Size-Exclusion Chromatography: A Path to Higher Yield and Reproducibility Compared to Sucrose Cushion Ultracentrifugation for Extracellular Vesicle Isolation in Multiple Myeloma.

Small extracellular vesicles (EVs) play a pivotal role in intercellular communication across various physiological and pathological contexts. Despite their growing significance as disease biomarkers and therapeutic targets in biomedical research, the lack of reliable isolation techniques remains challenging. This study characterizes vesicles that were isolated from conditioned culture media (CCM) sourced from three myeloma cell lines (MM.1S, ANBL-6, and ALMC-1), and from the plasma of healthy donors and multiple myeloma patients. We compared the efficacy, reproducibility, and specificity of isolating small EVs using sucrose cushion ultracentrifugation (sUC) vs. ultrafiltration combined with size-exclusion chromatography (UF-SEC). Our results demonstrate that UF-SEC emerges as a more practical, efficient, and consistent method for EV isolation, outperforming sUC in the yield of EV recovery and exhibiting lower variability. Additionally, the comparison of EV characteristics among the three myeloma cell lines revealed distinct biomarker profiles. Finally, our results suggest that HBS associated with Tween 20 improves EV recovery and preservation over PBS. Standardization of small EV isolation methods is imperative, and our comparative evaluation represents a significant step toward achieving this goal.

Rapid determination of toxic and essential metal binding proteins in biological samples by size exclusion chromatography-inductively coupled plasma tandem mass spectrometry.

Metalloproteins binding with trace elements play a crucial role in biological processes and on the contrary, those binding with exogenous heavy metals have adverse effects. However, the methods for rapid, high sensitivity and simultaneous analysis of these metalloproteins are still lacking. In this study, a fast method for simultaneously determination of both essential and toxic metal-containing proteins was developed by coupling size exclusion chromatography (SEC) with inductively coupled plasma tandem mass spectrometry (ICP-MS/MS). After optimization of the separation and detection conditions, seven metalloproteins with different molecular weight (from 16.0 to 443.0 kDa) were successfully separated within 10 min and the proteins containing iron (Fe), copper (Cu), zinc (Zn), iodine (I) and lead (Pb) elements could be simultaneously detected with the use of oxygen as the collision gas in ICP-MS/MS. Accordingly, the linear relationship between log molecular weight and retention time was established to estimate the molecular weight of unknown proteins. Thus, the trace metal and toxic metal containing proteins could be detected in a single run with high sensitivity (detection limits in the range of 0.0020-2.5 µg/mL) and good repeatability (relative standard deviations lower than 4.5 %). This method was then successfully used to analyze metal (e.g., Pb, Zn, Cu and Fe) binding proteins in the blood of Pb-intoxicated patients, and the results showed a negative correlation between the contents of zinc and lead binding proteins, which was identified to contain hemoglobin subunit. In summary, this work provided a rapid and sensitive tool for screening metal containing proteins in large number of biological samples.

Affinity-Resolved Size Exclusion Chromatography Coupled to Mass Spectrometry: A Novel Tool to Study the Attribute-and-Function Relationship in Therapeutic Monoclonal Antibodies.

Assessment of critical quality attributes (CQAs) is an important aspect during the development of therapeutic monoclonal antibodies (mAbs). Attributes that affect either the target binding or Fc receptor engagement may have direct impacts on the drug safety and efficacy and thus are considered as CQAs. Native size exclusion chromatography (SEC)-based competitive binding assay has recently been reported and demonstrated significant benefits compared to conventional approaches for CQA identification, owing to its faster turn-around and higher multiplexity. Expanding on the similar concept, we report the development of a novel affinity-resolved size exclusion chromatography-mass spectrometry (AR-SEC-MS) method for rapid CQA evaluation in therapeutic mAbs. This method features wide applicability, fast turn-around, high multiplexity, and easy implementation. Using the well-studied Fc gamma receptor III-A (FcγRIIIa) and Fc interaction as a model system, the effectiveness of this method in studying the attribute-and-function relationship was demonstrated. Further, two case studies were detailed to showcase the application of this method in assessing CQAs related to antibody target binding, which included unusual N-linked glycosylation in a bispecific antibody and Met oxidation in a monospecific antibody, both occurring within the complementarity-determining regions (CDRs).

Method for quantifying free hemoglobin, distinct from the hemoglobin-haptoglobin complex, in human serum.

The measurement of free hemoglobin (free Hb) in blood is crucial for assessing the risk of organ damage in patients with hemolytic diseases. However, the colorimetric method, commonly used in clinical practice, does not distinguish between free Hb and the hemoglobin-haptoglobin complex (Hb-Hp) in the blood, instead reflecting the total Hb level. Although size-exclusion high-performance liquid chromatography (SEC-HPLC) can specifically measure free Hb, its clinical use is limited by long assay times. Here, we developed a novel assay method for the rapid quantification of free Hb in serum, distinguishing it from Hb-Hp, using a latex agglutination immunoturbidimetric assay (LATIA). This method could be used to measure free Hb in sera in the range of 1-100 µg/mL in approximately 15 min using an automatic biochemistry analyzer. Using Hb-spiked serum samples from healthy adults, there was a high correlation with Hb levels determined using the newly developed method and SEC-HPLC, indicating a high specificity for free Hb. This novel assay can be used to monitor levels of free Hb in patients with various hemolytic diseases and to design therapeutic strategies based on measured values. However, further studies are required to assess its clinical performance.

Quality and efficiency assessment of five extracellular vesicle isolation methods using the resistive pulse sensing strategy.

Extracellular vesicles (EVs) have attracted great interest due to their great potential in disease diagnosis and therapy. The separation of EVs from complex biofluids with high purity is essential for the accurate analysis of EVs. Despite various methods, there is still no consensus on the best method for high-quality EV isolation and reliable mass production. Therefore, it is important to offer a standardized method for characterizing the properties (size distribution, particle concentration and purity) of EV preparations from different isolation methods. Herein, we employed a NanoCoulter Counter based on the resistive pulse sensing (RPS) strategy that enabled multi-parameter analysis of single EVs to compare the quality and efficiency of different EV isolation techniques including traditional differential ultracentrifugation, ultrafiltration, size exclusion chromatography, membrane affinity binding and polymer precipitation. The data revealed that the NanoCoulter Counter based on the RPS strategy was reliable and effective for the characterization of EVs. The results suggested that although higher particle concentrations were observed in three commercial isolation kits and ultrafiltration, traditional differential ultracentrifugation showed the highest purity. In conclusion, our results from the NanoCoulter Counter provided reliable evidence for the assessment of different EV isolation methods, which contributed to the development of EV-based disease biomarkers and treatments.

Monitoring of the disulfide scrambled species by mixed-mode SEC-HPLC during the purification of a bispecific antibody.

Size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is an analytical method routinely used for assessing aggregation content in protein samples. As SEC-HPLC separates analytes based on their hydrodynamic radius, it generally lacks the capability of differentiating species that are similar in size. Recently while purifying a bispecific antibody (bsAb), we noticed that SEC-HPLC can provide certain degree of resolution between the target bsAb and a disulfide scrambled form, although these two species were identical in molecular weight. In seeing the unexpected potential of SEC-HPLC at resolving species with similar size, we further tested Zenix SEC-300, a mixed-mode SEC-HPLC column from Sepax, which was reported to be capable of separating protein analytes based on other factors besides size. The Zenix column indeed provided resolution much better than the regular SEC-HPLC column. Upon further optimization, the Zenix column allowed close to baseline separation of the correctly folded and the disulfide scrambled species. The current study, as a complement to the previous reports, further demonstrates that mix-mode SEC-HPLC is capable of separating protein analytes that are close in size but are different in conformation and/or surface characteristics.

Size exclusion chromatography and asymmetrical flow field-flow fractionation for structural characterization of polysaccharides: A comparative review.

Natural polysaccharides exhibit a wide range of biological activities, which are closely related to their structural characteristics, including their molecular weight distribution, size, monosaccharide composition, glycosidic bond types and spatial conformation, etc. Size exclusion chromatography (SEC) and asymmetrical flow field-flow fractionation (AF4), as two potent separation techniques, both harbor potential for continuous development and enhancement. This manuscript reviewed the fundamental principles and separation applications of SEC and AF4. The structural information and spatial conformation of polysaccharides can be obtained using SEC or AF4 coupled with multiple detectors. In addition, this manuscript elaborates in detail on the shear degradation of samples such as polysaccharides separated by SEC. In addition, the abnormal elution that occurs during the application of the two methods is also discussed. Both SEC and AF4 possess considerable potential for ongoing development and refinement, thereby offering increased possibilities and opportunities for polysaccharide separation and characterization.

Quality control and validation of extracellular vesicles isolated from cultured human breast cancer cells.

OBJECTIVE: Extracellular vesicles (EVs) have been shown to play a critical role in promoting tumorigenesis. As EV research grows, it is of importance to have standardization of isolation, quality control, characterization and validation methods across studies along with reliable references to explore troubleshooting solutions. Therefore, our objective with this Research Note was to isolate EVs from multiple breast cancer cell lines and to describe and perform protocols for validation as outlined by the list of minimal information for studies of EVs (MISEV) from the International Society for Extracellular Vesicles. RESULTS: To isolate EVs, two techniques were employed: ultracentrifugation and size exclusion chromatography. Ultracentrifugation yielded better recovery of EVs in our hands and was therefore used for further validation. In order to satisfy the MISEV requirements, protein quantification, immunoblotting of positive (CD9, CD63, TSG101) and negative (TGFß1, ß-tubulin) markers, nanoflow cytometry and electron microscopy was performed. With these experiments, we demonstrate that yield of validated EVs varied between different breast cancer cell lines. Protocols were optimized to accommodate for low levels of EVs, and various technical and troubleshooting suggestions are included for potential application to other cell types that may provide benefit to investigators interested in future EV studies.

Purification and Quality Control of Recombinant Proteins Expressed in Mammalian Cells: A Practical Review.

In the recent years, there has been a rapid development of new technologies and strategies when it comes to protein purification and quality control (QC), but the basic technologies for these processes go back a long way, with many improvements over the past few decades. The purpose of this chapter is to review these approaches, as well as some other topics such as the advantages and disadvantages of various purification methods for intracellular or extracellular proteins, the most effective and widely used genetically engineered affinity tags, solubility-enhancing tags, and specific proteases for removal of nontarget sequences. Affinity chromatography (AC), like Protein A or G resins for the recovery of antibodies or Fc fusion proteins or immobilized metals for the recovery of histidine-tagged proteins, will be discussed along with other conventional chromatography techniques: ion exchange (IEC), hydrophobic exchange (HEC), mixed mode (MMC), size exclusion (SEC), and ultrafiltration (UF) systems. How to select and combine these different technologies for the purification of any given protein and the minimal criteria for QC characterization of the purity, homogeneity, identity, and integrity of the final product will be presented.

A native SEC-MS workflow and validation for analyzing drug-to-antibody ratio and drug load distribution in cysteine-linked antibody-drug conjugates.

The development and optimization of Antibody-Drug Conjugates (ADCs) hinge on enhanced analytical and bioanalytical characterization, particularly in assessing critical quality attributes (CQAs). The ADC's potency is largely determined by the average number of drugs attached to the monoclonal antibody (mAb), known as the drug-to-antibody ratio (DAR). Furthermore, the drug load distribution (DLD) influences the therapeutic window of the ADC, defining the range of dosages effective in treating diseases without causing toxic effects. Among CQAs, DAR and DLD are vital; their control is essential for ensuring manufacturing consistency and product quality. Typically, hydrophobic interaction chromatography (HIC) or reversed-phase liquid chromatography (RPLC) with UV detector have been used to quantitate DAR and DLD in quality control (QC) environment. Recently, Native size-exclusion chromatography-mass spectrometry (nSEC-MS) proves the potential as a platformable quantitative method for characterizing DAR and DLD across various cysteine-linked ADCs in research or early preclinical development. In this work, we established and assessed a streamlined nSEC-MS workflow with a benchtop LC-MS platform, to quantitatively monitor DAR and DLD of different chemotype and drug load level cysteine-linked ADCs. Moreover, to deploy this workflow in QC environment, complete method validation was conducted in three independent laboratories, adhering to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2(R1) guidelines. The results met the predefined analytical target profile (ATP) and performance criteria, encompassing specificity/selectivity, accuracy, precision, linearity, range, quantification/detection limit, and robustness. Finally, the method validation design offers a reference for other nSEC-MS methods that are potentially used to determine the DAR and DLD on cysteine-linker ADCs. To the best of our knowledge, this study is the first reported systematic validation of the nSEC-MS method for detecting DAR and DLD. The results indicated that the co-validated nSEC-MS workflow is suitable for DAR and DLD routine analysis in ADC quality control, release, and stability testing.

Aqueous size exclusion chromatography applied to polymer analysis: experimental conditions and molecular weight calibration curves.

Aqueous mode size exclusion chromatography (SEC) was employed for the analysis and construction of molecular weight (MW) calibration curves of three water-soluble polymers, namely, polyethylene glycol, polyethylene oxide, and polyacrylic acid sodium salt. Several calibration curves were obtained, varying chromatographic conditions such as columns arrangement, ionic strength, temperature and pH, in addition trends in polymeric chromatographic behavior were examined. The variation in SEC distribution coefficients at different temperatures was found to be below 10 %, indicating that the studied polymers follow an ideal SEC mechanism under the tested conditions. Thus, differences in chromatographic behavior were ascribed to changes in polymer configuration induced by media and/or temperature. These variations in morphology were consistent with the observed SEC behavior. Regarding MW calibration, polynomial regression models ranging from first to fifth order were applied, and the most adequate ones were selected based on their fit and prediction capabilities. Third order polynomials were the preferred models for polyethylene glycol and polyacrylic acid sodium salt, independently of chromatographic conditions. Meanwhile for polyethylene oxide, either third or fifth-order polynomial models were optimal depending on the chromatographic conditions. All the selected regression models presented coefficients of multiple determination (R2) above 0.990, while achieving relative errors of prediction (REP%) in MW ranging from 0.3 to 4 % for cross-validation.

Label-free single-vesicle based surface enhanced Raman spectroscopy: A robust approach for investigating the biomolecular composition of small extracellular vesicles.

Small extracellular vesicles (sEVs) are cell-released vesicles ranging from 30-150nm in size. They have garnered increasing attention because of their potential for both the diagnosis and treatment of disease. The diversity of sEVs derives from their biological composition and cargo content. Currently, the isolation of sEV subpopulations is primarily based on bio-physical and affinity-based approaches. Since a standardized definition for sEV subpopulations is yet to be fully established, it is important to further investigate the correlation between the biomolecular composition of sEVs and their physical properties. In this study, we employed a platform combining single-vesicle surface-enhanced Raman spectroscopy (SERS) and machine learning to examine individual sEVs isolated by size-exclusion chromatography (SEC). The biomolecular composition of each vesicle examined was reflected by its corresponding SERS spectral features (biomolecular "fingerprints"), with their roots in the composition of their collective Raman-active bonds. Origins of the SERS spectral features were validated through a comparative analysis between SERS and mass spectrometry (MS). SERS fingerprinting of individual vesicles was effective in overcoming the challenges posed by EV population averaging, allowing for the possibility of analyzing the variations in biomolecular composition between the vesicles of similar and/or different sizes. Using this approach, we uncovered that each of the size-based fractions of sEVs contained particles with predominantly similar SERS spectral features. Indeed, more than 84% of the vesicles residing within a particular group were clearly distinguishable from that of the other EV sub-populations, despite some spectral variations within each sub-population. Our results suggest the possibility that size-based EV fractionation methods produce samples where similarly eluted sEVs are correlated with their respective biochemical contents, as reflected by their SERS spectra. Our findings therefore highlight the possibility that the biogenesis and respective biological functionalities of the various sEV fractions may be inherently different.

Development of a Sampling and Storage Protocol of Extracellular Vesicles (EVs)-Establishment of the First EV Biobank for Polytraumatized Patients.

In the last few years, several studies have emphasized the existence of injury-specific EV "barcodes" that could have significant importance for the precise diagnosis of different organ injuries in polytrauma patients. To expand the research potential of the NTF (network trauma research) biobank of polytraumatized patients, the NTF research group decided to further establish a biobank for EVs. However, until now, the protocols for the isolation, characterization, and storage of EVs for biobank purposes have not been conceptualized. Plasma and serum samples from healthy volunteers (n = 10) were used. Three EV isolation methods of high relevance for the work with patients' samples (ultracentrifugation, size exclusion chromatography, and immune magnetic bead-based isolation) were compared. EVs were quantified using nanoparticle tracking analysis, EV proteins, and miRNAs. The effects of different isolation solutions; the long storage of samples (up to 3 years); and the sensibility of EVs to serial freezing-thawing cycles and different storage conditions (RT, 4/-20/-80 °C, dry ice) were evaluated. The SEC isolation method was considered the most suitable for EV biobanking. We did not find any difference in the quantity of EVs between serum and plasma-EVs. The importance of particle-free PBS as an isolation solution was confirmed. Plasma that has been frozen for a long time can also be used as a source of EVs. Serial freezing-thawing cycles were found to affect the mean size of EVs but not their amount. The storage of EV samples for 5 days on dry ice significantly reduced the EV protein concentration.

Optimizing Messenger RNA Analysis Using Ultra-Wide Pore Size Exclusion Chromatography Columns.

Biopharmaceutical products, in particular messenger ribonucleic acid (mRNA), have the potential to dramatically improve the quality of life for patients suffering from respiratory and infectious diseases, rare genetic disorders, and cancer. However, the quality and safety of such products are particularly critical for patients and require close scrutiny. Key product-related impurities, such as fragments and aggregates, among others, can significantly reduce the efficacy of mRNA therapies. In the present work, the possibilities offered by size exclusion chromatography (SEC) for the characterization of mRNA samples were explored using state-of-the-art ultra-wide pore columns with average pore diameters of 1000 and 2500 Å. Our investigation shows that a column with 1000 Å pores proved to be optimal for the analysis of mRNA products, whatever the size between 500 and 5000 nucleotides (nt). We also studied the influence of mobile phase composition and found that the addition of 10 mM magnesium chloride (MgCl2) can be beneficial in improving the resolution and recovery of large size variants for some mRNA samples. We demonstrate that caution should be exercised when increasing column length or decreasing the flow rate. While these adjustments slightly improve resolution, they also lead to an apparent increase in the amount of low-molecular-weight species (LMWS) and monomer peak tailing, which can be attributed to the prolonged residence time inside the column. Finally, our optimal SEC method has been successfully applied to a wide range of mRNA products, ranging from 1000 to 4500 nt in length, as well as mRNA from different suppliers and stressed/unstressed samples.

Quantitative description of the quality of size exclusion chromatography separations.

Size exclusion chromatography (SEC) is unique among chromatographic methods as it allows separation of non-retained analytes. However, such mechanism often put an analytical scientist in front of relatively poorly resolved set of peaks that may have strikingly different abundance. The description of such chromatograms needs a particular approach to accurately capture the overall quality of separation. Consequently, use of a single parameter description may not be accurate enough and therefore we introduce a dimensionless separation quality factor, which is based on five SEC specific measures (peak-to-valley, elution window width, peak widths, peak-positioning and recovery). Combining several factors allowed detailed differentiation of various simulated separations, clearly correlating column characteristics with specific contributions to separation quality whether they concern a single peak pair or entire peak landscape. The method could be further elaborated by the addition of normalized priority weighting allowing for flexible quality quantification of a relevant portion of real-life nucleic acid separation on different columns. With growing complexity of biotherapeutics to be separated, such a term is predicted to be a useful response function for purposes of factorial method optimization.

A fast and sensitive size-exclusion chromatography method for plasma extracellular vesicle proteomic analysis.

Extracellular vesicles (EVs) carry diverse biomolecules derived from their parental cells, making their components excellent biomarker candidates. However, purifying EVs is a major hurdle in biomarker discovery since current methods require large amounts of samples, are time-consuming and typically have poor reproducibility. Here we describe a simple, fast, and sensitive EV fractionation method using size exclusion chromatography (SEC) on a fast protein liquid chromatography (FPLC) system. Our method uses a Superose 6 Increase 5/150, which has a bed volume of 2.9 mL. The FPLC system and small column size enable reproducible separation of only 50 µL of human plasma in 15 min. To demonstrate the utility of our method, we used longitudinal samples from a group of individuals who underwent intense exercise. A total of 838 proteins were identified, of which, 261 were previously characterized as EV proteins, including classical markers, such as cluster of differentiation (CD)9 and CD81. Quantitative analysis showed low technical variability with correlation coefficients greater than 0.9 between replicates. The analysis captured differences in relevant EV proteins involved in response to physical activity. Our method enables fast and sensitive fractionation of plasma EVs with low variability, which will facilitate biomarker studies in large clinical cohorts.

Purified Pyropia yezoensis Pigment Extract-Based Tandem Dye Synthesis.

Red phycoerythrin (R-PE) is a highly valuable protein found in an edible seaweed, Pyropia yezoensis. It is used extensively in biotechnological applications due to its strong fluorescence and stability in diverse environments. However, the current methods for extracting and purifying R-PE are costly and unsustainable. The aim of the present study was to enhance the financial viability of the process by improving the extraction and purification of R-PE from dried P. yezoensis and to further enhance R-PE value by incorporating it into a tandem dye for molecular biology applications. A combination of ultrafiltration, ion exchange chromatography, and gel filtration yielded concentrated (1 mg·mL-1) R-PE at 99% purity. Using purified PE and Cyanine5 (Cy5), an organic tandem dye, phycoerythrin-Cy5 (PE-Cy5), was subsequently established. In comparison to a commercially available tandem dye, PE-Cy5 exhibited 202.3% stronger fluorescence, rendering it suitable for imaging and analyzes that require high sensitivity, enhanced signal-to-noise ratio, broad dynamic range, or shorter exposure times to minimize potential damage to samples. The techno-economic analysis confirmed the financial feasibility of the innovative technique for the extraction and purification of R-PE and PE-Cy5 production.

Glycogen ß-particles surface characterized by a combination of size exclusion chromatography and pyrene excimer fluorescence before and after ß-amylolysis.

Size exclusion chromatography (SEC) and pyrene excimer formation (PEF) experiments were conducted to characterize the local density profile inside a glycogen sample before (Glycogen) and after (Gly-ß-LD) treatment with ß-amylase. These experiments were conducted to assess whether the density at the periphery of the glycogen particles was very high to limit access to proteins involved in the metabolism of glycogen as predicted by the Tier model or low as suggested by the Gilbert model. SEC analysis indicated that the density inside the Glycogen and Gly-ß-LD samples remained constant with particle size and was not affected by ß-amylolysis. Analysis of the PEF experiments conducted on the Glycogen and Gly-ß-LD samples labeled with 1-pyrenebutyric acid showed that the particles have a dense interior and loose corona. The conclusions reached by the SEC and PEF experiments agree with the Gilbert model and have implications for the association of glycogen ß-particles into larger α-particles.

Recent research on material-based methods for isolation of extracellular vesicles.

Extracellular vesicles (EVs) are nanoparticles secreted by cells with a closed phospholipid bilayer structure, which can participate in various physiological and pathological processes and have significant clinical value in disease diagnosis, targeted therapy and prognosis assessment. EV isolation methods currently include differential ultracentrifugation, ultrafiltration, size exclusion chromatography, immunoaffinity, polymer co-precipitation and microfluidics. In addition, material-based biochemical or biophysical approaches relying on intrinsic properties of the material or its surface-modified functionalized monomers, demonstrated unique advantages in the efficient isolation of EVs. In order to provide new ideas for the subsequent development of material-based EV isolation methods, this review will focus on the principle, research status and application prospects of material-based EV isolation methods based on different material carriers and functional monomers.

Using molecular fine structure to identify optimal methods of extracting fungal glycogen.

Glycogen is a highly branched glucose polymer that is an energy storage material in fungi and animals. Extraction of glycogen from its source in a way that minimizes its molecular degradation is essential to investigate its native structure. In this study, the following extraction methods were compared: sucrose gradient density ultracentrifugation, thermal alkali, hot alcohol and hot water extractions. Molecular-size and chain-length distributions of glycogen were measured by size-exclusion chromatography and fluorophore-assisted carbohydrate electrophoresis, respectively. These two fine-structure features are the most likely structural characteristics to be degraded during extraction. The results show that the thermal alkali, hot alcohol and hot water extractions degrade glycogen molecular size and/or chain-length distributions, and that sucrose gradient density ultracentrifugation with neither high temperature nor alkaline treatment is the most suitable method for fungal glycogen extraction.