 2D SEC-CEX-MS allows multiplex analysis of mAb aggregates and charge variants in a single method. • 2D SEC-CEX-MS streamlines product development in industry. • 2D SEC-CEX-MS is applied to understand mAb forced degradation pathways. Monoclonal antibodies (mAbs) are extremely complex due to the presence of structural modifications resulting from enzymatic and chemical reactions such as glycosylation, glycation, deamidation, isomerisation, oxidation, aggregation and fragmentation. Size and charge variants analysis are carried out from the early stages of drug development throughout product lifetime to investigate product degradation pathways and optimise process conditions. However, conventional analytical workstreams for size and charge variant characterization are both time and sample demanding, requiring the application of multiple analytical methods. This study presents the development of a novel 2D-LC/MS approach combining both aggregate and charge variant profiling of a mAb candidate in a single method. Aggregate quantification was performed in the first dimension (1D) by size exclusion chromatography SEC, followed by online fraction transfer of the monomer peak to the second dimension (2D) by a heart-cutting for charge variant analysis by cation exchange chromatography (CEX). Aiming to maximise the information obtained from minimal sample and time required for analysis, a salt-based separation with UV detection was developed for supporting the processing of a large number of samples to facilitate high-throughput process development (HTPD). In addition, a mass spectrometry (MS) compatible SEC-CEX separation was developed enabling online charge variant peak identification. This study presented the ability to multiplex mAb size and charge variants analysis by coupling SEC with CEX in a 2D-LC set-up. To date, this is the first 2D SEC-CEX-UV(-MS) application for intact mAb analysis. [ABSTRACT FROM AUTHOR]

• Rapid purification of virus from cell lysate using an in-line AIEX and SEC setup. • 3.5-fold yield increase achieved by chromatography compared to conventional method. • New automated method yields 13 mg pure active virus per liter of cell lysate feed. • High potential for purification of viruses and nanoparticles, including adenovirus. • Easily scalable method provides a direct route to industrial use. An efficient chromatography-based virus purification method has been developed and validated for the non-pathogenic infectious virus PRD1. Compared to the conventional method that consists of relatively time-consuming and labour-intensive precipitation and density gradient ultracentrifugation steps, the method developed here is performed in a single flow using tandem-coupled anion exchange and size exclusion chromatography (AIEX-SEC) columns. This inline approach helps to minimize the loss of virus in the process and streamlines time consumption, since no physical transfer of the sample is required between purification steps. In the development process, sample feed composition, dynamic binding capacity and elution conditions for the AIEX resin as well as different exclusion limits for SEC resins were optimized to achieve maximal yield of pure infectious viruses. Utilizing this new approach, a high-quality virus sample was produced from a lysate feed in 320 min with a total yield of 13 mg purified particles per litre of cell lysate, constituting a 3.5-fold yield increase as compared to the conventional method, without compromising the high specific infectivity of the product (6 × 1012 to 7 × 1012 pfu/mg of protein). The yield of infectious viruses of the lysate feed was 54%. The easy scalability of chromatography-based methods provide a direct route to industrial usage without any significant changes needed to be made to the purification regime. This is especially interesting as the method has high potential to be used for purification of various viruses and nanoparticles, including adenovirus. [ABSTRACT FROM AUTHOR]

This study investigated the experimental conditions needed to obtain molecular weight distribution (MWD) of ultrahigh MW poly(ethylene oxide) (PEO) using size exclusion chromatography (SEC) hyphenated with a multiangle light scattering photometer (MALS) and a differential refractive index detector (RI). Ultrahigh MW components yielded non-linear angular dependency in scattered light intensities. The first-order linear fitting using Zimm formalism resulted in significant differences in MW depending on if the signals from detector 4 to 16 were used in the fitting or only five low-angles from 4 to 8 were used. In contrast, no significant differences in MW were obtained for lower MW PEO samples (equal or less than 1000 KDa) between the two fitting approaches. It was thus proposed to use only the five low-angles to derive MW for a sample with broad polydispersity including both ultrahigh and low MW components. The SEC separation was done using one column designed for ultrahigh MW polymer separation connected with another mixed-bed column. The ultrahigh MW column allowed separation and characterization of polymeric components in the MW range between 10 and 50 million Dalton (MDa) and the size range between 300 and 600 nm in radius of gyration (R g). Online calibration curves were obtained from the linear fittings of MW as a function of elution volume. MW polydispersity was derived from the online calibration curve showing that the ultrahigh MW PEO had higher polydispersity than the lower MW samples. The double logarithmic plot of radius of gyration versus MW indicated that both ultrahigh MW and low MW PEO would adopt expanded coil conformations in the aqueous solution. [ABSTRACT FROM AUTHOR]