Extracellular vesicles (EVs) are membranous vesicles secreted by both prokaryotic and eukaryotic cells and play a vital role in intercellular communication. EVs are classified into several subtypes ...based on their origin, physical characteristics, and biomolecular makeup. Exosomes, a subtype of EVs, are released by the fusion of multivesicular bodies (MVB) with the plasma membrane of the cell. Several methods have been described in literature to isolate exosomes from biofluids including blood, urine, milk, and cell culture media, among others. While differential ultracentrifugation (dUC) has been widely used to isolate exosomes, other techniques including ultrafiltration, precipitating agents such as poly-ethylene glycol (PEG), immunoaffinity capture, microfluidics, and size-exclusion chromatography (SEC) have emerged as credible alternatives with pros and cons associated with each. In this review, we provide a summary of commonly used exosomal isolation techniques with a focus on SEC as an ideal methodology. We evaluate the efficacy of SEC to isolate exosomes from an array of biological fluids, with a particular focus on its application to adipose tissue-derived exosomes. We argue that exosomes isolated via SEC are relatively pure and functional, and that this methodology is reproducible, scalable, inexpensive, and does not require specialized equipment or user expertise. However, it must be noted that while SEC is a good candidate method to isolate exosomes, direct comparative studies are required to support this conclusion.
A new optimized size exclusion chromatography small‐angle X‐ray scattering (SEC‐SAXS) system for biomolecular SAXS at the Australian Synchrotron SAXS/WAXS beamline has been developed. The compact ...configuration reduces sample dilution to maximize sensitivity. Coflow sample presentation allows an 11‐fold increase in flux on sample without capillary fouling, improving throughput and data quality, which are now primarily limited by the full flux available on the beamline. Multi‐wavelength fibre optic UV analysis in close proximity to the X‐ray beam allows for accurate concentration determination for samples with known UV extinction coefficients and thus estimation of the molecular weight of the scattering particle from the forward X‐ray scattering intensity. Fast‐flow low‐volume SEC columns provide sample throughput competitive with batch concentration series measurements, albeit with a concomitant reduction of potential resolution relative to lower flow rates and larger SEC columns. The performance of the system is demonstrated using a set of model proteins, and its utility to solve various challenges is illustrated with a diverse suite of protein samples. These developments increase the quality and rigor of SEC‐SAXS analysis and open new avenues for biomolecular solution SEC‐SAXS studies that have been challenged by low sample yields, temporal instability, radiation sensitivity and complex mixtures.
Size exclusion chromatography (SEC) small‐angle X‐ray scattering (SAXS) is a powerful structural biology tool where the best outcomes are obtained through an optimized experimental approach. Optimization of SEC and integration of a sheath flow sample environment into the Australian Synchrotron's SAXS/WAXS beamline has greatly improved data quality and the ability to deal with difficult protein samples.
Extracellular vesicles (EVs) are lipid membrane vesicles released by live cells that carry a variety of biomolecules, including nucleic acids, lipids, and proteins. Recently, proteins in ...plasma‐derived EVs have emerged as novel biomarkers with essential functions in the diagnosis and prognosis of human diseases. However, the current methods of isolating EVs from plasma often lead to coisolated impurities in biological fluids. Therefore, before performing any research protocol, the process of extracting EVs from plasma for proteomic analysis must be optimized. In this study, two EV isolation strategies, size exclusion chromatography (SEC) and SEC combined with ion exchange adsorption (SEC + IEA), were compared in terms of the purity and quantity of protein in EVs. Our results demonstrated that, compared to single‐step SEC, SEC combined with IEA could produce plasma‐derived EVs with a higher purity by decreasing the abundance of lipoprotein. Additionally, with MS analysis, we demonstrated that the combination approach maintained the stability and improved the purity of EVs in many plasma samples. Furthermore, by combining SEC with IEA, more cancer‐associated proteins were detected in the plasma of various cancer samples.
Monoclonal antibodies are tetrameric complex proteins, primarily produced using mammalian cell culture. Attributes such as titer, aggregates, and intact mass analysis are monitored during process ...development/optimization. In the present study, a novel workflow such that the Protein‐A affinity chromatography is performed in the first dimension for purification and titer estimation, whereas size exclusion chromatography is employed in the second dimension to characterize size variants using native mass spectrometry. The present workflow offers a significant advantage over the traditionally used standalone Protein‐A affinity chromatography followed by size exclusion chromatography analysis in that it can monitor these four attributes in 8 min while requiring a minimal sample size (10–15 μg) and not requiring any manual peak collection. In contrast, the traditional standalone approach requires manual collection of eluted peaks in Protein‐A affinity chromatography followed by buffer exchange to a mass‐compatible buffer, which can take up to 2–3 h with considerable risk of sample loss, degradation, and induced modifications. As the biopharma industry moves to make analytical testing efficient, we believe that the approach proposed here would be of significant interest due to its ability to monitor multiple process and product quality attributes in a single workflow and via rapid analysis.
The present study describes the possibilities offered by an innovative bioinert size exclusion chromatography column for size variant characterization of complex monoclonal antibody products. This ...size exclusion chromatography column includes a novel column hardware surface. The column was prepared from metallic hardware components that were treated to have prototype hydrophilically modified hybrid organic–inorganic silica surfaces called hybrid surface technology. This provides a significant reduction in nondesired hydrophobic and electrostatic interactions that can occur between column and analyte when performing size exclusion chromatography analysis with volatile mobile phase.
Compared to a reference stainless‐steel column packed with the same batch of packing material, peak tailing, band broadening, and above all recovery of high molecular weight species were distinctly improved for all types of monoclonal antibody products. Based on our observations, we found that 50 mM ammonium acetate in water was a suitable mobile phase offering good compromise in terms of liquid chromatography performance and mass spectrometry sensitivity. In addition, method repeatability (intra‐ and interday relative standard deviations) on elution times and high molecular weight species peak areas were found to be excellent.
By using this innovative size exclusion chromatography material, the low and high molecular weight species contained in various stressed and nonstressed monoclonal antibody products were successfully characterized with mass spectrometry detection.
Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ...ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively.
To develop a single-step protocol to isolate vesicles from human body fluids.
Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n=3). Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL) and protein were measured in each fraction.
Fractions 9-12 contained the highest concentrations of particles larger than 70 nm and platelet-derived vesicles (46%±6 and 61%±2 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8%±1 and 0.65%±0.3, respectively). HDL was present mainly in fractions 18-20 (32%±2 of total), and protein in fractions 19-21 (36%±2 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43%±23 in fractions 9-12, with an 8-fold and 70-fold enrichment compared to HDL and protein.
SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles.
The cyclic oligosaccharide β‐cyclodextrin (β‐CDx) is investigated regarding its ability on principle to reduce the debranching activity of pullulanase (PUL) or even terminate the enzymatic hydrolysis ...process of starch polymers completely. For this purpose, dissolved β‐CDx (aqueous solution) is mixed with the diluted PUL compound (solution) and conditioned (stirred at 40 °C for various durations 0, 20, 40, and 60 min; at 26.75 nmol β‐CDx U−1 and at various β‐CDx dosages 5.35, 13.375, 26.75, and 53.5 nmol β‐CDx U−1; reaction time 60 min). The PUL‐CDx‐mixtures are subsequently added to a 2.5% w/w starch solution (related to the initial specific debranching activity; 1593.6 NPUN g−1/557.8 U g−1) and gently stirred for 20 min at 40 °C before final thermal inactivation. The obtained samples are diluted and characterized molecularly, i.e., by means of size exclusion chromatography (SEC). An increasing β‐CDx dosage reduces the degree of molecular degradation systematically reflecting a successively reduced enzyme activity. However, the reaction time (PUL‐CDx‐mixture) has no impact on the enzyme's activity since the starch degradation is marginally and the SEC‐chromatograms similar to the one of the initial starch. Restrictions of the enzyme inhibiting effect of β‐CDx are found terminating the hydrolysis process in a starch suspension.
An increasing β‐‐CDx dosage reduces the degree of molecular degradation systematically reflecting a successively reduced enzyme activity. Restrictions of the enzyme inhibiting effect are found terminating the hydrolysis process in a starch suspension.
•Current SEC analysis of AAVs mimic protein analysis and are suboptimal.•SEC of AAVs with columns of smaller dimensions save analysis time and sample.•Smaller columns can be operated at high ...flowrates without affecting results.•Adequate AAV sample handling is critical to successful quantitation of aggregates.
Adeno-associated virus (AAV) analytical characterization is crucial to the well-defined and reproducible production of human gene therapies utilizing the AAV vector modality. The establishment of analytical methods based upon technology platforms currently widely used by bio-therapeutic manufacturers, namely HPLC, will assist efforts to produce high quality AAV reproducibly and decrease chemical manufacturing and control challenges in method portability and reliability. AAV analysis by size exclusion chromatography (SEC) is currently practiced with columns and mobile phase conditions traditional to SEC of proteins. Here, an improved method to measure multiple AVV critical quality attributes (CQA) rapidly by SEC is explored. The use of short columns made with small particles at high flow rates resulted in up to 80 % reduction in analysis time and 66 % in sample consumption while maintaining reliable quantitation of AAV aggregate or high molecular weight (HMW) content. These results were demonstrated across four different AAV serotypes. Furthermore, critical AAV sample handling learnings are shared.
In this study, three commercially available low‐density polyethylene (LDPE) polymers produced via a tubular reactor process, with varying melt flow rates at 190°C/2.16 kg (4.0, 1.9, and ...0.75 g/10 min), have been selected and subjected to high temperature‐size exclusion chromatography (SEC) analysis coupled with an infrared‐5 (IR‐5), viscometer (VISCO), and multiangle laser light‐scattering detectors. The molecular weight (MW), MW distribution, short‐chain branching (SCB), and long‐chain branching parameters were investigated. It was found that MW obtained by the universal technique (∼1.57–1.7 times) and multiangle laser light‐scattering detection technique is (∼1.43–1.55 times) higher than that of the conventional calibration technique, which could be attributed to structural complexity associated with LDPEs which is not clearly understood by conventional SEC mode alone. The bulk SCB per 1000 total carbon atoms estimated by IR‐5 detection was found to range from 16.50 to 17.80. On the other hand, long chain branching frequency per 1000 total carbon atoms obtained by online VISCO and multiangle laser light‐scattering detection ranged from 0.46 to 0.54 and 0.65 to 0.94, respectively. Further, the significance of long chain branching parameters on the polymer processing behavior was studied in correlation with rheological property (Die swell ratio).
Thermal modification of wood causes chemical changes that significantly affect the physical, mechanical and biological properties of wood; thus, it is essential to investigate these changes for ...better utilization of products. Fourier transform infrared spectroscopy and size exclusion chromatography were used for evaluation of chemical changes at thermal treatment of oak wood. Thermal modification was applied according to Thermowood process at the temperatures of 160, 180 and 210 °C, respectively. The results showed that hemicelluloses are less thermally stable than cellulose. Chains of polysaccharides split to shorter ones leading to a decrease of the degree of polymerization and an increase of polydispersity. At the highest temperature of the treatment (210 °C), also crosslinking reactions take place. At lower temperatures degradation reactions of lignin predominate, higher temperatures cause mainly condensation reactions and a molecular weight increase. Chemical changes in main components of thermally modified wood mainly affect its mechanical properties, which should be considered into account especially when designing various timber constructions.
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