Monomer Peak Research Articles

A label free ratiometric sensing of heparin using dynamic modulation of monomer–excimer equilibrium of coumarin 7

Heparin (Hp) is a multi-negatively charged polysaccharide with diverse physiological roles, particularly in regulating blood coagulation. Its clinical use as an anticoagulant requires a precise monitoring due to potential adverse effects at elevated levels. Therefore, there is a significant demand for an immediate, label-free, and user-friendly method to accurately quantify heparin (Hp). Herein we have explored a novel ratiometric sensing approach using coumarin-7 (C7) dye for precise Hp quantification. At pH 3, the protonated form of C7 undergoes aggregation upon interacting with polyanionic Hp via strong electrostatic forces. Consequently, there is alteration in the dynamics of the monomer-aggregate equilibrium for C7 in solution, resulting in the appearance of a new emission peak at ∼660 nm upon successive decline of the monomer peak at ∼512 nm. This leads to the generation of a ratiometric signal, which shows a nice linear response up to 6.8 μM of Hp to achieve limit of detection (LOD) of 89.6 nM for Hp in citrate buffer. Such ratiometric optical behaviour of C7 is not seen for any other interferants, which may be present in complex bio-matrices and thus, showcases a promising avenue for accurate and reliable Hp quantification even in complex biofluids. Additionally, the assembly C7 − Hp exhibits excellent responsiveness towards ionic strength of the medium, further extending its application towards the detection of an important bio-analyte, protamine (Pr). Importantly, this approach of ratiometric sensing strategy provides more accurate quantification due to its high reproducibility, minimal susceptibility to environmental effects, and self-calibration properties. The detailed spectroscopic analysis elucidates the dynamics of the C7 − Hp interaction, paving the way for advanced biosensing applications.

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.

Structural evolution of pea-derived albumins during pH and heat treatment studied with light and X-ray scattering

Pea albumins are found in the side stream during the isolation of pea proteins. They are soluble at acidic pH and have functional properties which differ from their globulin counterparts. In this study, we have investigated the aggregation and structural changes occurring to pea albumins under different environmental conditions, using a combination of size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALS) and small-angle X-ray scattering (SAXS). Albumins were extracted from a dry fractionated pea protein concentrate by precipitating the globulin fraction at acidic pH. The albumins were then studied at different pH (3, 4, 4.5, 7, 7.5, and 8) values. The effect of heating at 90 °C for 1, 3, and 5 min on their structural changes was investigated using SAXS. In addition, size exclusion of the albumins showed 4 distinct populations, depending on pH and heating conditions, with two large aggregates peaks (∼250 kDa): a dimer peak (∼24 kDa) containing predominantly pea albumin 2 (PA2), and a monomer peak of a molar mass of about 12 kDa (PA1). X-ray scattering intensities as a function of q were modeled as polydisperse spheres, and their aggregation was followed as a function of heating time. Albumins was most stable at pH 3, showing no aggregation during heat treatment. While albumins at pH 7.5 and 8 showed aggregation after heating, solutions at pH 4, 4.5, and 7 already contained aggregates even before heating. This work provides new knowledge on the overall structural development of albumins under different environmental conditions, improving our ability to employ these as future ingredients in foods.

A structural and functional comparison between two recombinant human lubricin proteins: Recombinant human proteoglycan-4 (rhPRG4) vs ECF843

Proteoglycan 4 (PRG4, lubricin) is a mucin-like glycoprotein present on the ocular surface that has both boundary lubricating and anti-inflammatory properties. Full-length recombinant human PRG4 (rhPRG4) has been shown to be clinically effective in improving signs and symptoms of dry eye disease (DED). In vitro, rhPRG4 has been shown to reduce inflammation-induced cytokine production and NFκB activity in corneal epithelial cells, as well as to bind to and inhibit MMP-9 activity. A different form of recombinant human lubricin (ECF843), produced from the same cell line as rhPRG4 but manufactured using a different process, was recently assessed in a DED clinical trial. However, ECF843 did not significantly improve signs or symptoms of DED compared to vehicle. Initial published characterization of ECF843 showed it had a smaller hydrodynamic diameter and was less negatively charged than native PRG4. Further examination of the structural and functional properties of ECF843 and rhPRG4 could contribute to the understanding of what led to their disparate clinical efficacy. Therefore, the objective of this study was to characterize and compare rhPRG4 and ECF843 in vitro, both biophysically and functionally.Hydrodynamic diameter and charge were measured by dynamic light scattering (DLS) and zeta potential, respectively. Size and molecular weight was determined for individual species by size exclusion chromatography (SEC) with in-line DLS and multi-angle light scattering (MALS). Bond structure was measured by Raman spectroscopy, and sedimentation properties were measured by analytical ultracentrifugation (AUC). Functionally, MMP-9 inhibition was measured using a commercial MMP-9 activity kit, coefficient of friction was measured using an established boundary lubrication test at a latex-glass interface, and collagen 1-binding ability was measured by quart crystal microbalance with dissipation (QCMD). Additionally, the ability of rhPRG4 and ECF843 to inhibit urate acid crystal formation and cell adhesion was assessed.ECF843 had a significantly smaller hydrodynamic diameter and was less negatively charged than rhPRG4, as assessed by DLS and zeta potential. Size was further explored with SEC-DLS-MALS, which indicated that while rhPRG4 had 3 main peaks, corresponding to monomer, dimer, and multimer as expected, ECF843 had 2 peaks that were similar in size and molecular weight compared to rhPRG4's monomer peak and a third peak that was significantly smaller in both size and molar mass than the corresponding peak of rhPRG4. Raman spectroscopy demonstrated that ECF843 had significantly more disulfide bonds, which are functionally determinant structures, relative to the carbon-carbon backbone compared to rhPRG4, and AUC indicated that ECF843 was more compact than rhPRG4. Functionally, ECF843 was significantly less effective at inhibiting MMP-9 activity and functioning as a boundary lubricant compared to rhPRG4, as well as being slower to bind to collagen 1. Additionally, ECF843 was significantly less effective at inhibiting urate acid crystal formation and at preventing cell adhesion.Collectively, these data demonstrate ECF843 and rhPRG4 are significantly different in both structure and function. Given that a protein's structure sets the foundation for its interactions with other molecules and tissues in vivo, which ultimately determine its function, these differences most likely contributed to the disparate DED clinical trial results.

Photoexcimerisation of pure acriflavine dye in water and alcohol

The light emission properties of acriflavine have been extensively utilised in its applications as dye laser active media, as photosensitizer, as fluorescent and FRET (Fluorescence Resonance Energy Transfer) agent for sensing mechanism etc. However, studies on the photochemical and photophysical properties of acriflavine and its aggregates are very limited and little is known about its emission characteristics in different media. The present study was undertaken to acquire a comprehensive understanding of the aggregation process and emission characteristics of the solution of pure acriflavine dye in water and alcohol, namely the propanol, methanol, ethanol and butanol. This study includes confirming the photoexcimerisation of acriflavine by concentration dependent UV–Visible spectroscopy, steady state fluorescence spectroscopy and time resolved fluorescence spectroscopy at room temperature. The present study shows the monomer and excimer fluorescence emission of acriflavine in the range 495–528 nm and 549–587 nm respectively (monomer and excimer emission peak wavelength varies for different solvents). The nature of the excimer emission band of acriflavine around 575 nm for all the solvents has been tested through temperature dependent steady state fluorescence spectroscopy and also from time resolved fluorescence spectra it has been found that the excimer band does not originate from any triplet excited state. The pH dependent emission spectra for fixed concentration of acriflavine in the solvents reveals red shifts in the monomer and the excimer fluorescence maxima. The excimerisation of acriflavine in water, propanol and methanol is found as static process from excited dimers, whereas in ethanol and butanol it is the result of dynamic diffusion process between a photoexcited and a ground state monomer. The excimerisation process of the acriflavine has been quantified by evaluating the monomer-excimer equilibrium concentration i.e., the critical concentration in the cited solvents. At the critical concentration the monomer and the excimer are found to be fluoresced with equal intensity. The acriflavine solutions specifically at the critical concentration in the cited solvents will enhance its photophysical and photochemical applications by extending them up to the new range of excimer emission radiation at the wavelength in-between 549 nm and 587 nm. Hence, this report shows that, by using acriflavine solutions of certain concentrations in any one of the cited solvents as an active medium of a dye laser and thereby obtaining both the monomer and excimer emission band, the wavelength of the laser can be tuned from 495 nm to 587 nm.

Polymer-mediated tuning of the monomer-aggregate equilibrium of a coumarin derivative for ratiometric sensing of protamine.

Amongst the various existing methods of analyte quantification, fluorescent-based methods, especially the ratiometric methods, continue to gain significant attention due to their high reproducibility, low environmental influence, and self-calibrating behavior. This paper presents the modulation in a monomer-aggregate equilibrium of coumarin-7 (C7) dye at pH ∼ 3, under the influence of a multi-anionic polymer, poly(styrene sulfonate) (PSS), leading to a significant modification in the ratiometric optical signal of the dye. At pH ∼ 3, cationic C7 formed aggregates in the presence of PSS via a strong electrostatic interaction, resulting in the development of a new emission peak at 650 nm at the expense of the monomer emission at 513 nm. Such contradicting changes in fluorescence intensities at two different wavelengths gave rise to a ratiometric signal, which was found to be highly sensitive towards external stimuli such as pH, and ionic strength. The stability of the C7-PSS complex was found to decrease as the pH of the solution was increased beyond 5, which indicated the decline in the electrostatic attraction between C7 and PSS due to the deprotonation of the C7 dye. Furthermore, an increase in the monomeric peak and a concomitant decrease of the aggregate peak with added salt in the solution (at pH ∼ 3) clearly justified the presence of an electrostatic attraction between C7 and PSS for the complex formation. This was further validated by the excited-state lifetime measurement of the C7-PSS complex, which showed a systematic increase in lifetime contribution from the monomeric species at the expense of aggregated species, as the concentration of NaCl increased in the solution. Thus, protamine (Pr), being a highly positively charged polypeptide, largely affected the monomer-aggregate equilibrium of the C7-PSS system, leading to a phenomenal change in the ratiometric signal, which was utilized to quantify with LOD as low as ∼2.8 nM in buffer for the bio-analyte Pr. Moreover, the ratiometric response of the C7-PSS assembly demonstrated excellent selectivity towards Pr, facilitating its practical relevance for the quantification of Pr in a 1% human serum matrix. Therefore, the studied C7-PSS can be utilized as a potential candidate for the quantification of the protamine even in complex biological media.

In-silico approach as a tool for selection of excipients for safer amphotericin B nanoformulations.

Safer and efficacious Amphotericin B (AmB) nanoformulations can be designed by augmenting AmB in the monomeric or super-aggregated state, and restricting the aggregated state, by choosing the appropriate excipient, which can be facilitated by employing in-silico prediction as a tool. Excipients selected for the study included linear fatty acids from caprylic (C8) to stearic(C18) and the stearate based amphiphilic surfactants polyoxyl-15-hydroxystearate (PS15) and polyoxyl-40-stearate (PS40). Blend module was employed to determine the two miscibility parameters mixing energy (Emix) and interaction parameter (χ). AmB-excipient interactions were modelled using molecular docking software. The fatty acids revealed a decrease in Emix and χ values with increase in carbon chain length, suggesting enhanced affinity with increase in fatty acid hydrophobicity. Significantly higher affinity was observed with amphiphilic surfactants, in particular PS40 which exhibited negative values of Emix and χ proposing very high degree of miscibility. Molecular docking study confirmed extensive interaction of all the excipients with the AmB polyene chain. PS15 and PS40 displayed in addition hydrophilic interactions with the mycosamine and polyol moieties with PS40 exhibiting complete wrapping of the AmB molecule. PS15 demonstrated only partial wrapping, attributed to the shorter ethylene oxide chain. AmB nanosuspensions (NS) were prepared by in situ nanoprecipitation using the excipients and the AmB state identified by UV scanning between 300-500nm. AmB NS with fatty acids and PS15-AmB NS revealed a high intensity peak between 330-350nm of aggregated AmB and low intensity monomeric peaks between 405-415nm reflecting predominance of the aggregated state. PS40-AmB NS on the other hand revealed complete absence of aggregated state and a high intensity peak between 321-325nm which corresponded to the super-aggregated state. Also, the super-aggregated state slowly released the safe monomeric form without aggregate formation. Furthermore, very low hemolysis seen with PS40-AmB NS confirmed low toxicity attributed to the safer super-aggregated state and while higher hemolysis as anticipated was seen with PS15-AmB NS (aggregated state). The basis for selection of the appropriate excipient for design of safer AmB nanoformulations would be those excipients that exhibit negative values of miscibility parameters Emix and χ, exhibit interaction with the hydrophobic and hydrophilic regions of AmB and demonstrate complete wrapping of AmB in the molecular docking study. Our study thus demonstrates feasibility of in-silico prediction as a practical tool for excipient selection for safer AmB nanoformulations.

Tractable Method for Rapid Quality Assessment of Therapeutic Antibodies in Harvested Cell Culture Fluid based on FcγRIIIa-Immobilized Magnetic Microspheres.

FcγRIIIa-binding affinity is one of the key factors to ensure the efficacy of many antitumor therapeutic antibodies, which should be monitored along with the titer, protein aggregation, and other critical quality attributes. The conventional workflow for the quality assessment of therapeutic antibodies in harvested cell culture fluid (HCCF) is time-consuming and costly nevertheless. In this study, a tractable method was established for rapid quality assessment of a HCCF sample through differentially extracting IgG with different FcγRIIIa affinity levels using FcγRIIIa-immobilized magnetic microspheres, followed by size exclusion chromatography (SEC) to determine the amount and monomer percentage of IgGs in the preceding eluate. FcγRIIIa-immobilized magnetic microspheres with polydopamine (PDA) and hydrophilic dendrimer (PAMAM) coating (denoted as Fe3O4@PDA@PAMAM-FcγRIIIa) were synthesized for the first time as magnetic adsorbents. The PDA cladding endowed the composites with good chemical stability in acidic elution buffer, and the PAMAM dendrimer empowered the composites of high ligand immobilization capacity and hydrophilic surface. The labile FcγRIIIa was immobilized under mild conditions. By directly applying a simple magnetic solid phase extraction procedure to treat HCCF, favored IgG species with high FcγRIIIa affinity would be selectively captured by Fe3O4@PDA@PAMAM-FcγRIIIa composites for subsequent SEC analysis. The monomer peak area value in SEC, which was set as the read-out of the proposed method, correlated directly with the theoretical overall quality of standard-spiked HCCF samples.

Effect of ion source polarity and dopants on the detection of auxin plant hormones by ion mobility-mass spectrometry.

Ion mobility spectrometry (IMS) equipped with a corona discharge (CD) ion source was used for measurement of three auxin plant hormones including indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), and indole-3-butyric acid (IBA). The measurements were performed in both positive and negative polarities of the CD ion source. Dopant gases NH3, CCl4, and CHBr3 were used to modify the ionization mechanism. A time-of-flight mass spectrometer (TOFMS) orthogonal to the IMS cell was used for identification of the product ions. Density functional theory was used to rationalize formation of the ions, theoretically. The mixtures of the auxins were analyzed by CD-IMS. The separation performance depended on the ion polarity and the dopants. In the positive polarity without dopants, auxins were ionized via protonation and three distinguished peaks were observed. Application of NH3 dopant resulted in two ionization channels, protonation, and NH4+ attachment leading to peak overlapping. In the negative polarity, two ionization reactions were operative, via deprotonation and O2- attachment. The separation of the monomer peaks was not achieved while the peaks of anionic dimers [2M-H]- were separated well. The best LOD (4ng) was obtained in negative polarity with CCl4 dopant. Methylation (esterification) of IAA improved LODs by about one order.

Effect of Buffer Salts on Physical Stability of Lyophilized and Spray-Dried Protein Formulations Containing Bovine Serum Albumin and Trehalose.

This study examined the effect of buffer salts on the physical stability of spray-dried and lyophilized formulations of a model protein, bovine serum albumin (BSA). BSA formulations with various buffers were dried by either lyophilization or spray drying. The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), powder X-ray diffraction (PXRD), size exclusion chromatography (SEC), solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS), and solid-state nuclear magnetic resonance spectroscopy (ssNMR). Particle characterizations such as Brunauer-Emmett-Teller (BET) surface area, particle size distribution, and particle morphology were also performed. Results from conventional techniques such as ssFTIR did not exhibit correlations with the physical stability of studied formulations. Deconvoluted peak areas of deuterated samples from the ssHDX-MS study showed a satisfactory correlation with the loss of the monomeric peak area measured by SEC (R2 of 0.8722 for spray-dried formulations and 0.8428 for lyophilized formulations) in the 90-day accelerated stability study conducted at 40°C. mDSC and PXRD was unable to measure phase separation in the samples right after drying. In contrast, ssNMR successfully detected the occurrence of phase separation between the succinic buffer component and protein in the lyophilized formulation, which results in a distribution of microenvironmental acidity and the subsequent loss of long-term stability. Moreover, our results suggested that buffer salts have less impact on physical stability for the spray-dried formulations than the lyophilized solids.

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

Size exclusion chromatography has become an essential tool for the protein therapeutics industry. Conceptually, it is a simple form of chromatography that is driven by entropy and sieving effects. An ideal size exclusion column would exhibit no adsorptive interactions between its internal surfaces and the solutes being analysed, but that is not easily achieved. To this end, we have studied the utility of three unique packing materials in pursuit of additional column chemistries that might be less prone to interacting with proteins. These packing materials were each prepared from bridged ethylene hybrid organic/inorganic particles but uniquely derivatized into either hydroxy terminated PEO bonded, methoxy terminated PEO bonded, or diol bonded packing materials. All three materials were packed into column hardware modified with hydrophilic hybrid surface technology (h-HST) so that packing material effects could be more clearly observed without any influence from the secondary interactions that can originate from metal hardware. Non-specific interactions were compared for various challenging protein samples in the presence of ammonium acetate (volatile) and phosphate buffered saline (non-volatile) buffers. It was reconfirmed that the h-HST column hardware mitigates a majority of non-desired secondary interactions. However, during studies on hydrophobic interactions, the new hydroxy terminated PEO packing material showed clear benefit to obtaining higher apparent recoveries to better ensure accurate aggregate quantitation. Further experiments were explored to show that a hydroxy terminated PEO column could be effectively paired with a mobile phase comprised of standard strength phosphate buffered saline to make a fast platform method capable of baseline resolving monoclonal antibody monomer and aggregate peaks within a 3 min analysis time.

Characterization of the Interactions between an Unassociated Cationic Pyrene-Labeled Gemini Surfactant and Anionic Sodium Dodecyl Sulfate.

The gemini surfactant PyO-3-12, made of two dimethylammonium bromides joined by a propyl linker and bearing a dodecyl pendant on one side and a 1-pyrenemethoxyhexyl group on the other side, was employed to probe the interactions between positively charged PyO-3-12 and negatively charged sodium dodecyl sulfate (SDS). PyO-3-12 was selected for its ability to respond to the polarity of its local environment through the fluorescence intensity ratio I1/I3 of the first-to-third fluorescence peaks of the pyrene monomer and the local pyrene concentration [Py]loc through the IE/IM ratio of the pyrene excimer-to-pyrene monomer fluorescence intensity. Furthermore, analysis of the fluorescence decays of aqueous solutions of PyO-3-12 and SDS yielded a measure of the internal dynamics, local concentration, and state (associated vs unassociated) of PyO-3-12 in solution. By following these parameters for aqueous solutions prepared with a constant PyO-3-12 concentration of either 1, 4, or 16 μM and SDS concentrations ranging from 0 to 200 mM, six SDS concentration regimes were identified to describe the interactions between PyO-3-12 and SDS in pure water. Sharp transitions of the parameters describing the fluorescence of pyrene marked the boundaries between the different regimes. Perhaps the most important transition was the one defining the formation of the PyO-3-12/SDS aggregates, which was completed at the equicharge point, implying that they were constituted of 1 meq of PyO-3-12 and 2 meq of SDS. The low I1/I3 ratio obtained for the PyO-3-12/SDS aggregates suggested that they were multilamellar aggregates, which would shield the pyrenyl labels from polar water. The formation of these multilamellar aggregates was confirmed by transmission electron microscopy (TEM), which demonstrated the existence of multilamellar vesicles, whose presence increased with decreasing PyO-3-12 concentration. This study suggests that the combination of pyrene excimer formation and TEM provides an interesting experimental means to probe the assemblies generated from oppositely charged surfactants at surfactant concentrations, which are much lower than their critical micelle concentration.

Anatomy of Protein Electrospray Ionization Mass Spectra by Superconducting Tunnel Junction Mass and Energy Spectrometry.

Cryogenic superconducting tunnel junction (STJ) detectors have the advantage of single-particle sensitivity, high quantum efficiency, low noise, and the ability to detect the time and relative impact energy of deposited ions. This makes them attractive for use in mass spectrometry (MS) and as a form of energy spectrometry. STJ cryodetectors have been coupled to time-of-flight (TOF) mass spectrometers equipped with a matrix-assisted laser desorption ionization (MALDI) source and to an electrospray ionization (ESI) TOF mass spectrometer. Here, a lab-made linear quadrupole ion trap (LIT) mass spectrometer system was coupled to an ESI source and a 16-channel Nb-STJ array with improved readout electronics. The goal was to investigate fundamentals of ESI-generated protein ions by further exploiting the advantage of resolving these ions in a third dimension of the relative energy deposited into the STJs. The proteins equine cytochrome c, bovine carbonic anhydrase, bovine serum albumin, and murine immunoglobulin G were studied using this ESI-LIT-STJ-MS instrument. Multiply charged monomers, multimers, and fragments from metastable ions were resolved from monomer peaks by differences in ion deposition energy even when these ions have the same mass-to-charge ratio as the corresponding monomer. The determination of a fragment mass from metastable decomposition is accomplished without knowing the charge state of the fragment. The average charge state of the multimers is reduced with each addition of a protein which is presumed to be a direct reflection of the surface area available for charging. Multiply charged in-source fragments have also been observed and distinguished in the mass spectrum of carbonic anhydrase by using the differences in the energy deposited in the STJs.

High-throughput multiplex analysis of mAb aggregates and charge variants by automated two-dimensional size exclusion-cation exchange chromatography coupled to mass spectrometry

Monoclonal antibodies (mAbs) are extremely complex due to the presence of structural modifications resulting from enzymatic and chemical reactions such as glycosylation, glycation, deamidation, isomerisation, oxidation, aggregation and fragmentation. Size and charge variants analysis are carried out from the early stages of drug development throughout product lifetime to investigate product degradation pathways and optimise process conditions. However, conventional analytical workstreams for size and charge variant characterization are both time and sample demanding, requiring the application of multiple analytical methods. This study presents the development of a novel 2D-LC/MS approach combining both aggregate and charge variant profiling of a mAb candidate in a single method. Aggregate quantification was performed in the first dimension (1D) by size exclusion chromatography SEC, followed by online fraction transfer of the monomer peak to the second dimension (2D) by a heart-cutting for charge variant analysis by cation exchange chromatography (CEX). Aiming to maximise the information obtained from minimal sample and time required for analysis, a salt-based separation with UV detection was developed for supporting the processing of a large number of samples to facilitate high-throughput process development (HTPD). In addition, a mass spectrometry (MS) compatible SEC-CEX separation was developed enabling online charge variant peak identification. This study presented the ability to multiplex mAb size and charge variants analysis by coupling SEC with CEX in a 2D-LC set-up. To date, this is the first 2D SEC-CEX-UV(-MS) application for intact mAb analysis.

Pyrolysis-GC–MS analysis of ingested polystyrene microsphere content in individual Daphnia magna

Microplastic (MP) pollution in the aquatic environment is a cause for increasing concern. However, analyzing MPs ingested by small organisms, such as zooplankton, is difficult because of the low content and small size of the ingested MPs. We attempted to determine the content of ingested MPs in individual zooplankton using pyrolysis–gas chromatography–mass spectrometry (Py-GC–MS). To establish zooplankton model of MP ingestion, individual Daphnia magna were cultivated separately in microplate cells with polystyrene (PS) microspheres (10 μm in diameter, 245,000 particles, 135 μg) under different conditions. To prepare calibration curves for determining ingested PS content, approximately 100–150 μg of commercially available Daphnia-based powdered fish food, roughly corresponding to the weight of a single D. magna organism, was mixed with PS microspheres (0.005–26 μg) and analyzed using Py-GC–MS at 600 °C. In the resulting pyrograms, peaks of the styrene monomer and trimer from PS were detected, and linear relationships were obtained between the relative peak area and the amount of added PS. Finally, the cultivated zooplankton were individually subjected to Py-GC–MS analysis, and the ingested PS content in each zooplankton was successfully determined. Individual zooplankton cultured with PS in the absence of food ingested 2.3–7.9 μg of PS particles, whereas that in the presence of food (Chlorella vulgaris) ingested only 0.1–0.2 μg of PS particles. This result suggests that zooplankton might preferentially ingest ordinary food when both food and MPs are present, although further systematic studies are necessary to validate this observation.

Supramolecular Polymerization Powered by Escherichia coli : Fabricating a Near-Infrared Photothermal Antibacterial Agent in Situ

Supramolecular Polymerization Powered by <i>Escherichia coli</i> : Fabricating a Near-Infrared Photothermal Antibacterial Agent in Situ

Isolation, physicochemical, and structure–function relationship of the hydrophobic variant of Fc-fusion proteins that bind to TNF-α receptor, HS002 and HS002A

HS002 is the recombinant human tumor necrosis factor-α receptor Ⅱ: IgG Fc fusion protein licensed in China to treat rheumatism and psoriasis. The aim of this study was to isolate and characterize the hydrophobic freeze-dried powder injection (HS002) and ampoule injection (HS002A) variants derived from proteins of the same sequence and then to explore the structure–function relationship. Extensive physicochemical and structural testing was performed during a side-by-side comparison of the monomer peak and variant. Then the TNF-α-related binding activity, cell biological activity and affinity with FcRn were analyzed. Finally, a transformation study of the hydrophobic variant was performed under serum-like redox conditions. This research revealed that HS002A has similar physicochemical and structure–function relationship profiles to those of HS002. The hydrophobic variant exhibited the presence of new incorrect disulfide bridging. At the same time, this novel disulfide scrambled species structure–function relationship was found to be the molecular basis for reduced TNF-α binding and cell biological activities. In addition, incorrect disulfide bridging was found to be reversible under serum-like redox conditions, restoring TNF-α binding and cell biological activities to almost normal levels, all of which indicate that the variant is probably irrelevant to clinical efficacy once the drug enters the bloodstream.

Characteristics of Dental Resin-Based Composites in Leukemia Saliva: An In Vitro Analysis.

Background: The aim was to analyze, in vitro, four resin based composite systems (RBCs) immersed in saliva of leukemia patients before starting chemotherapy regiments. Material and methods: Saliva was collected from 20 patients (4 healthy patients, 16 leukemia patients). Resin disks were made for each RBC and were immersed in the acute leukemia (acute lymphocytic (ALL), acute myeloid (AML)), chronic leukemia (chronic lymphocytic (CLL), chronic myeloid (CML)), Artificial saliva and Control environment, and maintained for seven days. At the end of the experiment, the characteristics and the effective response of saliva from the studied salivas’ on RBCs was assessed using water sorption, water solubility, residual monomer and scanning electron microscopy (SEM). Data analysis was performed and a p-value under 0.05 was considered statistically significant. Results: The behaviour of RBCs in different immersion environments varies according to the characteristics of the RBCs. RBCs with a higher filler ratio have a lower water sorption. The solubility is also deteriorated by the types of organic matrix and filler; the results of solubility being inversely proportional on the scale of negative values compared to sorption values. Chromatograms of residual monomers showed the highest amount of unreacted monomers in ALL and AML, and the Control and artificial saliva environments had the smallest residual monomer peaks. Because of the low number of differences between the experimental conditions, we further considered that there were no important statistical differences between experimental conditions and analysed them as a single group. Conclusion: The influence of saliva on RBCs depends on the type of leukemia; acute leukemia influenced the most RBCs by changing their properties compared to chronic leukemia.

Spectrophotometric study of the sodium dodecyl sulfate in the presence of methylene blue in the methanol–water mixed solvent system

The methylene blue (MB) dye is very sensitive to changes in the polarity of its surroundings. The interaction of MB with sodium dodecyl sulfate (SDS) was studied spectrophotometrically in 0.1, 0.2, 0.3 and 0.4 vol fractions of methanol in an aqueous medium. The dielectric constant value decreases with the addition of methanol in the water and hence there is the lower stabilization of the lowest unoccupied molecular orbital which results in the hypsochromic shift in the monomeric form of methylene blue. In a 0.1 vol fraction of methanol, the peaks are seen at 665 and 612 nm, which are monomeric and dimeric MB peaks, respectively. The dimerization of MB is reduced with the increasing volume fraction of methanol in the solvent and ceases at/above 0.3 vol fraction of methanol. In the 0.4 vol fraction of methanol, the turbidity appears with the addition of SDS and there were no significant changes in the absorbance. In the presence of methanol, MB prefers to stay with the sulfate groups of SDS at the peripheral regions of the premicelle and micelle. At very low surfactant concentrations, far below their critical micelle concentration (CMC), the formation of MB-MB dimers and/or MB-SDS ion pairs or clusters occurs, and a corresponding shift in absorption and decrease in absorbance is observed. On increasing the concentration of SDS, micellization occurs, which disrupts the MB-MB dimers and MB-SDS ion pairs are incorporated with the micelles. On further addition of SDS, lone MB monomers become associated with these micelles and the absorbance increases slightly higher than those of the original dye in the solvent. This is the case of dye solubilization where monomers are completely incorporated or bounded by micelles. The CMC of SDS decreases in the presence of dye suggesting the strong interaction between the oppositely charged dye-surfactant ion pairs. The value of binding constant and partition coefficient decreased with the increasing volume fraction of methanol. The negative value of ΔGb indicates that the interaction between SDS and MB is spontaneous but the negative values of ΔGb decreased with the increasing volume fraction of methanol.

Electrostatic spray drying for monoclonal antibody formulation

This study explored the feasibility of electrostatic spray drying for producing a monoclonal antibody (mAb) powder formulation at lower drying temperatures than conventional spray drying and its effect on protein stability. A mAb formulation was dried by either conventional spray drying or electrostatic spray drying with charge (ESD). The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), size exclusion chromatography (SEC), and solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). Particle characterizations such as BET surface area, particle size distribution, and particle morphology were also performed. Conventional spray drying of the mAb formulation at the inlet temperature of 70 °C failed to generate dry powders due to poor drying efficiency; electrostatic spray drying at the same temperature and 5 kV charge enabled the formation of powder formulation with satisfactory moisture contents. Deconvoluted peak areas of deuterated samples from the ssHDX-MS study showed a good correlation with the loss of the monomeric peak area measured by size exclusion chromatography in the 90-day accelerated stability study conducted at 40 °C. Low-temperature (70 °C inlet temperature) drying with an electrostatic charge (5 kV) led to better protein physical stability as compared with the samples spray-dried at the high temperature (130 °C inlet temperature) without charge. This study shows that electrostatic spray drying can produce solid monoclonal antibody formulation at lower inlet temperature than traditional spray drying with better physical stability.