Size Exclusion Column Research Articles

Microflow size exclusion chromatography to preserve micromolar affinity complexes and achieve subunit separations for native state mass spectrometry

For high throughput native mass spectrometry (MS) protein characterization, it is advantageous to desalt and separate proteins by size exclusion chromatography (SEC). Sensitivity, resolution, and speed in these methods remain limited by standard SEC columns. Moreover, the efficient packing of small bore columns is notoriously difficult. SEC sensitivity is inherently limited because solutes are not focused into concentrated bands and low affinity native complexes may dissociate on column. Recent work evaluated the suitability of crosslinked gel media in small bore formats for online desalting. Here, small bore format online SEC for native MS studies is again investigated but with alternative materials. We systematically studied the utility of diol and hydroxy terminated polyethylene oxide (PEO) bonded 1.7 µm organosilica particles as packed into 1 mm ID stainless steel (SS) hardware and hardware treated with hydrophilic hybrid surface technology (h-HST). For the equivalent diol-bonded particle and hardware, UV limits of detection (LODs) were reduced 32 to 89% with a microflow separation (15 µL/min) on a 1 × 50 mm column as compared to a 4.6 × 150 mm high-flow separation (300 µL/min) at the same linear velocity. Run times were also shortened by 45%. A switch from SS to h-HST hardware led to a significant reduction in secondary interactions and a corresponding improvement in detection limits for trastuzumab, myoglobin, IgG and albumin for both UV and MS. Coupling of the small bore columns to multichannel microflow emitters resulted in 10 to 100-fold gains in MS sensitivity, depending on the analyte. MS LOD values were significantly reduced into the low attomole ranges. Columns were then evaluated for their effects on the preservation of complexes, including concanavalin A, in its apo and ligand-bound states, and three therapeutically relevant noncovalent systems previously undetected on large column formats. The results suggest that the detection of large complexes by SEC is not just a function of sensitivity but is directly affected by chemical secondary interactions. The ability to detect 0.1 to 1 MDa complexes, with between 1 and 40 micromolar dissociation constants, represents a critical advancement for high-throughput native MS workflows as applied to the analysis of therapeutics.

Calibration of high performance size exclusion chromatography (HPSEC) for molecular weight estimation of aquatic humic substances

Three high performance size exclusion chromatography (HPSEC) columns were compared, along with mobile phase chemistry and calibration standards, for the determination of apparent molecular weight (AMW) of aquatic humic substances using a UV absorbance detector (λ = 254 nm). The addition of 10 % organic solvent did not influence peak elution times of sodium polystyrene sulfonate (PSS) standards or chromatograms of Suwannee River humic acid (SRHA) or fulvic acid (SRFA) but did result in an additional peak being detected in a treated municipal wastewater sample (WWT). Mobile phase concentration was found to have a greater effect on peak position of PSS standards and SRHA than mobile phase pH or sample injection volume. Several chemicals of known molecular weight were tested alongside PSS standards, with salicylic acid (138.1 Da) and 4-chlorobenzoic acid (156.6 Da) resulting in retention times that aligned with the PSS MW standards. Of the three columns tested, the two with comparable pore sizes (i.e., 250 and 300 Å) were found to result in similar peak elution times and highly linear calibration curves over the range of 210–17,000 Da, while the column with the smaller pore size (i.e., 100 Å) gave better peak separation in the low AMW range (i.e., < 1000 Da). Estimations of AMW of SRHA using various sets of calibration standards on one of the three columns tested ranged from 1080 to 2799 Da using 0.02 M NH4CH3CO2 as eluent and 60-µL injections. AMW of SRFA ranged from 939 to 2434 Da for the same conditions.

Characterization of bitter peptides in casein hydrolysates using comprehensive two-dimensional liquid chromatography

Casein hydrolysates are important additives to foods for elderly and sports nutrition. However, due to the enzymatic generation of so-called bitter peptides, their application is limited. Therefore, the procedure needs to be optimized in order to restrict their occurrence. For this, extensive sensory evaluations are necessary. By combining two separation techniques using comprehensive two-dimensional liquid chromatography, we present a novel method for estimating the bitter taste of hydrolysate samples on the basis of their elution pattern. Using a size exclusion column in the first and a reversed phase column in the second dimension allows for a detailed sample evaluation regarding peptide size and relative hydrophobicity. The results obtained for different casein hydrolysates were correlated with the sensory evaluation. We found that hydrolysates with increasing bitterness contain a higher amount of peptides of high hydrophobicity and a molecular weight less than 6.5 kDa.

Rapid Isolation of Extracellular Vesicles Using a Hydrophilic Porous Silica Gel-Based Size-Exclusion Chromatography Column.

Extracellular vesicles (EVs) are nanoscale lipid bilayer vesicles released by almost all cell types and can be found in biological fluids, such as blood and urine. EVs play an important role in various physiological and pathological processes via cell–cell communication, highlighting their potential applications as diagnostic markers for diseases and therapeutic drug delivery carriers. Although various methods have been developed for the isolation of EVs from biological fluids, most of them exhibit major limitations, including low purity, long processing times, and high cost. In this study, we developed a size-exclusion chromatography (SEC) column device using hydrophilic porous silica gel (PSG). Owing to the resistance to pressure of the device, a rapid system for EV isolation was developed by connecting it to a flash liquid chromatography system furnished with a UV detector and a fraction collector. This system can be used for the real-time monitoring of eluted EVs by UV absorption without further analysis and separation of high-purity EVs from urine samples with high durability, reusability, and reproducibility. In addition, there were no significant differences between the PSG column- and conventional SEC column-isolated EVs in the proteome profiles and cellular uptake activities, suggesting the good quality of the EVs isolated by the PSG column. These findings suggest that the PSG column device offers an effective and rapid method for the isolation of intact EVs from biological fluids.

High in-vivo stability in preclinical and first-in-human experiments with [18F]AlF-RESCA-MIRC213: a 18F-labeled nanobody as PET radiotracer for diagnosis of HER2-positive cancers.

[18F]AlF-RESCA was introduced as a core particularly useful for 18F-labeling of heat-sensitive biomolecules. However, no translational studies have been reported up to now. Herein, we reported the first-in-human evaluation of an 18F-labeled anti-HER2 nanobody MIRC213 as a PET radiotracer for imaging HER2-positive cancers. MIRC213 was produced by E. coli and conjugated with ( ±)-H3RESCA-Mal. [18F]AlF-RESCA-MIRC213 was prepared at room temperature. Its radiochemical purity and stability of were determined by radio-HPLC with the size-exclusion chromatographic column. Cell uptake was performed in NCI-N87 (HER2 +) and MCF-7 (HER2-) cells and the cell-binding affinity was verified in SK-OV-3 (HER2 +) cells. Small-animal PET/CT was performed using SK-OV-3, NCI-N87, and MCF-7 tumor-bearing mice at 30min, 1h, and 2h post-injection. For blocking experiment, excess MIRC213 was co-injected with radiotracer. Biodistribution were performed on SKOV-3 and MCF-7 tumor-bearing mice at 2h post-injection. For clinical study, PET/CT images were acquired at 2h and 4h after injection of [18F]AlF-RESCA-MIRC213 (1.85-3.7MBq/kg) in six breast cancer patients (3 HER2-positive and 3 HER2-negative). All patients underwent [18F]-FDG PET/CT within a week for tissue selection purpose. Distribution and dosimetry were calculated. Standardized uptake values (SUV) were measured in tumors and normal organs. MIRC213 was produced with > 95% purity and modified with RESCA to obtain RESCA-MIRC213. [18F]AlF-RESCA-MIRC213 was prepared within 20min at room temperature with the radiochemical yield of 50.48 ± 7.6% and radiochemical purity of > 98% (n > 10), and remained stable in both PBS (88%) and 5% HSA (92%) after 6h. The 2h cellular uptake of [18F]AlF-RESCA-MIRC213 in NCI-N87 cells was 11.22 ± 0.60 AD%/105 cells. Its binding affinity Kd value was determined to be 1.23 ± 0.58nM. Small-animal PET/CT with [18F]AlF-RESCA-MIRC213 can clearly differentiate SK-OV-3 and NCI-N87 tumors from MCF-7 tumors and background with a high uptake of 4.73 ± 1.18 ID%/g and substantially reduced to 1.70 ± 0.13 ID%/g for the blocking group (p < 0.05) in SK-OV-3 tumors at 2h post-injection. No significant bone radioactivity was seen in the tumor-bearing animals. In all six breast cancer patients, there was no adverse reaction during study. The uptake of [18F]AlF-RESCA-MIRC213 was mainly in lacrimal gland, parotid gland, submandibular gland, thyroid gland, gallbladder, kidneys, liver, and intestines. There was no significant bone radioactivity accumulation in cancer patients. [18F]AlF-RESCA-MIRC213 had significantly higher tumor uptake in lesions from HER2-positive patients than that lesions from HER2-negative patients (SUVmax of 3.62 ± 1.56 vs. 1.41 ± 0.41, p = 0.0012) at 2h post-injection. The kidneys received the highest radiation dose of 2.42 × 10-1mGy/MBq, and the effective dose was 1.56 × 10-2mSv/MBq. [18F]AlF-RESCA-MIRC213 could be prepared with high radiolabeling yield under mild conditions. [18F]AlF-RESCA-MIRC213 has relatively high stability both in vitro and in vivo. The results from clinical transformation suggest that [18F]AlF-RESCA-MIRC213 PET/CT is a safe procedure with favorable pharmacokinetics and dosimetry profile, and it is a promising new PET radiotracer for noninvasive diagnosis of HER2-positive cancers.

Clip Formation in the Complementarity Determining Region of Bevacizumab Lowers Monomer Stability and Affinity for Both FcRn and FcγR: A Comprehensive Characterization of the Clipped Variant Including its Higher Order Structure

The presence of monoclonal antibody (mAb) fragments in pharmaceutical mAb products is a critical quality attribute and should be controlled for safety. Several mAb fragments derived from clip formation in the complementarity determining regions (CDRs), as well as from cleavage in the hinge region, have been reported. However, the properties of CDR-clipped variants are not fully understood because of difficulties in separating them from intact mAbs under non-denaturing conditions due to similarities in size. We have established a method for separating CDR-clipped variants under non-denaturing conditions using an appropriate size exclusion chromatography column.1 In this report, we provide a comprehensive characterization of a CDR-clipped variant from bevacizumab. The variant exhibited a lower pI, a higher tendency to form dimers, and a lower affinity for both neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR). The effects of clip formation in CDR H3 on the higher order structure were analyzed by hydrogen/deuterium exchange mass spectrometry, and the observed changes in the structures of the VH, CH2, and VL domains were in agreement with the lowered affinity for antigen, FcRn, and FcγR. These findings suggest that clip formation in the CDR may affect the efficacy, safety, and pharmacokinetics of pharmaceutical mAbs.

An Online-SPE/SEC/LCMS Method for the Detection of N-Nitrosamine Disinfection Byproducts in Wastewater Plant Tailwater

N-nitrosamines have recently attracted attention as a class of disinfection byproducts and are also a hot spot in environmental studies. Current N-nitrosamine analytical methods typically involve manual solid phase extraction (SPE) of samples followed by quantitative analysis using liquid chromatography-mass spectrometry (LCMS), which is time-consuming and may also fail to eliminate complex matrix effects. Size exclusion chromatography (SEC) is a technique that can separate compounds according to their molecular size. For the first time, this study developed an Online-SPE/SEC/LCMS quantitative analysis method to detect and analyze nine common N-nitrosamine disinfection byproducts in wastewater plant tailwater, including N-dimethylnitrosamine (NDMA) and N-nitrosodiethylamine (NDEA), etc. The samples of 1.0 mL can be directly injected after the simple 0.22 μm membrane filtration. This method reports the combination of SPE, SEC, and RP C18 columns to achieve several functions in a processing time of 20 min, including online enrichment, desalination, and matrix separation for the first time. The method provides good linearity (R2 &gt; 0.999), recoveries ranging from 91.67% to 105.88%, relative standard deviation (RSD) lower than 4.17%, and the limits of detection (LOD) are 0.12–6.60 ng/L. This method alleviates tedious human labor and can effectively overcome the matrix effect (ME &lt; 20%). This method allows for the accurate quantitative analysis of N-nitrosamines with high compatibility in wastewater plant tailwater, rivers, and lakes with a high background matrix. Interested researchers can also use this method as a reference in the online analysis of other specific pollutants after necessary optimization. It can also be utilized for non-targeted screening and targeted analysis of contaminants in water with a wide range of applications, giving valuable information for environmental monitoring.

Size exclusion chromatography using large pore size media induces adverse conformational changes of inactivated foot-and-mouth disease virus particles

Size exclusion chromatography (SEC) of biomacromolecules using large pore size media and long column are usually necessary to obtain a satisfactory separation. However, the SEC separation of inactivated foot and mouth disease virus (FMDV) was found to induce some subtle but important conformational changes of FMDV in a pore-size and column length dependent manner. Here three Sephacryl media including S-300 HR, S-400 HR, and S-500 HR were tested, whose pore sizes were smaller than, similar to, and larger than the FMDV particles, respectively. High performance size exclusion chromatography (HPSEC) analyses showed that the FMDV after all these three SEC processes had earlier retention time, compared with that before SEC, but had no detectable difference in particle integrity. Longer SEC column led to more significant peak shifting in subsequent HPSEC analysis of FMDV. Further analyses indicated the SEC using larger pore size media induced more remarkable conformational changes and decrease in thermostability of FMDV, as well as decrease in immunogenicity in animal test. Fluorescence probe diffusion study suggested compared to SEC by S300, the compactness of the viral capsid after SEC by S400 and S500 was decreased, possibly due to more shear-induced FMDV particle rotation and inter-particle collision inside the media pores, as well as their interactions with the pore walls of the media during flowing through the column. Finally, a stabilization strategy by appending 5 mM CaCl2 in mobile phase of SEC separation was proposed and proved to efficiently maintain the conformation of the FMDV.

P-196 Assessing human embryo implantation competence through analysis of extracellular vesicles and their contents

Abstract Study question Could extracellular vesicles (EVs) derived from spent media serve as a non-invasive quality marker for embryo implantation and pregnancy? Summary answer EVs isolated from spent media derived from human blastocysts that either did or didn’t establish a pregnancy after transfer differ in size, concentration, miRNA expression. What is known already EVs are important for communication from one cell to another, influencing the translation of genes to proteins. They play a regulating role during biological processes, including pregnancy. We hypothesized that EVs released by a high-quality human embryo, which has the potential to create a pregnancy after the transfer, differ from EVs released by poor quality non-implanting embryos. Study design, size, duration Patients scheduled for a fresh single blastoycst transfer in IVF/ICSI cycles using own gametes consented to sampling of spent culture media (n = 102). Spent media from the transferred blastocyst (25 μl) were collected from implanted (hCG+/ET) (n = 45) and non-implanted (hCG-/ET) (n = 51). Spent media from degenerated embryos (n = 21) were collected from 6 patients. Media samples were pooled randomly into three replicates for each group (implanted (I), non-implanted (NI), degenerated (DG)). Participants/materials, setting, methods Extracellular vesicles were isolated by qEV single size exclusion column(SEC):a maximum of 200 µl of implanted or non-implanted or degenerated spent media was loaded onto the qEV SEC. Identification and characterization of EVs was performed by nanoparticle tracking analysis, transmission electron microscopy, and western blotting (CD9, CD63). Next, EV-RNA isolation was performed on these three groups (three replicates from each group) by Norgen Biotek Exosomal RNA Purification Kit, followed by RT-qPCR, and further data analysis. Main results and the role of chance Extracellular vesicles isolated from the SEC column are referred to as I-EVs, NI –EVs, and DG-EVs (implanted, non-implanted, and degenerated, respectively). Western blot confirmed the presence of EV-specific markers (CD63, CD9) in all EV samples (I-EVs, NI-EVs, and DG-EVs). By NTA, a higher particle concentration was observed in I-EVs (2.69±1.9 × 1010 particles/mL) compared to NI-EVs (6.13±0.2 × 1010 particles/mL) (p &amp;lt; 0.05), whereas DG-EVs particle concentration (4.97±0. 2 × 109 particles/mL) was significantly (P &amp;lt; 0.01). I-EVs were also characterized by a larger particle size with an average mean diameter of 180.3±5.4 nm, whereas NI-EVs displayed a smaller particle size with a mean diameter of 141.5±2.5 nm (p &amp;lt; 0.05). TEM confirmed the presence of larger-sized vesicles in I-EVs (≥ 200 nm) compared with NI and DG samples. During earlier work in EVs from bovine blastocysts we found miR-378a-3p to be typically present in blastocyst EVs, with a prominent role in the hatching process of the blastocyst. Here, we compared miR-378a-3p expression levels by RT-qPCR in I-EVs vs. NI-EVs vs DG-EVs. Higher expression of miR-378a-3p was identified in NI-EVs and I-EVs, compared to DG-EVs (log2fc= 30.10, log2fc = 8.94 p &amp;lt; 0.001 respectively), but was not correlated with implantation rate. Limitations, reasons for caution Currently, the methodology is unable to study EVs from a single droplet of spent media, hence pooling of the samples is necessary. As a consequence, data on EV-miRNA from individual blastocyst is lost. Wider implications of the findings Our data demonstrated that EVs and their cargo (miRNAs) have potential to be used as a non-invasive method to predict embryo implantation:miR-378a-3p has been associated with hatching in bovine embryos, but was not related with human implantion. Other embryo-derived miRNAs may be tested as a possible biomarker for human pregnancy. Trial registration number EC 2018/0266 – B670201835577

Aspidopterys obcordata vine inulin fructan affects urolithiasis by modifying calcium oxalate crystallization

Aspidopterys obcordata vine is a Chinese Dai ethnic herb used to treat urolithiasis. However, the material basis and underlying mechanisms remain undefined. In this study, a 2.3 kD inulin-like A. obcordata fructan (AOFOS) was isolated by size exclusion column chromatography and characterized by ultrahigh-performance liquid chromatography-ion trap-time of flight mass spectrometry (UPLC-IT-TOF-MS), nuclear magnetic resonance (NMR) spectroscopy, gas chromatography mass spectrometry (GC–MS) and high-performance gel permeation chromatography (HGPC). In addition, AOFOS showed unique anti-urolithiasis activity in Drosophila kidney stone models. Mechanism study indicated that AOFOS reduced the size of calcium oxalate crystals by inhibiting the formation of large size crystals and the generation rate of calcium oxalate crystals as well as the crystal form conversion from calcium oxalate monohydrate (COM) to calcium oxalate dihydrate (COD).

Investigating the secondary interactions of packing materials for size-exclusion chromatography of therapeutic proteins

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 5326: Targeted extracellular vesicle to deliver combined chemotherapies to treat cancers

Abstract Extracellular vesicles (EV), facilitated with cancer-targeted antibody or peptide, have been demonstrated as an effective delivery vehicle of chemotherapy and gene therapy. This study aimed to develop and evaluate a targeted EV to deliver a newly identified natural cytotoxic marine compound verrucarin A (Ver-A) or combination with highly potent microtubule polymerization inhibitor mertansine (DM1). The tumor models, glioblastoma (GBM), triple-negative breast cancer (TNBC) and others, were used to validate the developed targeted therapy. First, the stirred-tank bioreactor-based EV biomanufacturing was optimized by developing a two-step purification procedure using size exclusion (300 kDa) and affinity (CD63) columns in liquid chromatography system. Second, both single and dual tumor-targeting monoclonal antibodies (mAbs), such as anti-SSTR2, CD276, EGFR and/or CD47 mAb either developed in house or used in clinics, were tagged on the surface of PEGylated EV to construct mAb-EV-drugs. The flow cytometry analysis, live-cell confocal images and IC50 assay revealed the effective surface binding, intracellular drug delivery and high anti-cancer cytotoxicity of mAb-EV-drugs in cancer cell lines, and the biodistribution study confirmed the tumor targeting in xenograft mouse models. Furthermore, the maximum tolerated dose study showed minimal systemic toxicity, as confirmed by hematoxylin and eosin staining of multiple important organs. Finally, the anti-tumor efficacy study demonstrated significant inhibition of tumor growth by mAb-EV-Ver-A or Ver-A/DM1 in cell line-derived xenograft models, patient-derived xenograft models, and/or immunocompetent xenograft models. This study suggested that the targeted EV is an efficient platform to deliver combined potent chemotherapies to treat cancer or tumor. Citation Format: Kai Chen, Yingnan Si, Seulhee Kim, Zhuoxin Zhou, Lufang Zhou, X. Margaret Liu. Targeted extracellular vesicle to deliver combined chemotherapies to treat cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5326.

Modifying the Metal Surfaces in HPLC Systems and Columns to Prevent Analyte Adsorption and Other Deleterious Effects

Interactions of certain analytes with metal surfaces in high performance liquid chromatography (HPLC) instruments and columns cause a range of deleterious effects, including peak broadening and tailing, low peak areas, and the formation of new peaks due to chemical reactions. To mitigate these effects, we have developed a novel surface modification technology in which a hybrid organic/inorganic surface based on an ethylene-bridged siloxane chemistry is applied to the metal components in HPLC instruments and columns. We demonstrate the impact of this technology on peak symmetry, peak area, and injection-to-injection and column-to-column reproducibility for several metal-sensitive analytes. We also show an example of the mitigation of an on-column oxidation reaction. A variant of this technology has recently been developed for size-exclusion chromatography of proteins. An example is shown demonstrating the use of this variant applied to size-exclusion columns for the separation of a monoclonal antibody monomer and higher molecular weight species. Together, these results highlight the importance of preventing interactions of analytes with metal surfaces in HPLC in order to achieve accurate and precise results.

Bioactive constituents isolated from the Sri Lankan endemic plant Artocarpus nobilis and their potential to use in novel cosmeceuticals

Two novel compounds, dicarbonyl ester (2) and artolankanin A (3) were isolated from the sequential ethyl acetate and ethanol extracts of stem bark of A. nobilis along with the known compounds, artonin E (1) and artobiloxanthone (4) by bioassay-guided fractionation using a combination of normal phase and size exclusion column chromatography. Their structures were elucidated by spectroscopic techniques, including NMR (1H, 13C, APT, 1H–1H COSY, HSQC, and HMBC), Mass, IR, and UV. The isolated compounds were evaluated for DPPH free radical scavenging activity, tyrosinase, elastase, hyaluronidase, and A5-lipoxygenase enzyme inhibitory activities lipopolysaccharide-induced nitric oxide production inhibitory activity in SIM-A9 microglial cells. Artonin E exhibited marked tyrosinase and A5-lipoxygenase enzyme inhibitory activities and NO inhibition activity in LPS-stimulated microglia cells having IC50 values of 60.92 ± 3.45, 56.38 ± 1.05, and 23.94 ± 1.08 µg/mL, respectively. Kinetic studies showed that the tyrosinase inhibitory activity of artonin E is via a noncompetitive inhibition mechanism. Further, artolankanin A and artobiloxanthone exhibited good A5-lipoxygenase enzyme inhibitory activity having IC50 values of 59.48 ± 1.06 and 62.45 ± 2.25 µg/mL, respectively. Insights from molecular docking and molecular dynamics simulations align with these experimental observations and propose plausible mechanisms of inhibition. Collectively these results suggest that these compounds have the potential to be used as depigmenting and anti-inflammatory agents.

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media.

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

Investigating the Application of Size‐Exclusion Chromatography to Purify Bacterial Extracellular Vesicles

Sepsis is a leading cause of death as well as the most expensive condition to treat in U.S. Hospitals. Sepsis occurs when host proinflammatory response is unusually elevated, in part due to bacterial molecules secreted in bacterial extracellular vesicles (EVs). Due to their abundance, robustness, and unique characteristics between bacterial species, these bacterial EVs can potentially serve as a molecular diagnostic biomarker for Sepsis. Thus, considering the importance of diagnosing Sepsis accurately and early, a high‐purity purification technique is necessary for isolating bacterial EVs for further characterization. We propose that size exclusion chromatography (SEC) can be used to isolate bacterial EVs from the sera of sepsis patients. Additionally, we hypothesize that SEC columns packed with CL‐4B Sepharose beads will isolate bacterial EVs from heterogeneous samples with a finer resolution than columns packed with CL‐2B Sepharose beads. Previous literature has shown that CL‐2B SEC columns successfully separate human EVs from smaller particles according to size in heterogeneous samples. We have extended this work to investigate bacterial EV separation and CL‐4B Sepharose beads using three different systems: fluorescent beads and rhodamine, simulated sera, and bacterial EVs. Together, these preliminary experiments have shown that both CL‐2B and CL‐4B successfully separate bacterial EVs from smaller particles in heterogeneous samples. Future work will likely show that CL‐4B SEC columns separate bacterial EVs from other particles in patient samples with a higher resolution compared to CL‐2B beads due to their smaller pore sizes. Additional future studies will combine the use of these SEC approaches with ultrathin membranes in a single‐pass microfluidic device.

Front Cover: Comparison of the separation of proteins of wide‐ranging molecular weight via trilobal polypropylene capillary‐channeled polymer fiber, commercial superficiously porous, and commercial size exclusion columns

DOI: 10.1002/jssc.202100891 The reversed-phase (RP) separation of proteins, without size-exclusion contributions, is affected on capillary-channeled polymer (C-CP) fiber stationary phases. Polyester C-CP fibers are demonstrated to perform the rapid RP separation of peptides and proteins ranging in molecule weight of 1.44-to-660 kDa. Comparisons with commercial RP superficially porous and size exclusion columns designed for protein separations show that the across-the-board separation quality realized by the fibers is superior to the other column formats, with column material costs being far less than the standard columns.

Direct coupling of size exclusion chromatography and mass spectrometry for the characterization of complex monoclonal antibody products.

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.

Extracellular Vesicles from Human Cerebrospinal Fluid Are Effectively Separated by Sepharose CL-6B-Comparison of Four Gravity-Flow Size Exclusion Chromatography Methods.

Extracellular vesicles (EVs) are a versatile group of cell-secreted membranous nanoparticles present in body fluids. They have an exceptional diagnostic potential due to their molecular content matching the originating cells and accessibility from body fluids. However, methods for EV isolation are still in development, with size exclusion chromatography (SEC) emerging as a preferred method. Here we compared four types of SEC to isolate EVs from the CSF of patients with severe traumatic brain injury. A pool of nine CSF samples was separated by SEC columns packed with Sepharose CL-6B, Sephacryl S-400 or Superose 6PG and a ready-to-use qEV10/70 nm column. A total of 46 fractions were collected and analysed by slot-blot followed by Ponceau staining. Immunodetection was performed for albumin, EV markers CD9, CD81, and lipoprotein markers ApoE and ApoAI. The size and concentration of nanoparticles in fractions were determined by tunable resistive pulse sensing and EVs were visualised by transmission electron microscopy. We show that all four SEC techniques enabled separation of CSF into nanoparticle- and free protein-enriched fractions. Sepharose CL-6B resulted in a significantly higher number of separated EVs while lipoproteins were eluted together with free proteins. Our data indicate that Sepharose CL-6B is suitable for isolation of EVs from CSF and their separation from lipoproteins.

Physico-chemical and functional characterization of polysaccharide purified from mangrove Rhizophora mucronata leaves having potent biological activity

Rhizophora mucronata Leaves Polysaccharide (RMLP) was extracted and purified from the mangrove Rhizophora mucronata leaves by anion-exchange and size-exclusion column chromatography. Physicochemical characterization of RMLP was evaluated by spectrtoscopic analysis (FT-IR, 1H & 13C NMR) and RP-HPLC. The molecular weight of RMLP was found to be 2.69 kDa, comprised of monosaccharides such as arabinose, galacturonic acid, galactose, mannose, xylose, rhamnose and glucose. The scanning electron microscope (SEM) images showed the appearance of irregular stacks of crystal blocks with a compact structure of RMLP at 5000x fold magnification. The viscosity analysis revealed the pseudoplastic fluid behavior of the polysaccharide with shear-thinning properties. The result of x-ray diffraction (XRD) and Congo red analysis confirmed the semicrystalline nature of RMLP and has triple-helical conformation. Thermal analysis (TGA and DSC) results proved high thermal stability of the RMLP fraction as its degradation temperature was found to be 310 C. The RMLP showed tremendous antibiofilm activity in a dose-dependent manner against the gram positive and gram negative pathogens. The RMLP showed effective antioxidant ability on DPPH scavenging, superoxide, ABTS, hydroxyl anion radical and Fe2+ chelating activities. These findings indicated that RMLP has promising antioxidant and antibiofilm properties against human pathogens, which makes it a better therapeutic agent in the field of functional foods and medicines.