Multi-angle Light Scattering Research Articles

Chromatographic analysis of branched and debranched starch structure: Variability of their results

Starch structure determination is crucial for understanding its properties and applications. However, method-dependent variations in size determination can lead to ambiguous interpretations. This study investigates the ambiguities in starch structure determination by evaluating the variation in size of four commercial branched starches as determined by average molar mass, gyration radius, hydrodynamic radius, and chain-length distribution. The starches were analyzed using high-performance size-exclusion chromatography (HPSEC) coupled with multi-angle laser light scattering (MALLS) and refractive index detector (RI), and after debranching by HPSEC-RI and high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detector (PAD). MALLS-derived MW values for amylose and amylopectin were higher than those obtained using the hydrodynamic volume method. The molecular weight of amylopectin chains, determined by the degree of polymerization (DP), ranged between 2.1 and 2.9 × 103 g/mol for short chains, and between 4.1 × 103 and 1.1 × 104 g/mol for long chains. Differences in amylopectin chain content were observed between HPSEC-RI and HPAEC-PAD, highlighting the complementary nature of these techniques. The study underscores the need for standardizing chromatography-based methodologies in starch research, particularly with the advent of new technologies.

Asymmetric Flow Field-Flow Fractionation for Comprehensive Characterization of Hetero-aggregates made of nano-Silver and Extracellular Polymeric Substances

The present study explores the capability of asymmetrical flow field-flow fractionation (AF4) coupled online with diode array (DAD), fluorescence detectors (FLD), multi-angle light scattering (MALS) and dynamic light scattering (DLS) to characterize silver nanoparticles (nAg) hetero-aggregates formed with diatoms derived extracellular polymeric substances (EPS). The content of EPS varied from 10.5 to 105 mgC L-1 and nAg were dispersed at 4 mg L-1 in a freshwater medium. Good recoveries (∼ 76.9 ± 8.4%) of nAg-EPS were obtained from AF4-DAD signals, but an anomalous elution was observed as EPS concentration increased: AF4 retention times decreased despite average gyration radii measured by MALS for nAg-EPS increased (from 16 nm to 24 nm), which suggests a change in the aggregation state, as evaluated by UV-Vis scans obtained from DAD. A regular Brownian relaxation of nAg-EPS was proven for each EPS concentration using these 2 detectors. The comparison of on-line and batch DLS measurements validated in addition, that no (dis)aggregation occurs upon injections. After a thorough comparison with classical AF4 using standards, the frit-inlet-AF4 was used. Slightly higher recovery (79.6±4.6 %) was obtained but similar deviation of nAg-EPS elution occurred, excluding the implication of membrane differential fouling of nAg-EPS /conditioning effects of EPS. The investigation of physico-chemical parameters controlling the Brownian relaxation of nAg-EPS suggests the influence of nAg-EPS structure and EPS loading. This study demonstrates the suitable use of AF4 coupled to multiple detectors to probe-out eco-corona formation and characterize polydisperse systems containing NPs, EPS and their hetero-aggregates in freshwaters, even under a non-ideal size-fractionation scenario.

Assessment of the impact of pasteurisation on protein denaturation in sheep sweet whey by asymmetrical flow field-flow fractionation according to sampling period

Whey recovery is limited in artisanal cheese manufacture. This by-product of cheese manufacture was traditionally considered as waste and is highly polluting. However, interest in the nutritional properties of native whey and its proteins has recently increased. Pretreatment is required to preserve highly perishable whey for processing. Only a few studies have focused on sheep whey, despite its considerable nutritional and technological potential. Here, we investigated the effect of heat treatment on the denaturation and formation of protein aggregates in sweet sheep whey collected in January, April and July. Two pasteurisation protocols were studied (72 °C for 1 min and 80 °C for 15 s). Microbiological quality was assessed by checking for the presence of microbes after five days of storage at 4 °C. Asymmetrical flow field-flow fractionation (AF4) coupled to UV detector, multiangle light scattering (MALS) and refractometer (dRi) is a mild, non-naturing technique for determining the extent of denaturation of whey proteins and the size of whey protein aggregates. Greater whey protein denaturation and larger whey protein aggregates were observed for the 80 °C/15 s protocol than for the 72 °C/1 min protocol, for the January and April samples. A decrease in Immunoglobulin G (IgG) content was observed after heat treatments in the samples from July but not significantly for the other proteins. Moreover, the retention times of the monomeric whey protein peaks on the AF4 fractogram were higher for this period, indicating that the proteins were larger. Microbiological testing showed that both pasteurisation treatments were sufficiently effective to ensure good sanitary quality. The pasteurisation schedule best preserving native proteins was heating at 72 °C for 1 min.

Determination of particle number concentration for biological particles using AF4-MALS: Dependencies on light scattering model and refractive index

Determining accurate counts and size distributions for biological particles (bioparticles) is crucial in wide-ranging fields, but current methods to this end are susceptible to bias from polydispersity in size. This bias can be mitigated by incorporating a separation step prior to characterization. For this reason, asymmetrical flow field-flow fractionation (AF4) with on-line multiangle light scattering (MALS) has become an important platform for determining particle size. AF4-MALS has also been increasingly used to report particle concentration, particularly for complex biological particles, yet the impact of light scattering models and particle refractive indices (RI) have not been quantitatively evaluated. Here, we develop an analysis workflow using AF4-MALS to simultaneously separate and determine particles sizes and concentrations. The impacts of the MALS particle counting model used to process data and the chosen RI value(s) on particle counts are systematically assessed for polystyrene latex (PSL) particles and bacterial outer membrane vesicles (OMVs) in the 20-500 nm size range. Across spherical models, PSL and OMV particle counts varied up to 13% or 200%, respectively. For the coated-sphere model used in the analysis of OMV samples, the sphere RI value greatly impacts particle counts. As the sphere RI value approaches the RI of the suspending medium, the model becomes increasingly sensitive to the light scattering signal-to-noise ratio ultimately causing erroneous particle counts. Overall, this work establishes the importance of selecting appropriate MALS models and RI values for bioparticles to obtain accurate counts and provides an AF4-MALS method to separate, enumerate, and size polydisperse bioparticles.

Chemometric approaches for polysaccharide viscosity profiling

Traditional viscosity measurements for carrageenan are laborious, present practical and environmental challenges, and fail to provide structure-property understanding for application and manufacturing development. We hypothesize that integrating Size Exclusion Chromatography (SEC) with Multi-Angle Light Scattering (MALS) and online viscometry, combined with chemometric techniques, can develop a more efficient and environmentally friendly method for determining the apparent viscosity of carrageenan solutions.To test this hypothesis, predictive chemometric models were developed using SEC-MALS data for carrageenan extracted from four different seaweed species. By integrating SEC-MALS with Partial Least Squares (PLS) regression, key molecular parameters such as hydrodynamic radius, intrinsic viscosity, and molecular mass were identified as significant influencers of viscosity. The model for carrageenan from Eucheuma denticulatum yielded the lowest prediction error (RMSEP 8.4), while those for carrageenan extracted from Kappaphycus alvarezii or from several species of the Chondrus genus showed higher errors due to κ-carrageenan sensitivity. For carrageenan extracted from seaweed of the Gigartina genus, incorporating the root mean square radius resulted in a low prediction error of 10.This study concludes that integrating SEC-MALS with PLS regression effectively identifies key molecular parameters influencing carrageenan viscosity, enhancing structure-property understanding and providing a reliable analytical method for optimizing quality control and application in various industries.

Multivalent Protein-Nucleic Acid Interactions Probed by Composition-Gradient Multiangle Light Scattering.

Many RNA-binding proteins, such as TDP-43 or CELF1, interact multivalently with nucleic acid repetitive elements. The molecular stoichiometry of protein to nucleic acid is often difficult to assess, particularly by standard electrophoretic mobility shift assays (EMSAs). Here, we investigate the use of composition-gradient multiangle light scattering (CG-MALS) for quantifying binding affinity and stoichiometry for two RNA-binding proteins with their nucleic acid partners of varied sequence and length: TDP43's N-terminal RNA recognition motifs with both TG/GU-repeat ssDNA and ssRNA, respectively, and CELF1's two N-terminal RNA recognition motifs with (TG/UGUU/GU) repeats and an experimentally defined cognate GU-rich element (GRE). Our CG-MALS data derived from each of these interactions is consistent with expected ranges of binding affinity and stoichiometry for proteins binding to shorter nucleic acid repeats. Furthermore, we conclude that CG-MALS can be an excellent method for obtaining quantitative estimates even for high (>2) protein-nucleic acid stoichiometric ratios.

Scaling laws for nanoparticles – Online shape heterogeneity analysis by size exclusion chromatography coupled with multi-angle light scattering

Nanoparticle-based drug delivery systems are rising technologies to access challenging therapeutic targets. Following commercial success of lipid-based nanoparticles (LBNP), accruing understandings of nanoparticle structures and critical quality attributes through advanced analytics are beneficial to future clinical development and generalization of this delivery platform. The morphological attributes of nanoparticles, such as shape, can affect uptake, cell-interaction, drug release, circulation, and flow. Gaining an understanding of these structure-activity relationships in early-stage formulation development is important because mix morphologies can affect quality and potency but often exist before process control strategies are fully implemented. In this study, we used shape heterogeneous nanoparticle mixtures, containing various populations of liposomes and lipodisks, as a model system and developed an online semi-quantitative method to characterize the nanoparticle shape heterogeneity by size exclusion chromatography (SEC) coupled with multi-angle light scattering (MALS). The liposomes and lipodisks were separated in SEC when their sizes were ∼3 fold different. When the particles of different shapes were in similar sizes, size-based separation was not always feasible. Instead, light scattering data distinguished liposomes and lipodisks by the scaling law linking radius of gyration and molecular weight of the nanoparticles, enabling morphological identification. A semi-quantitative model was built based on the exponential correlation between the scaling law exponents and the ratios of liposomes and lipodisks. The model was applied to test 6 random formulations made with different compositions and manufacturing processes, and the predicted liposome percentage for 5 formulations was within 25 % absolute difference from the percentage determined by cryogenic electron microscopy (cryo-EM). We envision this method being routinely used to characterize liposome and lipodisk shape heterogeneity during formulation screening as well as on stability studies. Potentially, the method can be converted to in-process control method and extended to other categories of nanoparticles beyond liposomes.

Pharmaceutical Applications and Advances with Zetasizer: An Essential Analytical Tool for Size and Zeta Potential Analysis

: Zetasizer is an advanced device that measures various properties of particles or molecules suspended in a liquid medium. It is extensively used for evaluating the size of nanoparticles, colloids, and biomolecular particles, and for determining particle charge. There are several analytical techniques by which the size, zeta potential, and molecular weight can be determined, like Dynamic Light Scattering (DLS) that measures the size of particles in dispersed systems, which can range from sub-nanometers to several micrometers in diameter. Electrophoretic Light Scattering (ELS) analyzes the mobility and charge of particles, also known as the zeta potential. Static Light Scattering (SLS) determines the molecular weight of particles in a solution. The Zetasizer is part of the Zetasizer Advance range of benchtop systems available for laboratory use. The Zetasizer Ultra model offers unique measurement capabilities, such as Multi-angle Dynamic Light Scattering (MADLS) and particle concentration. These features offer a deeper understanding of samples, making the Zetasizer a vital instrument in numerous scientific and industrial applications. In this review, we have discussed Zetasizer’s principles for the determination of particle size, zeta potential, and molecular weight, along with its qualification and applications in different formulations.

Characterization of Nanoparticles in Drinking Water Using Field-Flow Fractionation Coupled with Multi-Angle Light Scattering and Inductively Coupled Plasma Mass Spectrometry

The current absence of well-established and standardized methods for characterizing submicrometer- and nano-sized particles in water samples presents a significant analytical challenge. With the increasing utilization of nanomaterials, the potential for unintended exposure escalates. The widespread and persistent pollution of water by micro- and nanoplastics globally is a concern that demands attention, not only to reduce pollution but also to develop methods for analyzing these pollutants. Additionally, the analysis of naturally occurring nano entities such as bubbles and colloidal matter poses challenges due to the lack of systematic and consistent methodologies. This study presents Asymmetric Flow Field-Flow Fractionation (AF4) separation coupled with a UV-VIS spectrometer followed by Multi-Angle Light Scattering (MALS) for detection and size characterization of nanometric entities. It is coupled with an Inductively Coupled Plasma Mass Spectrometer (ICP-MS) for elemental analysis. Water samples from different sources, such as untreated mountain spring water, groundwater, and bottled drinking water, were analyzed. The system was calibrated using pure particle standards of different metallic compositions. Our study demonstrates the capability of AF4-UV-MALS-ICP-MS to detect metals such as Al, Ba, Cu, and Zn in particles of around 200 nm diameter and Mg associated with very small particles between 1.5 and 10 nm in different drinking water samples.

Development of an advanced separation and characterization platform for mRNA and lipid nanoparticles using multi-detector asymmetrical flow field-flow fractionation.

In recent years, the use of lipid nanoparticles (LNPs) for delivery of messenger RNA (mRNA)-based therapies has gained substantial attention in the field of drug development. In such an application, multiple LNP attributes have to be carefully characterized to ensure product safety and quality, whereas accurate and efficient characterization of these complex mRNA-LNP formulations remains a challenging endeavor. Here, we present the development and application of an online separation and characterization platform designed for the isolation and in-depth analysis of mRNAs and mRNA-loaded LNPs. Our asymmetrical flow field-flow fractionation with a multi-detector (MD-AF4) method has demonstrated exceptional resolution between mRNA-LNPs and mRNAs, delivering excellent recoveries (over 70%) for both analytes and exceptional repeatability. Notably, this platform allows for comprehensive and multi-attribute LNP characterization, including online particle sizing, morphology characterization, and determination of encapsulation efficiency, all within a single injection. Furthermore, real-time online sizing by synchronizing multi-angle light scattering (MALS) and dynamic light scattering (DLS) presented higher resolution over traditional batch-mode DLS, particularly in differentiating heterogeneous samples with a low abundance of large-sized particles. Additionally, our method proves to be a valuable tool for monitoring LNP stability under varying stress conditions. Our work introduces a robust and versatile analytical platform using MD-AF4 that not only efficiently provides multi-attribute characterizations of mRNA-LNPs but also holds promise in advancing studies related to formulation screening, quality control, and stability assessment in the evolving field of nanoparticle delivery systems for mRNAs.

Intrinsic CO2 nanobubbles in alkaline aqueous solutions

Nanobubbles (NBs) and their derivative, bulk nanobubbles (BNBs), have been widely studied due to their potential wide range of applications. However, the stable existence of intrinsic NBs and BNBs in solutions is still controversial. This study investigates the existence and behaviour of unknown nano-entities in dilute alkaline solutions using Field-Flow Fractionation Multi-Angle Light Scattering (FFF-MALS), resulting in the first report of the presence, size distribution, and size distribution changes under different treatments. The results suggest that neutral and acidic pure solutions of salts at concentrations below the solubility product do not contain detectable nano entities. Alkaline solutions, on the other hand, such as carbonate buffer and sodium hydroxide, do contain nano scatterers in the range of ∼100 nm diameter that cannot be removed by boiling or filtration. Reducing the pH of the solutions or freezing reduces their number densities. The freeze-thaw procedure strongly suggests that these nano entities are BNBs. Zeta Nanoparticle Tracking Analysis (ZNTA) shows the zeta potential and the concentration in agreement with previous literature. It can be concluded that intrinsic BNBs in alkaline solutions are an integral part of the total nano-entity population. This study highlights the importance of considering intrinsic BNBs when investigating nanoparticles in alkaline solutions with analytical methods such as Static Light Scattering (SLS), which can detect low number densities in the original liquid state that may not be plausible by other detection methods due to their detection thresholds or acquisition of sample preparation methods of drying or freezing that do not allow real-time observations.

USP’s characterization of commercial poly(lactic-co-glycolic acid) utilizing SEC-Multi-Angle Light Scattering and Refractive Index techniques via Absolute Method approach

This research focuses on evaluating the data quality and reliability of Size Exclusion Chromatography with Multi-Angle Light Scattering and Refractive Index (SEC-MALS/RI) chromatography in determining molecular weight-related parameters in poly(lactic-co-glycolic) acid (PLGA) polymers. The study utilizes key metrics, particularly percent relative standard deviation (%RSD), to evaluate the precision across different data processing conditions. Exploring baseline selection of the chromatograms reveals that selection of the baseline based on the intrinsic shape of the chromatogram, rather than relying on elution peaks, improves consistency. The study emphasizes the critical role of result fitting in addressing systematic errors and sample heterogeneity, recommending the minima of the elution peak selection range for realistic molecular weight distribution representation. Analysis of light-scattering (LS) detector chromatograms highlights varying effects on different PLGA polymers, emphasizing the need for careful assessment and potential exclusion for precision improvement, especially in the presence of experimental artifacts. Across diverse PLGA polymers, the utilization of different column types, along with considerations such as baseline determination and peak selection, shows minimal variations in Mn, Mw, and polydispersity index (PDI), affirming the consistency of the methodology. The %RSD analysis further supports the methodology's precision, with values well within acceptable limits. In summary, this comprehensive evaluation of methodological conditions, including baseline selection, result fitting, LS detector data management, and column types, establishes a reliable SEC-MALS/RI chromatographic approach by Absolute Method for determining molecular weight parameters in PLGA polymers.

Exploring the structural, functional, and biocompatibility aspects of marine bacterial extracellular polysaccharides for biopharmaceutical applications

Marine bacterial extracellular polysaccharides (EPS) represent a unique class of biopolymers with vast potential in biopharmaceutical research due to their diverse structural characteristics, functional properties, and biocompatibility. This study delves into the comprehensive analysis of the monosaccharide composition, molecular weight distribution, and conformational dynamics of marine bacterial EPS, employing advanced analytical techniques such as Nuclear Magnetic Resonance (NMR) spectroscopy, Mass Spectrometry (MS), and Size-Exclusion Chromatography coupled with Multi-Angle Laser Light Scattering (SEC-MALLS). Furthermore, we explore the functional applications of these EPS in biofilm inhibition, immunomodulation, and as innovative drug delivery systems, highlighting their role in enhancing antibiotic efficacy and mitigating resistance. Through quantitative analysis and mathematical modeling, including rheological studies and kinetic modeling of biodegradation, this work assesses the EPSs biocompatibility and biodegradability, essential for their safe use in biomedicine. Our findings underscore the potential of marine bacterial EPS in developing novel biopharmaceutical formulations that are not only effective but also environmentally sustainable and biocompatible, paving the way for future advancements in medical and therapeutic applications.

Multiangle Light Scattering as a Lentivirus Purification Process Analytical Technology.

The limited biomolecular and functional stability of lentiviral vectors (LVVs) for cell therapy poses the need for analytical tools that can monitor their titers and activity throughout the various steps of expression and purification. In this study, we describe a rapid (25 min) and reproducible (coefficient of variance ∼0.5-2%) method that leverages size exclusion chromatography coupled with multiangle light scattering detection (SEC-MALS) to determine size, purity, and particle count of LVVs purified from bioreactor harvests. The SEC-MALS data were corroborated by orthogonal methods, namely, dynamic light scattering (DLS) and transmission electron microscopy. The method was also evaluated for robustness in the range of 2.78 × 105-2.67 × 107 particles per sample. Notably, MALS-based particle counts correlated with the titer of infectious LVVs measured via transduction assays (R2 = 0.77). Using a combination of SEC-MALS and DLS, we discerned the effects of purification parameters on LVV quality, such as the separation between heterogeneous LV, which can facilitate critical decision-making in the biomanufacturing of gene and cell therapies.

Synthesis and end-group control of soluble, high-molecular-weight hyperbranched poly(phenylene thienylene)s by non-stoichiometric A2 + B3 Suzuki-Miyaura polycondensation without gelation

Non-stoichiometric Suzuki-Miyaura polycondensation of tribromo monomer 1, containing a thienylene group, with diboronic acid (ester) monomer 7 in the presence of tBuPPd catalyst, which has a propensity for intramolecular catalyst transfer on a π-electron face, was investigated. We anticipated that the presence of thienylene groups in the obtained hyperbranched polymer would increase the detection sensitivity in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and enable the analysis of the polymer end groups up to the high-molecular-weight region. We first conducted Suzuki-Miyaura reaction of several kinds of 1 with phenylboronic acid 2 in the presence of tBuPPd precatalyst 3. The reaction of tris(5-bromo-4-hexylthien-2-yl)benzene 1c with 2 afforded exclusively tri-substituted product 4c, indicating that the tBuPPd catalyst undergoes intramolecular catalyst transfer on 1c after the first and second substitutions of 1c with 2. The polycondensation of equimolar 1c and pinacol phenylenediboronate 7a (reaction site ratio of Br:BPin = 1.5:1.0 (BPin = pinacol boronate)) was then carried out in the presence of 3 for 3 h, affording hyperbranched polymer with Mn = 37500 without gelation. The product was purified by precipitation. The degree of polymerization (DP) was calculated from the absolute molecular weight, determined by multi-angle light scattering (MALLS) analysis, to be 200, which is a far higher than would be predicted from the Flory-Stockmayer theory at the critical gel point (DP = 6.6). Furthermore, the MALDI-TOF mass spectra showed two series of peaks due to polymer with boronate ends and boronate ends along with one cyclic unit up to m/z of about 7500, as we had expected. The absence of peaks due to polymer with Br ends indicates that the degree of branching (DB) of this hyperbranched polymer is 100 %.

An HPLC-SEC-based rapid quantification method for vesicular stomatitis virus particles to facilitate process development

Virus particle (VP) quantification plays a pivotal role in the development of production processes of VPs for virus-based therapies. The yield based on total VP count serves as a process performance indicator for evaluating process efficiency and consistency. Here, a label-free particle quantification method for enveloped VPs was developed, with potential applications in oncolytic virotherapy, vaccine development, and gene therapy. The method comprises size exclusion chromatography (SEC) separation based on high-performance liquid chromatography (HPLC) instruments. Ultraviolet (UV) was used for particle quantification and multi-angle light scattering (MALS) for particle characterization. Consistent recoveries of over 97% in the SEC were achieved upon mobile phase screenings and addition of bovine serum albumin (BSA) as sample stabilizer. A calibration curve was generated, and the method's performance and applicability to in-process samples were characterized. The assay’s repeatability variation was < 1% and its intermediate precision variation was < 3%. The linear range of the method spans from 7.08×108 to 1.72×1011 VP/mL, with a limit of detection (LOD) of 7.72×107 VP/mL and a lower limit of quantification (LLOQ) of 4.20×108 VP/mL. The method, characterized by its high precision, requires minimal hands-on time and provides same-day results, making it efficient for process development.

In-line coupling of capillary-channeled polymer fiber columns with optical absorbance and multi-angle light scattering detection for the isolation and characterization of exosomes

Extracellular vesicles (EVs) have garnered much interest due to their fundamental role in intracellular communication and their potential utility in clinical diagnostics and as biotherapeutic vectors. Of particular relevance is the subset of EVs referred to as exosomes, ranging in size from 30 to 150 nm, which contain incredible amounts of information about their cell of origin, which can be used to track the progress of disease. As a complementary action, exosomes can be engineered with therapeutic cargo to selectively target diseases. At present, the lack of highly efficient methods of isolation/purification of exosomes from diverse biofluids, plants, and cell cultures is a major bottleneck in the fundamental biochemistry, clinical analysis, and therapeutic applications. Equally impactful, the lack of effective in-line means of detection/characterization of isolate populations, including concentration and sizing, is limiting in the applications. The method presented here couples hydrophobic interaction chromatography (HIC) performed on polyester capillary-channeled polymer (C-CP) fiber columns followed by in-line optical absorbance and multi-angle light scattering (MALS) detection for the isolation and characterization of EVs, in this case present in the supernatant of Chinese hamster ovary (CHO) cell cultures. Excellent correlation was observed between the determined particle concentrations for the two detection methods. C-CP fiber columns provide a low-cost platform (< $5 per column) for the isolation of exosomes in a 15-min workflow, with complementary absorbance and MALS detection providing very high-quality particle concentration and sizing information.

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.

Evaluation of size-exclusion chromatography, multi-angle light scattering detection and mass photometry for the characterization of mRNA

The recent approval of messenger ribonucleic acid (mRNA) as vaccine to combat the COVID-19 pandemic has been a scientific turning point. Today, the applicability of mRNA is being demonstrated beyond infectious diseases, for example in cancer immunotherapy, protein replacement therapy and gene editing. mRNA is produced by in vitro transcription (IVT) from a linear DNA template and modified at the 3′ and 5′ ends to improve translational efficiency and stability. Co-existing impurities such as RNA fragments and double-stranded RNA (dsRNA), amongst others, can drastically impact mRNA quality and efficacy. In this study, size-exclusion chromatography (SEC) is evaluated for the characterization of IVT-mRNA. The effect of mobile phase composition (ionic strength and organic modifier), pH, column temperature and pore size (300 Å, 1000 Å, and 2000 Å) on the separation performance and structural integrity of IVT-mRNA varying in size is described. Non-replicating, self-amplifying (saRNA), temperature degraded, and ribonuclease (RNase) digested mRNA, the latter to characterize the 3′ poly(A) tail, were included in the study. Beyond ultraviolet (UV) detection, refractive index (RI) and multi-angle light scattering (MALS) detection were implemented to accurately determine molecular weight (MW) of mRNA. Finally, mass photometry is introduced as a complementary methodology to study mRNA under native conditions.

Advancing Molecular Weight Determination of Lignin by Multi-Angle Light Scattering.

Due to the complexity and recalcitrance of lignin, its chemical characterization is a key factor preventing the valorization of this abundant material. Multi-angle light scattering (MALS) is becoming a sought-after technique for absolute molecular weight (MW) determination of polymers and proteins. Lignin is a suitable candidate for MW determination via MALS, yet further investigation is required to confirm its absolute MW values and molecular size. Studies aiming to break down lignin into a variety of renewable products will benefit greatly from a simple and reliable determination method like MALS. Recent pioneering studies, discussed in this review, addressed several key challenges in lignin's MW characterization. Nevertheless, some lignin-specific issues still need to be considered for in-depth characterization. This study explores how MALS instrumentation manages the complexities of determining lignin's MW, e.g., with simultaneous fractionation and fluorescence interference mitigation. Additionally, we rationalize the importance of a more detailed light scattering analysis for lignin characterization, including aspects like the second virial coefficient and radius of gyration.