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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]

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]

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]

• 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]

• 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]

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]