Size Exclusion Chromatography Fractions Research Articles

Analysis of aggregation profile of glucagon using SEC-HPLC and FFF-MALS methods.

Recently, the first generic glucagon for injection was approved for the treatment of severe hypoglycemia. Unlike its brand name recombinant glucagon, the generic glucagon is synthetic. Since glucagon has a high propensity to form aggregates in solution, it is essential to assess the aggregation profile of the synthetic glucagon compared to the recombinant glucagon. In this study, two robust separation methods, size-exclusion chromatography (SEC-HPLC) and field-flow fractionation coupled with a multi-angle light scattering detector (FFF-MALS), were employed to characterize generic and brand glucagon aggregation in six lots (three newly released, three expired). The presence of aggregation in samples was determined from the generated chromatograms and analyzed. The study showed that both products have comparable aggregation profiles. The SEC-HPLC demonstrated that in both glucagon versions, the expired lots had a higher percentage of dimers than the newly released lots, but even at expiration, the amount was negligible (∼0.1%). The FFF-MALS method did not detect any dimers or higher molecular weight aggregates. Further evaluation of the detection limit found that FFF-MALS was unable to detect aggregates at amounts lower than 0.5% of total glucagon. The negligible amounts of dimer detected in the generic and brand glucagon indicate that both versions are physically stable and are not prone to aggregation under clinically relevant conditions.

Global Interactome Mapping Reveals Pro-tumorigenic Interactions of NF-κB in Breast Cancer

NF-κB pathway is involved in inflammation; however, recent data shows its role also in cancer development and progression, including metastasis. To understand the role of NF-κB interactome dynamics in cancer, we study the complexity of breast cancer interactome in luminal A breast cancer model and its rearrangement associated with NF-κB modulation. Liquid chromatography-mass spectrometry measurement of 160 size-exclusion chromatography fractions identifies 5460 protein groups. Seven thousand five hundred sixty eight interactions among these proteins have been reconstructed by PrInCE algorithm, of which 2564 have been validated in independent datasets. NF-κB modulation leads to rearrangement of protein complexes involved in NF-κB signaling and immune response, cell cycle regulation, and DNA replication. Central NF-κB transcription regulator RELA co-elutes with interactors of NF-κB activator PRMT5, and these complexes are confirmed by AlphaPulldown prediction. A complementary immunoprecipitation experiment recapitulates RELA interactions with other NF-κB factors, associating NF-κB inhibition with lower binding of NF-κB activators to RELA. This study describes a network of pro-tumorigenic protein interactions and their rearrangement upon NF-κB inhibition with potential therapeutic implications in tumors with high NF-κB activity.

Unfolding of Lignin Structure Using Size-Exclusion Fractionation.

The heterogeneous and recalcitrant structure of lignin hinders its practical application. Here, we describe how new approaches to lignin characterization can reveal structural details that could ultimately lead to its more efficient utilization. A suite of methods, which enabled mass balance closure, the evaluation of structural features, and an accurate molecular weight (MW) determination, were employed and revealed unexpected structural features of the five alkali lignin fractions obtained with preparative size-exclusion chromatography (SEC). A thermal carbon analysis (TCA) provided quantitative temperature profiles based on sequential carbon evolution, including the final oxidation of char. The TCA results, supported with thermal desorption/pyrolysis gas chromatography-mass spectrometry (TD-Py-GC-MS) and 31P NMR spectroscopy, revealed the unfolding of the lignin structure as a result of the SEC fractionation, due to the disruption of the interactions between the high- and low-MW components. The "unraveled" lignin revealed poorly accessible hydroxyl groups and showed an altered thermal behavior. The fractionated lignin produced significantly less char upon pyrolysis, 2 vs. 47%. It also featured a higher occurrence of low-MW thermal evolution products, particularly guaiacol carbonyls, and more than double the number of OH groups accessible for phosphitylation. These observations indicate pronounced alterations in the lignin intermolecular association following size-exclusion fractionation, which may be used for more efficient lignin processing in biorefineries.

Analytical characterization of host-cell-protein-rich aggregates in monoclonal antibody solutions.

Host-cell proteins (HCPs) and high molecular weight (HMW) species have historically been treated as independent classes of impurities in the downstream processing of monoclonal antibodies (mAbs), but recent indications suggest that they may be partially linked. We have explored this connection with a shotgun proteomic analysis of HMW impurities that were isolated from harvest cell culture fluid (HCCF) and protein A eluate using size-exclusion chromatography (SEC). As part of the proteomic analysis, a cross-digest study was performed in which samples were analyzed using both the standard and native digest techniques to enable a fair comparison between bioprocess pools. This comparison reveals that the HCP profiles of HCCF and protein A eluate overlap substantially more than previous work has suggested, because hundreds of HCPs are conserved in aggregates that may be up to ~50 nm in hydrodynamic radius and that persist through the protein A capture step. Quantitative SWATH proteomics suggests that the majority of the protein A eluate's HCP mass is found in such aggregates, and this is corroborated by ELISA measurements on SEC fractions. The SWATH data also show that intra-aggregate concentrations of individual HCPs are positively correlated between aggregates that were isolated from HCCF and protein A eluate, and species that have generally been considered difficult to remove tend to be more concentrated than their counterparts. These observations support prior hypotheses regarding aggregate-mediated HCP persistence through protein A chromatography and highlight the importance of this persistence mechanism.

Defining the influence of size‐exclusion chromatography fraction window and ultrafiltration column choice on extracellular vesicle recovery in a skeletal muscle model

AbstractExtracellular vesicles (EVs) have the potential to provide new insights into skeletal muscle (SM) physiology and pathophysiology. However, current isolation protocols often do not eliminate co‐isolated components such as lipoproteins and RNA binding proteins that could confound outcomes and hinder downstream clinical translation. In this study, we validated an EV isolation protocol that combined size‐exclusion chromatography (SEC) with ultrafiltration (UF) to increase sample throughput, scalability and purity, while providing the very first analysis of the effects of UF column choice and fraction window on EV recovery. C2C12 myotube conditioned medium was pre‐concentrated using either Amicon® Ultra 15 or Vivaspin®20 100 KDa UF columns and processed by SEC (IZON, qEV 70 nm). The resulting thirty fractions obtained were individually analysed to identify an optimal fraction window for EV recovery. The EV marker TSG101 could be detected from fractions 5 to 14, while CD9 and Annexin A2 only up to fraction 6. ApoA1+ lipoprotein co‐isolates were detected from fraction 6 onwards for both protocols. Strikingly, Amicon and Vivaspin UF concentration protocols led to qualitative and quantitative variations in EV marker profiles and purity. Eliminating lipoprotein co‐isolation by reducing the SEC fraction window resulted in a net loss of particles, but increased measures of sample purity and had only a negligible impact on the presence of EV marker proteins. In conclusion, our study developed an effective UF+SEC protocol for the isolation of EVs based on sample purity (fractions 1–5) and total EV abundance (fractions 2–10). We provide evidence to demonstrate that the choice of UF column can affect the composition of the resulting EV preparation and needs to be considered when being applied in EV isolation studies in SM. The resulting protocols will be valuable in isolating highly pure EV preparations for applications in a range of therapeutic and diagnostic studies.

Cell-Free RNA from Plasma in Patients with Neuroblastoma: Exploring the Technical and Clinical Potential

Neuroblastoma affects mostly young children, bearing a high morbidity and mortality. Liquid biopsies, e.g., molecular analysis of circulating tumor-derived nucleic acids in blood, offer a minimally invasive diagnostic modality. Cell-free RNA (cfRNA) is released by all cells, especially cancer. It circulates in blood packed in extracellular vesicles (EV) or attached to proteins. We studied the feasibility of analyzing cfRNA and EV, isolated by size exclusion chromatography (SEC), from platelet-poor plasma from healthy controls (n = 40) and neuroblastoma patients with localized (n = 10) and metastatic disease (n = 30). The mRNA content was determined using several multiplex droplet digital PCR (ddPCR) assays for a neuroblastoma-specific gene panel (PHOX2B, TH, CHRNA3) and a cell cycle regulation panel (E2F1, CDC6, ATAD2, H2AFZ, MCM2, DHFR). We applied corrections for the presence of platelets. We demonstrated that neuroblastoma-specific markers were present in plasma from 14/30 patients with metastatic disease and not in healthy controls and patients with localized disease. Most cell cycle markers had a higher expression in patients. The mRNA markers were mostly present in the EV-enriched SEC fractions. In conclusion, cfRNA can be isolated from plasma and EV and analyzed using multiplex ddPCR. cfRNA is an interesting novel liquid biopsy-based target to explore further.

Laser scattering centrifugal liquid sedimentation method for the accurate quantitative analysis of mass-based size distributions of colloidal silica.

This paper proposes a laser scattering centrifugal liquid sedimentation (LS-CLS) method for the accurate quantitative analysis of the mass-based size distributions of colloidal silica. The optics comprised a laser diode light source and multi-pixel photon-counting detector for detecting scattered light intensity. The unique optics can only detect the light scattered by a sample through the interception of irradiated light. The developed centrifugal liquid sedimentation (CLS) method comprised a light-emitting diode and silicon photodiode detector for detecting transmittance light attenuation. The CLS apparatus could not accurately measure quantitative volume- or mass-based size distribution of poly-dispersed suspensions, such as colloidal silica, because the detecting signal includes both transmitted and scattered light. The LS-CLS method exhibited improved quantitative performance. Moreover, the LS-CLS system allowed the injection of samples with concentrations higher than that permitted by other particle size distribution measurement systems with particle size classification units using size-exclusion chromatography or centrifugal field-flow fractionation. The proposed LS-CLS method achieved an accurate quantitative analysis of the mass-based size distribution using both centrifugal classification and laser scattering optics. In particular, the system could measure the mass-based size distribution of approximately 20mgmL-1 poly-dispersed colloidal silica samples, such as in a mixture of the four mono-dispersed colloidal silica, with high resolution and precision, thereby demonstrating high quantitative performance. The measured size distributions were compared with those observed through transmission electron microscopy. The proposed system can be used in practical setups to achieve a reasonable degree of consistency for determining particle size distribution in industrial applications.

Investigating serum extracellular vesicles in Cystic Fibrosis

BackgroundExtracellular vesicles (EVs) are emerging as biomarkers of disease with diagnostic potential in CF. With the advent of highly effective modulator therapy, sputum production is less common and there is a need to identify novel markers of CF disease progression, exacerbation and response to therapies in accessible fluids such as serum. MethodsWe used size exclusion chromatography (SEC) to isolate and characterise EVs from the blood of PWCF of different ages and compared to ultracentrifugation (UC). We used nanoparticle tracking analysis to measure the number of EVs present in serum obtained from children and adults with CF. Mass spectrometry based proteomics was used to characterise protein expression changes between the groups. ResultsEVs were successfully isolated in SEC fractions from 250 µl serum from PWCF in greater numbers (p <0.01) than density ultracentrifugation. There was not a significant difference in EV numbers between young children with CF and controls. However, there was significantly more EVs in adults compared to children (<6yrs) (p < 0.05). EVs from PWCF before and after Kaftrio treatment were also analysed. Significant protein expression changes were observed within all 3 group. The largest changes detected were between children and adults with CF (57 proteins had a 1.5 fold change in expression with 19 significant changes p < 0.05) and PWCF taking Kaftrio (24 significant changes in EV protein expression was observed 12 months post treatment). ConclusionIn this pilot study, we performed an initial characterisation of EVs in serum from PWCF demonstrating the potential of serum EVs for further diagnostic investigation.

High-Throughput Proteome Profiling of Plasma and Native Plasma Complexes Using Native Chromatography.

We describe a high-throughput method for co-fractionation mass spectrometry (CF-MS) profiling for native plasma protein profiling. CF-MS allows the profiling of endogenous protein complexes between samples. Proteins often interact with other proteins and form macromolecular complexes that are different in disease states as well as cell states and cell types. This protocol describes an example for the sample preparation of 954 individual size exclusion chromatography (SEC) fractions, derived from 18 plasma samples that were separated into 53 fractions. Eighteen plasma samples were chosen based on the TMTpro multiplexing, but this methodology can be adapted for fewer or larger numbers of samples as appropriate. Our automated sample preparation method allows for high-throughput native plasma profiling, and we provide detailed methods for both a label-free and an isobaric labeling approach, discuss the merits of each approach, and detail the advantages of combining these strategies for comprehensive native plasma proteome profiling.

Thermal desorption gas chromatography-mass spectrometry for investigating the thermal degradation of polyurethanes.

Thermal Desorption Gas Chromatography-Mass Spectrometry (TD-GC-MS) was used to investigate the thermal degradation of two different polyurethanes (PUs). PU samples were heated at different heating rates and the desorbed products were collected in a cold injection system and thereafter submitted to GC-MS. The prospects and limitations of the detection and quantification of semi-volatile degradation products were investigated. Temperature dependent PU depolymerization was observed at temperatures above 200 °C, proved by an increased release of 1,4-butanediol and methylene diphenyl diisocyanate (MDI) representing the main building blocks of both polymers. Their release was monitored quantitatively based on external calibration with authentic compounds. Size Exclusion Chromatography (SEC) of the residues obtained after thermal desorption confirmed the initial competitive degradation mechanism indicating an equilibrium of crosslinking and depolymerization as previously suggested. Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry of SEC fractions of thermally degraded PUs provided additional hints on the degradation mechanism.

Large-scale top-down proteomics of the Arabidopsis thaliana leaf and chloroplast proteomes.

We present a large-scale top-down proteomics (TDP) study of plant leaf and chloroplast proteins, achieving the identification of over 4700 unique proteoforms. Using capillary zone electrophoresis coupled with tandem mass spectrometry analysis of offline size-exclusion chromatography fractions, we identify 3198 proteoforms for total leaf and 1836 proteoforms for chloroplast, with 1024 and 363 proteoforms having post-translational modifications, respectively. The electrophoretic mobility prediction of capillary zone electrophoresis allowed us to validate post-translational modifications that impact the charge state such as acetylation and phosphorylation. Identified modifications included Trp (di)oxidation events on six chloroplast proteins that may represent novel targets of singlet oxygen sensing. Furthermore, our TDP data provides direct experimental evidence of the N- and C-terminal residues of numerous mature proteoforms from chloroplast, mitochondria, endoplasmic reticulum, and other sub-cellular localizations. With this information, we suggest true transit peptide cleavage sites and correct sub-cellular localization signal predictions. This large-scale analysis illustrates the power of top-down proteoform identification of post-translational modifications and intact sequences that can benefit our understanding of both the structure and function of hundreds of plant proteins.

Determination of Short Chain Branching in LLDPE by Rheology

AbstractThe relationship between the structure and the final properties of materials is an important issue in polymer science. In particular, the properties of polyolefins are impacted by their branching contents. A simple rheological method is presented to estimate the degree of short chain branching (SCB) in linear low‐density polyethylene (LLDPE). A range of ethylene‐1‐hexene copolymers is synthetized using a metallocene catalyst to obtain homogenous LLDPE models with various comonomer contents. The polymers are analyzed by size exclusion chromatography, 13C NMR, and crystallization elution fractionation to determine both their molar masses and their chemical composition distributions. Subsequently, they are measured by rheology to assess the impact of SCB. This work provides calibration curves for rheology and proves that it is possible to predict the SCB content in LLDPE from the melt viscosity analysis.

Analysis of Host Cell Proteins in Monoclonal Antibody Therapeutics Through Size Exclusion Chromatography.

Host cell proteins (HCPs) are impurities derived from expression systems during the manufacturing of biotherapeutics. Even trace amounts of certain HCPs can potentially compromise product safety and quality. Therefore, comprehensive analytical characterization is necessary. In particular, understanding how each HCP co-purifies with the biotherapeutics throughout the purification process would help guide process development to avoid further contamination. We developed a new strategy based on size exclusion chromatography (SEC) fractionation followed by mass spectrometry (MS) analysis to study HCPs. Through an optimized experimental procedure, HCPs were effectively separated from monoclonal antibody (mAb) drug substances via SEC fractionation and sensitively detected with MS. Many HCPs were enriched in the high molecular weight fraction, thus indicating the formation of HCP-mAb complexes. SEC separation under mild denaturing conditions was demonstrated to disrupt weak interactions between certain HCPs and mAbs. The binding profiles of HCPs to mAbs were further characterized through comparison of the relative abundance of HCPs in each fraction under either native or mild denaturing SEC conditions. This new method not only achieves improved identification of HCPs in biotherapeutic drug substances but also offers an effective means to evaluate the binding properties between biotherapeutics and a wide range of HCPs.

Effect of cell culture media on extracellular vesicle secretion from mesenchymal stromal cells and neurons

BackgroundExtracellular vesicles (EVs) secreted by neuronal cells in vitro have promising therapeutic potential for brain diseases. Optimization of cell culture conditions and methodologies for high-yield isolation of EVs for preclinical and clinical applications, however, remains a challenge. ObjectiveTo probe the cell culture conditions required for optimal EV secretion by human-derived neuronal cells. MethodologyFirst, we optimized the EV purification protocol using human mesenchymal stromal cell (MSC) cultures. Next, we compared the effects of different variables in human pluripotent stem cell (hPSC)-derived neuronal cultures on EV secretion. EVs were isolated from cell conditioned media (CCM) and control media with no cells (NCC) using ultrafiltration combined with size-exclusion chromatography (SEC). The hPSC neurons were cultured in 2 different media from which EVs were collected at 2 maturation time-points (days 46 and 60). Stimulation with 25 mM KCl was also evaluated as an activator of EV secretion by neurons. The collected SEC fractions were analyzed by nanoparticle tracking analysis (NTA), protein concentration assay, and blinded transmission electron microscopy (TEM). ResultsA peak in cup-shaped particles was observed in SEC fractions 7–10 of MSC samples, but not corresponding media controls, indicating successful isolation of EVs. Culture medium had no significant effect on EV yield. The EV yield of the samples did not differ significantly according to the culture media used or the cell maturation time-points. Stimulation of neurons with KCl for 3 h reduced rather than increased the EV yield. ConclusionsWe demonstrated successful EV isolation from MSC and neuronal cells using an ultrafiltration-SEC method. The EV yield from MSC and neuronal cultures exhibited a large batch effect, apparently related to the culture media used, highlighting the importance of including NCC as a negative control in all cell culture experiments.

Investigation of the Multimer Cyclization Effect during Click Step-Growth Polymerization of AB-Type Macromonomers

In this work, by combining size exclusion chromatography (SEC) and liquid chromatography at the critical condition (LCCC) techniques, we have studied the multimer cyclization effect during click step-growth polymerization of AB-type macromonomers. First, by atom transfer radical polymerization (ATRP), we have synthesized a series of alkynyl and azide functional AB-type polystyrene macromonomers with different molar masses, i.e., PSn. Second, via the click step-growth polymerization of a PSn macromonomer in dimethylformamide (DMF) at varied reaction concentrations (Cs), we have prepared polyaddition products [(PSn)m] with large polydispersity indices, where m represents the average degree of polymerization of the macromonomer. Further, narrowly distributed fractions have been obtained by SEC fractionation. Third, we have quantitatively analyzed how the macromonomer molar mass, reaction concentration, and multimer molar mass influence the percentage of a cyclic topoisomer (wtcyclic) for (PSn)m samples. In particular, we have established the relation between wtcyclic and C for unfractionated (PSn)m, i.e., wtcyclic = 0.86–0.32 log C, and the relation between wtcyclic and m for fractionated (PSn)m prepared at a constant C = 100 g/L, i.e., log wtcyclic = −0.46m + 0.16. For the first time, our result has unambiguously pointed out that neither a cyclic topoisomer nor a linear topoisomer can be ignored when considering the structure–property quantitative relationship for polyaddition products.

Circulating extracellular vesicles provide valuable protein, but not DNA, biomarkers in metastatic breast cancer

AbstractDetection of cell‐free circulating tumour DNA (ctDNA) and cancer‐specific extracellular vesicles (EVs) in patient blood have been widely explored as non‐invasive biomarkers for cancer detection and disease follow up. However, most of the protocols used to isolate EVs co‐isolate other components and the actual value of EV‐associated markers remain unclear. To determine the optimal source of clinically‐relevant circulating biomarkers in breast cancer, we applied a size exclusion chromatography (SEC) procedure to analyse separately the content in nucleic acids of EV‐enriched and EV‐depleted fractions, in comparison to total plasma. Both cellular and mitochondrial DNA (cellDNA and mtDNA) were detected in EV‐rich and EV‐poor fractions. Analysing specific mutations identified from tumour tissues, we detected tumour‐specific cellular alleles in all SEC fractions. However, quantification of ctDNA from total plasma was more sensitive than from any SEC fractions. On the other hand, mtDNA was preferentially enriched in EV fractions from healthy donor, whereas cancer patients displayed more abundant mtDNA in total plasma, and equally distributed in all fractions. In contrast to nucleic acids, using a Multiplexed bead‐based EV‐analysis assay, we identified three surface proteins enriched in EVs from metastatic breast cancer plasma, suggesting that a small set of EV surface molecules could provide a disease signature. Our findings provide evidence that the detection of DNA within total circulating EVs does not add value as compared to the whole plasma, at least in the metastatic breast cancer patients used here. However, analysis of a subtype of EV‐associated proteins may reliably identify cancer patients. These non‐invasive biomarkers represent a promising tool for cancer diagnosis and real‐time monitoring of treatment efficacy and these results will impact the development of therapeutic approaches using EVs as targets or biomarkers of cancer.

Enzymatic modification of oat globulin enables covalent interaction with procyanidin B2

The effect of enzyme treatment on protein–tannin interactions was investigated using up-to-date analytical approaches for improving their physical properties. The formation of ligands between procyanidin B2 and native oat globulin (OG) was observed to be affected by the ratio of procyanidin B2 to OG and the availability of tryptophan. For the transglutaminase-treated OG, the results obtained from circular dichroism (CD) and size exclusion chromatography (SEC) revealed that procyanidin B2 acted as an acyl acceptor in the process of OG deamidation. Procyanidin B2 also inhibited the non-covalent protein–protein interactions occurring between the aromatic side-chains or sedimentation of tryptophan aggregates. For trypsin-treated OG, procyanidin B2 interacted with phenylalanine and the tryptophan side-chain of OG. The inhibition of procyanidin B2 towards protein–protein aggregation was proved by the observation of CD, SEC and asymmetric flow field-flow fractionation.

Unveiling the Native Morphology of Extracellular Vesicles from Human Cerebrospinal Fluid by Atomic Force and Cryogenic Electron Microscopy.

Extracellular vesicles (EVs) are membranous structures in biofluids with enormous diagnostic/prognostic potential for application in liquid biopsies. Any such downstream application requires a detailed characterization of EV concentration, size and morphology. This study aimed to observe the native morphology of EVs in human cerebrospinal fluid after traumatic brain injury. Therefore, they were separated by gravity-driven size-exclusion chromatography (SEC) and investigated by atomic force microscopy (AFM) in liquid and cryogenic transmission electron microscopy (cryo-TEM). The enrichment of EVs in early SEC fractions was confirmed by immunoblot for transmembrane proteins CD9 and CD81. These fractions were then pooled, and the concentration and particle size distribution were determined by Tunable Resistive Pulse Sensing (around 1010 particles/mL, mode 100 nm) and Nanoparticle Tracking Analysis (around 109 particles/mL, mode 150 nm). Liquid AFM and cryo-TEM investigations showed mode sizes of about 60 and 90 nm, respectively, and various morphology features. AFM revealed round, concave, multilobed EV structures; and cryo-TEM identified single, double and multi-membrane EVs. By combining AFM for the surface morphology investigation and cryo-TEM for internal structure differentiation, EV morphological subpopulations in cerebrospinal fluid could be identified. These subpopulations should be further investigated because they could have different biological functions.

Abstract 523: Lipid-associated Plasminogen Is A Ribonucleoprotein Carrier Of Extracellular Small RNAs In Atherosclerosis

Small RNAs (sRNA) are exchanged between cells and tissues by various lipid and protein carriers. Extracellular sRNAs regulate gene expression in recipient cells, including activation of pattern recognition receptors and inflammatory signaling pathways. Based on size-exclusion chromatography (SEC) fractionation of human plasma, a large proportion of extracellular sRNAs are detected in small lipid-associated ribonucleoproteins (50-150 kDa), a class of carriers that have been largely overlooked. Sequential fractionation approaches, mass spectrometry, enzymatic activity assays, and western blotting all support that lipid-associated human plasminogen (PLG) co-purifies with sRNAs in plasma. PLG transports approximately 10 μg total RNA/ mg protein in plasma. Depletion of PLG from plasma by lysine-sepharose resin was also found to reduce plasma miRNA levels. Based on microscale thermophoresis assays, PLG was determined to have moderate binding to single-stranded sRNAs (K d =1.8μM). Immune cells may be a source of sRNAs, as macrophages were found to export both host and non-host sRNAs to PLG in vitro . To identify PLG-associated sRNAs, sRNA sequencing was completed, and results showed that PLG host sRNA are predominantly derived from parent rRNAs (rDR), tRNAs (tDR), yRNA (yDRs), and microRNAs (miRNA). Furthermore, we found that host sRNAs on PLG are significantly altered in subjects with familial hypercholesterolemia (hetFH), including 22 miRNAs, 5 tDRs, and 10 yDRs. PLG was also found to transport many non-host microbial sRNAs derived from bacteria, fungi, and viruses in the microbiome and environment; and genome counts for many of these microbial species were significantly altered on PLG isolated from hetFH subjects. PLG was found to cause dramatic gene (mRNA) expression changes in treated-macrophages; however, it is unknown what fraction of these changes are associated with PLG-sRNA delivery. Collectively, results suggest that PLG transports high levels of extracellular sRNAs from host and non-host species that are sensitive to hypercholesterolemia and disease associated changes. Although the impact of PLG-sRNAs remains to be determined, they hold great potential as therapeutic targets for inflammation.

Multi angle light scattering as a process analytical technology tool for real-time monitoring of molar mass of protein-polysaccharide conjugate fractions

Current polysaccharide conjugate vaccine manufacturing process involves conjugation of polysaccharide with protein followed by separation/purification of the conjugate from reaction components and free polysaccharide/protein, by preparative Size-Exclusion Chromatography (SEC). Each SEC fraction is analyzed and pooled the conjugate based on its molar mass and absence of free polysaccharide. In the present work, we have evaluated in-line Multi Angle Light Scattering (MALS) as a Process Analytical Technology (PAT) tool to eliminate the need for offline analysis of individual fractions after purification. The preparatory SEC unit is coupled to a MALS detector, i.e. preparatory SEC-MALS. In SEC, the molecules are separated based on size, where polysaccharide-protein conjugate elutes first followed by free polysaccharide and then free protein. The MALS detector monitors in-line weight-average molar mass (Mw) during SEC-fractionation. Except the first fraction, the molar mass distribution data from in-line MALS enables decision on preparative SEC fractions to be pooled. We found that offline analysis of preparative fractions using analytical SEC-MALS was in good agreement with the >2000 kDa cutoff criterion. Thus in-line process analysis is effective, time-saving, and simple to implement in manufacturing processes of conjugate vaccines.