Size Exclusion Column Research Articles

Optimized method for fluorine-18 radiolabeling of Affibody molecules using RESCA

BackgroundIn recent years, the interest in Al[18F]F as a labeling agent for Positron Emission Tomography (PET) radiotracers has risen, as it allows for fast and efficient fluorine-18 labeling by harnessing chelation chemistry. The introduction of Restrained Complexing Agent (RESCA) as a chelator has also shown that chelator-based radiolabeling reactions can be performed in mild conditions, making the radiolabeling process attractively more facile than most conventional radiofluorination methods. The aim of the study was to establish optimized conditions for Al[18F]F labeling of Affibody molecules using RESCA as a complexing agent, using Z09591 and Z0185, two Affibody proteins targeting PDGFRβ and TNFα, respectively, as model compounds.ResultsThe Al[18F]F labeling of RESCA-conjugated Z09591 was tested at different temperatures (rt to 60 °C) and with varying reaction times (12 to 60 min), and optimal conditions were then implemented on RESCA-Z0185. The optimized synthesis method was: 1.5–2.5 GBq of cyclotron produced fluorine-18 were trapped on a QMA cartridge and eluted with saline solution to react with 12 nmol of AlCl3 and form Al[18F]F. The respective RESCA-conjugated Affibody molecule (14 nmol) in NaOAc solution was added to the Al[18F]F solution and left to react at 60 °C for 12 min. The mixture was purified on a NAP5 size exclusion column and then analyzed by HPLC. The entire process took approximately 35 min, was highly reproducible, indicating the efficiency and reliability of the method. The labeled compounds demonstrated retained biological function for their respective targets after purification.ConclusionsWe present a general and optimized method for Al[18F]F labeling of RESCA-conjugated Affibody molecules, which can be widely applied to this class of peptide-based imaging agents. Moreover, radiochemical yields were improved when the labeling was conducted at 37 °C or above. In vitro and in vivo assessment of the respective tracers was promising, showing retained binding capacity as well as moderate defluorination, which is usually regarded as a potential downside for RESCA-conjugated tracers.Graphical abstract

Comprehensive chromatographic profiling and structural analysis of key anticoagulant components in enoxaparin

Heparin is the most widely used anticoagulant in clinical practice, with enoxaparin being one of the most important low molecular weight heparins (LMWHs). In this study, an antithrombin III (ATIII) affinity column was used. Enoxaparin and its oligosaccharides of varying sizes, prepared using preparative size exclusion chromatography (SEC), were fractionated through the ATIII affinity column. The different affinity fractions from each oligosaccharide size were profiled using strong anion exchange (SAX) chromatography. Each peak was automatically transferred to an SEC column for desalting prior to mass spectrometry (MS) analysis, which enabled structural identification using a multiple heart-cut (MHC) 2D LC-MS system (SAX-SEC-MS). The high-affinity fraction from enoxaparin was further analyzed using the MHC 2D LC system (SEC-SAX). SAX profiles of the high-affinity oligosaccharides, prepared by both size and affinity fractionation, were consistent with those obtained by direct SEC-SAX analysis. The possible sequences of several high-affinity hexasaccharides and the domain compositions of high-affinity octa- and decasaccharides in enoxaparin were further elucidated by disaccharide analysis after manual collection of the oligosaccharides. This work advances the understanding of enoxaparin's structural features and offers a potential approach to improve the quality of enoxaparin, as well as to identify key structural motifs in heparin/LMWHs that contribute to protein binding.

Antimalarial evaluation of selected medicinal plants traditionally used for treatment of fever, by inhibition test of heme polymerization

World Health Organization (WHO) has reported 249 million cases infected by malaria worldwide and 608 thousands deaths in 2022. Investigations for new antimalarial drugs from traditional medicine have proven to be more effective and less expensive. The medicinal plants that have been used for treatment of malaria, generally known as types of fevers in traditional medicine, can be suitable candidates for evaluating antimalarial effects. The aim of the present study was to evaluate the in-vitro mechanism of antimalarial action of selected medicinal plants by inhibition test of heme detoxification (ITHD). The methanol extract and fractions were prepared through maceration of the dry powdered plants. The ITHD method was carried in 96-wells plate and the percentage of heme polymerization inhibition was determined. The cytotoxicity of the effective plants were examined on MDBK cell line by MTT assay. Bioassay guided fractionation was performed exposing to size exclusion column chromatography and liquid-liquid fractionation and was assayed by the ITHD method for the sample with the least IC50 value and lowest cytotoxic effect. The methanol fraction of Viola odorata whole plant showed the most considerable results among the tested plants with IC50 171.8 μg.mL-1 beside the lowest cytotoxic effects. This fraction by the bioassay guided fractionation led to fraction SB2 and this fraction demonstrated the most effective result with lowest IC50 = 14.8 ± 3µg.mL-1 in ITHD assay. Regarding the results of the present study and the traditional use of V. odorata for overcoming fever in Iranian traditional medicine, the final fraction of the plant could be proper candidate for further phytochemical and antimalarial studies.

Unified and Versatile Multiplex Platform for Expedited Method Development and Comprehensive Characterization of Therapeutic Peptides.

Agile analytical approaches are needed for fast and comprehensive characterization of peptide drug candidates. In this study, a unified and versatile multiplex platform was developed to expedite method development and enable the routine determination of multiple quality attributes simultaneously. The platform integrates the automation of size exclusion chromatography (SEC), reversed phase liquid chromatography coupled to reversed phase liquid chromatography (RPLC-RPLC), and hydrophilic interaction liquid chromatography hyphenated to charged aerosol detection (HILIC-CAD). Various therapeutic peptide constructs, including macrocyclic peptides and disulfide constrained peptides, across different lots were studied. The effect of the mobile phase acetonitrile content on the impurity profiles was systematically studied using two SEC columns. A prototype MaxPeak Premier SEC 125 Å column packed with BEH PEO particles achieved the separation of impurities (>2.0% area), whereas no impurities could be observed with an ACQUITY UPLC Protein BEH SEC 125 Å column packed with BEH diol particles. Comprehensive impurity profiling and expedited method development was performed utilizing RPLC-RPLC. Each peptide was analyzed by a combination of 12 conditions in the second dimension, using four columns with octadecyl, phenyl-hexyl, and cyano bonded phases, and three mobile phases with various solvents, modifiers, and pH compositions. Additionally, a HILIC-CAD method was developed for the quantification of TFA, commonly present in peptide products.

Regioselective Ligand Substitution of Thiolate-Protected Gold Nanoclusters with Porphyrins

Hybridization of photofunctional organic molecules like porphyrins with inorganic nanomaterials provides interesting photophysical properties like photo-induced electron transfer, energy transfer, or photoemission from charge transfer states. For fine tuning of these photo-functionalities, precise control over organic-inorganic interfaces, including spatial distances and orientations of organic molecules against inorganic nanomaterial, should be crucial. However, control and evaluation of the interfaces are quite difficult, mainly due to heterogeneous and unclear environments on the surface of inorganic nanomaterials like metal nanoparticles and metal nanosheet, which basically have size and/or composition distributions.Among the inorganic nanomaterials, atomically-precise metal nanoclusters (MNCs) are one of the homogeneous materials with uniform composition and distinct surface structure at an atomic level. Thus, MNCs can be an appropriate model to evaluate the effects of organic-inorganic interfaces on photophysical properties of the hybridized materials. Nevertheless, even in one of the most common and simplest MNCs like thiolate-protected Au25 cluster ([Au25(SR)18]–, SR = thiolate), two kinds of inequivalent ligands (S1, S2 in Figure 1) are present on surface. Thus, control over the regioselectivity should be required to archive precise substitution of photo-functional molecules, which has yet to be reported. In this work, we have controlled regioselectivity in the modification of [Au25(SC2Ph)18]– (SC2Ph = phenylethanethiolate) with porphyrinthiol ligands (Por) through ligand-exchange reactions. An advantage of Por as a photofunctional ligand for Au25 cluster is more bulkiness than that of original ligands (SC2Ph), which aids the isolation of mono-porphyrin-coordinated Au25 cluster ([Au25(SC2Ph)17(Por)1]–, Au25-Por1 ). Moreover, synthetic flexibility of porphyrins enabled us to control the regioselectivity in Au25-Por1 systematically.We sythesized a series of Por with a thiol moiety at different positions to evaluate the steric effect on the regioselectivity in ligand-exchange reactions (Figure 1). Porphyrin thiol with different substituents at meso-phenyl groups were also synthesized to evaluate the electronic effect. Substitution of porphyrins onto Au25 clusters was conducted by ligand-exchange reactions of [Au25(SC2Ph)18]– with 1 equivalent of Por in tetrahydrofuran. After the careful purification of the reaction mixture through size-exclusion column chromatography, we have successfully isolated Au25-Por1 in ca. 20% yield. The characterizations of Au25-Por1 were performed by MALDI-TOF-MS and 1H NMR spectroscopy, which confirmed the high purities of a series of Au25-Por1 .The regioselectivity in Au25-Por1 was evaluated through 1H NMR measurements. When using para-substituted porphyrin (PorSH) as an incoming ligand, 1H NMR spectrum of Au25-SPor1 in acetone-d 6 indicated the presence two kinds of regioisomers with the ratio of 2.5:1. Interestingly, the regioselectivity increased up to 80% (4:1) in Au25 cluster with more steric meta-substituted porphyrin (Au25-S m Por1 ), whereas the ratio reached to almost 1:1 in the case of Au25 cluster with less steric phenyl-spacer introduced porphyrin (Au25-SPhPor1 ). Because S2 position in the Au25 clusters was less steric than S1 position, we concluded that more steric ligands like m PorSH tend to be introduced at S2 position favorably. On the other hand, we hardly observed the electronic effect of Por on the regioselectivity in Au25-Por1 , indicating the importance of steric factor of an incoming ligand for precise control over the exchanged position (Figure 1). This is the first report to control over ligand-exchange position of MNCs, which could be achieved through ligand design.Finally, we briefly evaluated the photophysical properties of Au25-Por1 in toluene solution. When the Soret band of coordinated-porphyrins was photoexcited, the fluorescence quenching was clearly observed in each Au25-Por1 . Judging from the energy diagram of Au25-Por1 , electron transfer from Au25 to singlet excited state of porphyrin ligand would occur to form charge separated state. It should be noted that the quenching efficiency depended on not only the distance between electron donor and acceptor, but also the position of the coordinated chromophore. These results suggest the importance of precise modification of organic-inorganic interfaces to tune photodynamics of chromophore-nanocluster systems.In conclusion, we have successfully controlled the regioselectivity in the ligand-exchange reactions of thiolate-protected gold nanoclusters with porphyrinthiol derivatives through the steric effect. The essential roles of the ligand-introduced position on altering photodynamics of porphyrin-coordinated MNCs were also clarified, which provides an interesting insight into fine-tuning of photofunctional materials.Figure 1.Reaction scheme of ligand-exchange reaction of [Au25(SC2Ph)18]– with Por. The R groups of SR ligands were omitted for clarity. Figure 1

P-040 The characteristic of human seminal plasma extracellular vesicle (SPEVs) profiles with different sperm features in a large cohort of 309 Taiwanese men

Abstract Study question To investigate human seminal plasma extracellular vesicle profiles in different sperm characteristics and their potential clinical application. Summary answer Normozoospermic semen has significantly higher SPEV concentration than asthenozoospermic or oligozoospermic samples. Moreover, SPEV concentration was highly correlated with total motile sperm count. What is known already Seminal plasma extracellular vesicles (SPEVs) are cell-derived membrane structures that carry proteins, nucleic acid, and lipids, which can modify the constitution and biological function of sperm. However, the characteristics of SPEVs in different sperm features remained unclear. Study design, size, duration From January 2021 to September 2023, 309 men who visited the NCKUH andrology clinic agreed to collect semen samples for this study, which the Institutional Review Board has approved. Participants/materials, setting, methods Semen parameters were measured by the X1 CASA system (Bonraybio, Taiwan). SPEVs were isolated by size exclusion chromatography column and ExoQuick™. Next, the SPEVs’ concentration and size distribution were performed with nanoparticle tracking analysis. The Kruskal-Wallis test calculated comparison in multiple groups, and Dunn’s multiple comparison tests accessed group-group comparison. The Pearson correlation coefficient r of exosome concentration with individual sperm parameters was calculated. A p-value less than 0.05 was set as statistical significance. Main results and the role of chance Among them, 59 semen samples were presented with normozoospermia, 36 were oligozoospermia, 73 were asthenozoospermia, and 141 samples had no sperm in semen. Among these 141 azoospermic men, 89 men are characterized by non-obstructive azoospermia (NOA), while the other 52 men had a vasectomy. Our data showed that normozoospermic semen has significantly higher SPEV concentration than asthenozoospermic or oligozoospermic semen samples (26 x109 [IQR 17.25 x109-56.25 x109] versus 12 x109 [IQR 5.6 x109-19.5 x109] versus 15.5 x109 [IQR 8.575 x109-26.75 x109], p = 0.0001). Nevertheless, azoospermic semen has the lowest SPEV concentrations. The concentration of SPEVs from infertile men diagnosed with NOA is slightly lower than vasectomized men (3.9 x109 [IQR 1.875 x109-7.675 x109] versus 5.4 x109 [IQR 2.9 x109-8 x109], p = 0.0001). The Pearson r coefficient of SPEV concentration with sperm concentration, progressive motility, and total motile sperm count was 0.238, 0.303, and 0.451, respectively. (P < 0.0001) Limitations, reasons for caution Because our data were collected from Taiwanese men, it is not yet known whether SPEV profiles differ among races. Wider implications of the findings Human SPEV concentration and its bio-content may be a potential non-invasive biomarker to predict the male reproductive outcome. Trial registration number not applicable

On-line coupling of hollow-fiber flow field-flow fractionation and depolarized multi-angle static light scattering (HF5/D-MALS). Proof of principle

Introduced here is the on-line coupling of hollow-fiber flow field-flow fractionation (HF5) to depolarized multi-angle static light scattering (D-MALS). HF5 is a size-based separation alternative to size-exclusion and hydrodynamic chromatography and asymmetric flow field-flow fractionation. HF5 can separate larger sizes than its chromatographic counterparts and provides several advantages over its fractionation counterpart, including reduced sample consumption and greater ease of operation. D-MALS is a variant of MALS in which the depolarized scattering from the analyte solution is measured at a variety of angles simultaneously. Measurements of depolarized scattering have previously been employed in studying the optical properties of solutions or suspensions, to determine the length of rod-like analytes, and to gain increased accuracy in the determination of analyte molar mass. The coupling HF5/D-MALS allows for the depolarization ratio of a solution or suspension to be measured continuously across the fractogram. This is demonstrated here for a Teflon latex the size range of which extends beyond that accessible to commercial size-exclusion columns. The results presented provide the first reported on-line HF5/D-MALS coupling, showing the feasibility of the technique as well as its realized potential for providing continuous depolarization measurements, inter alia.

Amniotic fluid-derived stem cells: potential factories of natural and mimetic strategies for congenital malformations.

Mesenchymal stem cells (MSCs) from gestational tissues represent promising strategies for in utero treatment of congenital malformations, but plasticity and required high-risk surgical procedures limit their use. Here we propose natural exosomes (EXOs) isolated from amniotic fluid-MSCs (AF-MSCs), and their mimetic counterparts (MIMs), as valid, stable, and minimally invasive therapeutic alternatives. MIMs were generated from AF-MSCs by combining sequential filtration steps through filter membranes with different porosity and size exclusion chromatography columns. Physiochemical and molecular characterization was performed to compare them to EXOs released from the same number of cells. The possibility to exploit both formulations as mRNA-therapeutics was explored by evaluating cell uptake (using two different cell types, fibroblasts, and macrophages) and mRNA functionality overtime in an in vitro experimental setting as well as in an ex vivo, whole embryo culture using pregnant C57BL6 dams. Molecular and physiochemical characterization showed no differences between EXOs and MIMs, with MIMs determining a 3-fold greater yield. MIMs delivered a more intense and prolonged expression of mRNA encoding for green fluorescent protein (GFP) in macrophages and fibroblasts. An ex-vivo whole embryo culture demonstrated that MIMs mainly accumulate at the level of the yolk sac, while EXOs reach the embryo. The present data confirms the potential application of EXOs for the prenatal repair of neural tube defects and proposes MIMs as prospective vehicles to prevent congenital malformations caused by in utero exposure to drugs.

Triethanolamine-based esterquat in sediments: New analytical method, environmental occurrence, and homologue composition

The cationic surfactant triethanolamine-based esterquat (TEAQ) is a main ingredient in commercial fabric softeners and is produced and used in large quantities. However, little information is available for its occurrence in the environment, particularly in sediments. Here, we developed an analytical method for quantifying TEAQ in sediment and investigated TEAQ contamination in Japanese river and lake sediments. In our analytical method, TEAQ concentrations were measured by liquid chromatography–tandem mass spectrometry using a polymer-based size-exclusion column, which resulted in excellent peak shapes. TEAQ was detected at significant levels in procedural blanks, resulting in a method limit of detection in the sediment of 8.9–97 μg/kg-dry for TEAQ monoesters and 0.6–24 μg/kg-dry for TEAQ diesters. TEAQ was detected in 22 out of 26 sediment samples, with the sum of all homologue concentrations being up to 1340 μg/kg-dry. The concentration of TEAQ in sediments was high at locations where the concentrations of benzalkoniums and the organic carbon content were also high. TEAQ was detected in 8 out of 14 commercial fabric softeners at concentrations of 1.7–7.4 wt%. TEAQ homologues containing only saturated fatty acids accounted for 83 ± 5% of the total TEAQ in the sediments, whereas those with unsaturated fatty acids accounted for 71 ± 14% of the total TEAQ in a commercial technical mixture and the softener products. The results of this study will be useful for the environmental risk assessment of esterquats.

Intact quantitation of cysteine-conjugated antibody-drug conjugates using native mass spectrometry.

A common strategy for antibody-drug conjugate (ADC) quantitation from in vivo study samples involves measurement of total antibody, conjugated ADC, and free payload concentrations using multiple reaction monitoring (MRM) mass spectrometry. This not only provides a limited picture of biotransformation but can also involve lengthy method development. Quantitation of ADCs directly at the intact protein level in native conditions using high-resolution mass spectrometers presents the advantage of measuring exposure readout as well as monitoring the change in average drug-to-antibody ratio (DAR) and in vivo stability of new linker payloads with minimal method development. Furthermore, site-specific cysteine-conjugated ADCs often rely on non-covalent association to retain their quaternary structure, which highlights the unique capabilities of native mass spectrometry (nMS) for intact ADC quantitation. We developed an intact quantitation workflow involving three stages: automated affinity purification, nMS analysis, and data processing in batch fashion. The sample preparation method was modified to include only volatile ion-pairing reagents in the buffer systems. A capillary size-exclusion chromatography (SEC) column was coupled to a quadrupole time-of-flight high-resolution mass spectrometer for high-throughput nMS analysis. Samples from two mouse pharmacokinetic (PK) studies were analyzed using both intact quantitation workflow and the conventional MRM-based approach. A linear dynamic range of 5-100 μg/mL was achieved using 20 μL of serum sample volume. The results of mouse in vivo PK measurement using the intact quantitation workflow and the MRM-based approach were compared, revealing excellent method agreement. We demonstrated the feasibility of utilizing nMS for the quantitation of ADCs at the intact protein level in preclinical PK studies. Our results indicate that this intact quantitation workflow can serve as an alternative generic method for high-throughput analysis, enabling an in-depth understanding of ADC stability and safety in vivo.

Analytical Quality by Design as applied to the development of a SEC-HPLC platform procedure for the determination of monoclonal antibody purity without mobile phase additives

This work presents the application of AQbD principles to the development of a size exclusion chromatography (SEC) HPLC procedure for the determination of monoclonal antibody (mAb) product purity using state-of-the-art column technology available via the Waters™ XBridge Premier Protein SEC column. Analytical Quality by Design (AQbD) emphasizes a systematic, risk-based lifecycle approach to analytical procedure development based on sound statistical methodologies. It has recently become increasingly recommended by regulatory agencies as a response to the need for greater efficiency, improved reliability, and increased robustness among modern analytical procedures in the pharmaceutical industry. Use of an Analytical Target Profile (ATP) and formal risk assessments informed the application of Design of Experiments (DoE) to optimize this analytical procedure, as well as assess its robustness and ruggedness. Importantly, our ruggedness results demonstrated the transferability of this procedure between two laboratories within the Catalent Biologics Global Network. Application of this analytical procedure as a platform approach for evaluating mAb purity is expected to support expedited, first-in-human timelines of mAb molecules by enabling great quantitative performance with simple mobile phase buffer compositions. Taken together, this case study demonstrates the utility of adopting AQbD principles in analytical procedure development.

Effect of the 35 nm and 70 nm Size Exclusion Chromatography (SEC) Column and Plasma Storage Time on Separated Extracellular Vesicles.

The technical difficulty of separating extracellular vesicles (EVs) from plasma proteins in human blood presents a significant hurdle in EV research, particularly during nano ultra-high-performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) analysis, where detecting "vesicular" proteins among abundant plasma proteins is challenging. Standardisation is a pressing issue in EV research, prompting collaborative global efforts to address it. While the MISEV guidelines offer valuable recommendations, unanswered questions remain, particularly regarding sample storage. We compared size exclusion chromatography (SEC) columns with pore sizes of 35 nm and 70 nm to identify fractions with minimal contaminating proteins and the highest concentration of small EVs (sEVs). Following column selection, we explored potential differences in the quality and quantity of sEVs isolated from platelet-free plasma (PFP) after long-term storage at -80 °C (>2.5 years) compared to freshly drawn blood. Our methodologically rigorous study indicates that prolonged storage, under correct storage and processing conditions, does not compromise sEV quality. Both columns effectively isolated vesicles, with the 70 nm column exhibiting a higher abundance of "vesicular" proteins. We propose a relatively rapid and moderately efficient protocol for obtaining a comparatively pure sEV fraction from plasma, facilitating sEV processing in clinical trials.

A magnetic separation method for isolating and characterizing the biomolecular corona of lipid nanoparticles

Lipid nanoparticle (LNP) formulations are a proven method for the delivery of nucleic acids for gene therapy as exemplified by the worldwide rollout of LNP-based RNAi therapeutics and mRNA vaccines. However, targeting specific tissues or cells is still a major challenge. After LNP administration, LNPs interact with biological fluids (i.e., blood), components of which adsorb onto the LNP surface forming a layer of biomolecules termed the "biomolecular corona (BMC)" which affects LNP stability, biodistribution, and tissue tropism. The mechanisms by which the BMC influences tissue- and cell-specific targeting remains largely unknown, due to the technical challenges in isolating LNPs and their corona from complex biological media. In this study, we present a new technique that utilizes magnetic LNPs to isolate LNP-corona complexes from unbound proteins present in human serum. First, we developed a magnetic LNP formulation, containing >40 superparamagnetic iron oxide nanoparticles (IONPs)/LNP, the resulting LNPs containing iron oxide nanoparticles (IOLNPs) displayed a similar particle size and morphology as LNPs loaded with nucleic acids. We further demonstrated the isolation of the IOLNPs and their corresponding BMC from unbound proteins using a magnetic separation (MS) system. The BMC profile of LNP from the MS system was compared to size exclusion column chromatography and further analyzed via mass spectrometry, revealing differences in protein abundances. This new approach enabled a mild and versatile isolation of LNPs and its corona, while maintaining its structural integrity. The identification of the BMC associated with an intact LNP provides further insight into LNP interactions with biological fluids.

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

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

Polyethylene Glycol -Mediated Exosome Isolation: A Method for Exosomal RNA Analysis.

: Exosomal RNAs (ExoRNAs) offer valuable insights into their cellular origin. ExoRNA studies were faced with challenges in obtaining sufficient amounts of high-quality RNA. Herein, we aimed to compare three traditional exosome isolation methods to introduce an appropriate strategy to extract RNA from cancer-derived exosomes for further RNA analysis. Exosomes were isolated through ultracentrifugation, precipitation kit, and size exclusion column chromatography, and then characterized by dynamic light scattering and transmission electron microscopy, followed by extracting total RNA. The quality and quantity of the extracted RNAs were assessed by a NanoDrop and 2.5% agarose gel electrophoresis. Extracted exosomes displayed a similar range of size and morphology. We found that polyethylene glycol-precipitation method resulted in a higher RNA yield with a 260/280 ratio of 1.9. The obtained exoRNA appeared as a smear in the agarose gel, indicative of small exoRNAs. We provide researchers a suitable approach to isolate exosomes based on yield and purity of exoRNA.

Identification of Hypoglycemic Glycolipids from Ipomoea murucoides by Affinity-Directed Fractionation, In Vitro, In Silico and Dynamic Light Scattering Analysis.

In the pursuit of identifying the novel resin glycoside modulators glucose-6-phosphatase and α-glucosidase enzymes, associated with blood sugar regulation, methanol-soluble extracts from the flowers of Ipomoea murucoides (cazahuate, Nahuatl), renowned for its abundance of glycolipids, were employed. The methanol-soluble extracts were fractionated by applying the affinity-directed method with glucose-6-phosphatase enzymes from a rat's liver and α-glucosidase enzymes from its intestines. Mass spectrometry and nuclear magnetic resonance were employed to identify the high-affinity compound as a free ligand following the release from the enzymatic complex. Gel permeation through a spin size-exclusion column allowed the separated high-affinity molecules to bind to glucose-6-phosphatase and α-glucosidase enzymes in solution, which led to the identification of some previously reported resin glycosides in the flowers of cazahuate, where a glycolipid mainly structurally related to murucoidin XIV was observed. In vitro studies demonstrated the modulating properties of resin glycosides on the glucose-6-phosphatase enzyme. Dynamic light scattering revealed conformational variations induced by resin glycosides on α-glucosidase enzyme, causing them to become more compact, akin to observations with the positive control, acarbose. These findings suggest that resin glycosides may serve as a potential source for phytotherapeutic agents with antihyperglycemic properties.

In-line buffer exchange in the coupling of Protein A chromatography with weak cation exchange chromatography for the determination of charge variants of immunoglobulin G derived from chinese hamster ovary cell cultures

Immunoglobulin G (IgG) is the most common monoclonal antibody (mAb) grown for therapeutic applications. While IgG is often selectively isolated from cell lines using protein A (ProA) chromatography, this is only a stepping stone for complete characterization. Further classification can be obtained from weak cation exchange chromatography (WCX) to determine IgG charge variant distributions. The charge variants of monoclonal antibodies can influence the stability and efficacy in vivo, and deviations in charge heterogeneity are often cell-specific and sensitive to upstream process variability. Current methods to characterize IgG charge variants are often performed off-line, meaning that the IgG eluate from the ProA separation is collected, diluted to adjust the pH, and then transferred to the WCX separation, adding time, complexity, and potential contamination to the sample analysis process. More recently, reports have appeared to streamline this separation using in-line two-dimensional liquid chromatography (2D-LC). Presented here is a novel, 2D-LC coupling of ProA in the first dimension (1D) and WCX in the second dimension (2D) chromatography. As anticipated, the initial direct column coupling proved to be challenging due to the pH incompatibility between the mobile phases for the two stages. To solve the solvent compatibility issue, a size exclusion column was placed in the switching valve loop of the 2D-LC instrument to act as a means for the on-line solvent exchange. The efficacy of the methodology presented was confirmed through a charge variant determination using the NIST monoclonal antibody standard (NIST mAb), yielding correct acidic, main, and basic variant compositions. The methodology was employed to determine the charge variant profile of IgG from an in-house cultured Chinese hamster ovary (CHO) cell supernatant. It is believed that this methodology can be easily implemented to provide higher-throughput assessment of IgG charge variants for process monitoring and cell line development.

Production of Bioactive Porcine Lactoferrin through a Novel Glucose-Inducible Expression System in Pichia pastoris: Unveiling Antimicrobial and Anticancer Functionalities.

Lactoferrin (LF) stands as one of the extensively investigated iron-binding glycoproteins within milk, exhibiting diverse biological functionalities. The global demand for LF has experienced consistent growth. Biotechnological strategies aimed at enhancing LF productivity through microbial expression systems offer substantial cost-effective advantages and exhibit fewer constraints compared to traditional animal bioreactor technologies. This study devised a novel recombinant plasmid, wherein the AOX1 promoter was replaced with a glucose-inducible G1 promoter (PG1) to govern the expression of recombinant porcine LF (rpLF) in Pichia pastoris GS115. High-copy-number PG1-rpLF yeast clones were meticulously selected, and subsequent induction with 0.05 g/L glucose demonstrated robust secretion of rpLF. Scaling up production transpired in a 5 L fermenter, yielding an estimated rpLF productivity of approximately 2.8 g/L by the conclusion of glycerol-fed fermentation. A three-step purification process involving tangential-flow ultrafiltration yielded approximately 6.55 g of rpLF crude (approximately 85% purity). Notably, exceptional purity of rpLF was achieved through sequential heparin and size-exclusion column purification. Comparatively, the present glucose-inducible system outperformed our previous methanol-induced system, which yielded a level of 87 mg/L of extracellular rpLF secretion. Furthermore, yeast-produced rpLF demonstrated affinity for ferric ions (Fe3+) and exhibited growth inhibition against various pathogenic microbes (E. coli, S. aureus, and C. albicans) and human cancer cells (A549, MDA-MB-231, and Hep3B), similar to commercial bovine LF (bLF). Intriguingly, the hydrolysate of rpLF (rpLFH) manifested heightened antimicrobial and anticancer effects compared to its intact form. In conclusion, this study presents an efficient glucose-inducible yeast expression system for large-scale production and purification of active rpLF protein with the potential for veterinary or medical applications.

Physical origin of the peak tailing of monoclonal antibodies in size-exclusion chromatography using bio-compatible systems and columns.

The analysis of mixtures containing monoclonal antibody (mAb) (approximately 150 kDa molecular weight) and sub-unit impurities (approximately 100 kDa) is challenging, even when adopting the latest ultra-high-pressure liquid chromatography (UHPLC) columns (4.6 mm [Formula: see text] 150mm coated hardware, 1.7 [Formula: see text]m 250 BEH[Formula: see text] Surface-modified Particles) and systems (ACQUITY[Formula: see text] UPLC[Formula: see text] I-class Bio Plus). The main issue still encountered is a persistent tail of the mAb peak. Here, the physical origin(s) of such peak tailing in size-exclusion chromatography (SEC) are investigated from both fundamental and practical approaches. Up to five relevant physical origins are analyzed: sample heterogeneity (glycoforms), UHPLC system dispersion, strong residual binding of the mAb to the SEC particles (via hydrophobic and/or electrostatic interactions) and to the stainless steel column/system hardware, slow escape kinetics of the mAb from the SEC particles, and flow heterogeneity caused by the non-ideal slurry packing of SEC columns. Experiments (testing sample heterogeneity, system dispersion, and strong residual interactions) and calculations (predicting the transient absorption/escape kinetics in a single SEC particle and the two-dimensional peak concentration profiles) altogether unambiguously demonstrate that the observed mAb peak tailing is caused primarily by the long-range velocity biases across the SEC column combined with the slow transverse dispersion of mAbs. Therefore, improvement in the resolution between mAb and sub-unit fragment impurities can only be achieved by increasing the column length, e.g., by applying recycling chromatography at acceptable pressures.

Intracellular and Extracellular Peptidomes of the Model Plant, Physcomitrium patens.

Here, we report our approach to peptidomic analysis of the plant model Physcomitrium patens. Intracellular and extracellular peptides were extracted under conditions preventing proteolytic digestion by endogenous proteases. The extracts were fractionated on size exclusion columns to isolate intracellular peptides and on reversed-phase cartridges to isolate extracellular peptides, with the isolated peptides subjected to LC-MS/MS analysis. Mass spectrometry data were analyzed for the presence of peptides derived from the known proteins or microproteins encoded by small open reading frames (<100 aa, smORFs) predicted in the moss genome. Experimental details are provided for each step.