A Hemoglobinopathy That Produces an Array of Different Hemoglobin A1c Values.

Hemoglobin A1c (HbA1c) refers to non-enzymatically glycated hemoglobin and reflects the patient's glycemic status over approximately 3 months. An elevated HbA1c over 6.5% National Glycohemoglobin Standardization Program (NGSP) (48 mmol/mol the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)) can be used to diagnose diabetes mellitus. In our laboratory, HbA1c is determined by ion-exchange chromatography which has the advantage of detecting common Hb variants such as Hb S, C, E and D without adversely affecting the HbA1c determination. Certain homozygous or compound heterozygous hemoglobinopathies such as homozygous sickle disease and Hb SC disease can significantly lower the HbA1c by reducing red cell lifespan. Occasionally however, rare and mostly benign hemoglobinopathies can interfere with this technique resulting in an apparent elevation of HbA1c in an otherwise non-diabetic patient. In this report, we describe such a hemoglobinopathy termed Hb Wayne that resulted in a significant HbA1c elevation in a normoglycemic individual. HbA1c was determined by multiple methods including immunoassay, a modified capillary electrophoresis and an alternative ion-exchange system. These techniques yielded significantly lower A1c results, more in keeping with the patient's clinical background. The alternative ion-exchange system resulted in a low A1c that was qualified by warning flags on the chromatogram that indicated the result was not reportable. The hemoglobinopathy in question, Hb Wayne, is a frameshift mutation in the alpha globin gene that results in an extended alpha globin polypeptide that can form two variants Hb Wayne I and Wayne II. Hb Wayne is a clinically silent asymptomatic disorder with no hematologic consequences. The artifactual elevation of HbA1c is, in contrast, very significant because it may result in a misdiagnosis of diabetes mellitus leading to unnecessary treatment. In this report, we compare our findings with other descriptions of Hb Wayne in the literature and corroborate a number of previous observations and conclusions.

Real-time monitoring of gradient chromatography using dual Kalman-filters.

Real-time state estimation in chromatography is a useful tool to improve monitoring of biopharmaceutical downstream processes, combining mechanistic model predictions with real-time data acquisition to obtain an estimation that surpasses that of either approach individually. One common technique for real-time state estimation is Kalman filtering. However, non-linear adsorption isotherms pose a significant challenge to Kalman filters, which are dependent on fast algorithm execution to function. In this work, we apply Kalman filtering of non-constant elution conditions using a non-linear adsorption isotherm using a novel approach where dual Kalman filters are used to estimate the states of the adsorption modifier, salt, and the components to be separated. We performed offline tuning of the Kalman filters on real chromatogram data from a linear gradient, ion-exchange separation of two proteins. The tuning was then validated by running the Kalman filters in parallel with a chromatographic separation in real time. The resulting, tuned, dual Kalman filters improved the L2 norm by 53 % over the open-loop model prediction, when compared to the true elution profiles. The Kalman filters were also applicable in real-time with a signal sampling frequency of 5 s, enabling accurate and robust estimation and paving the way for future applications beyond monitoring, such as real-time optimal pooling control.

[Applications of high performance liquid chromatography-mass spectrometry in proteomics].

Proteomics profiling plays an important role in biomedical studies. Proteomics studies are much more complicated than genome research, mainly because of the complexity and diversity of proteomic samples. High performance liquid chromatography-mass spectrometry (HPLC-MS) is a fundamental tool in proteomics research owing to its high speed, resolution, and sensitivity. Proteomics research targets from the peptides and individual proteins to larger protein complexes, the molecular weight of which gradually increases, leading to sustained increases in structural and compositional complexity and alterations in molecular properties. Therefore, the selection of various separation strategies and stationary-phase parameters is crucial when dealing with the different targets in proteomics research for in-depth proteomics analysis. This article provides an overview of commonly used chromatographic-separation strategies in the laboratory, including reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), hydrophobic interaction chromatography (HIC), ion-exchange chromatography (IEC), and size-exclusion chromatography (SEC), as well as their applications and selectivity in the context of various biomacromolecules. At present, no single chromatographic or electrophoretic technology features the peak capacity required to resolve such complex mixtures into individual components. Multidimensional liquid chromatography (MDLC), which combines different orthogonal separation modes with MS, plays an important role in proteomics research. In the MDLC strategy, IEC, together with RPLC, remains the most widely used separation mode in proteomics analysis; other chromatographic methods are also frequently used for peptide/protein fractionation. MDLC technologies and their applications in a variety of proteomics analyses have undergone great development. Two strategies in MDLC separation systems are mainly used in proteomics profiling: the "bottom-up" approach and the "top-down" approach. The "shotgun" method is a typical "bottom-up" strategy that is based on the RPLC or MDLC separation of whole-protein-sample digests coupled with MS; it is an excellent technique for identifying a large number of proteins. "Top-down" analysis is based on the separation of intact proteins and provides their detailed molecular information; thus, this technique may be advantageous for analyzing the post-translational modifications (PTMs) of proteins. In this paper, the "bottom-up" "top-down" and protein-protein interaction (PPI) analyses of proteome samples are briefly reviewed. The diverse combinations of different chromatographic modes used to set up MDLC systems are described, and compatibility issues between mobile phases and analytes, between mobile phases and MS, and between mobile phases in different separation modes in multidimensional chromatography are analyzed. Novel developments in MDLC techniques, such as high-abundance protein depletion and chromatography arrays, are further discussed. In this review, the solutions proposed by researchers when encountering compatibility issues are emphasized. Moreover, the applications of HPLC-MS combined with various sample pretreatment methods in the study of exosomal and single-cell proteomics are examined. During exosome isolation, the combined use of ultracentrifugation and SEC can yield exosomes of higher purity. The use of SEC with ultra-large-pore-size packing materials (200 nm) enables the isolation of exosomal subgroups, and proteomics studies have revealed significant differences in protein composition and function between these subgroups. In the field of single-cell proteomics, researchers have addressed challenges related to reducing sample processing volumes, preventing sample loss, and avoiding contamination during sample preparation. Innovative methods and improvements, such as the utilization of capillaries for sample processing and microchips as platforms to minimize the contact area of the droplets, have been proposed. The integration of these techniques with HPLC-MS shows some progress. In summary, this article focuses on the recent advances in HPLC-MS technology for proteomics analysis and provides a comprehensive reference for future research in the field of proteomics.

A combination of multiple LC-MS approaches for the comprehensive characterization of cysteine-linked ADCs.

Antibody-drug conjugates (ADCs) represent the forefront of the next generation of biopharmaceuticals. An ADC typically comprises an antibody covalently linked to a cytotoxic drug via a linker, resulting in a highly heterogeneous product. This study focuses on the analysis of a custom-made cysteine-linked ADC. Initially, we developed a LC-MS-based characterization workflow using brentuximab vedotin (Adcetris®), encompassing native intact MS, analysis of reduced chains and subunits under denaturing condition, peptide mapping and online strong cation exchange chromatography coupled with UV and mass spectrometry detection (SCX-UV-MS) applied for brentuximab vedotin first time reported. Subsequently, we applied this in-depth characterization workflow to a custom-made cysteine-linked ADC. The measured drug-to-antibody ratio(DAR) of this ADC is 6.9, further analysis shown that there is a small amount of unexpected over-conjugation. Over-conjugation sites were successfully identified using multiple UHPLC-MS based characterization techniques. Also, one competitively cysteine-conjugated impurity was observed in native intact MS results, by combing native intact MS, reduced chains, subunit analysis and peptide mapping results, the impurity conjugation sites were also identified. Since this molecule is at early development stage, this provides important information for conjugation process improvement and link-drug material purification. SCX-UV-MS approach can separate the custom-made cysteine-linked ADC carrying different payloads and reduce the complexity of the spectra. The integrated approach underscores the significance of combining the SCX-UV-MS online coupling technique with other characterization methods to elucidate the heterogeneity of cysteine-linked ADCs.

Proteomics-based method to comprehensively model the removal of host cell protein impurities.

Mechanistic models mostly focus on the target protein and some selected process- or product-related impurities. For a better process understanding, however, it is advantageous to describe also reoccurring host cell protein impurities. Within the purification of biopharmaceuticals, the binding of host cell proteins to a chromatographic resin is far from being described comprehensively. For a broader coverage of the binding characteristics, large-scale proteomic data and systems level knowledge on protein interactions are key. However, a method for determining binding parameters of the entire host cell proteome to selected chromatography resins is still lacking. In this work, we have developed a method to determine binding parameters of all detected individual host cell proteins in an Escherichia coli harvest sample from large-scale proteomics experiments. The developed method was demonstrated to model abundant and problematic proteins, which are crucial impurities to be removed. For these 15 proteins covering varying concentration ranges, the model predicts the independently measured retention time during the validation gradient well. Finally, we optimized the anion exchange chromatography capture step in silico using the determined isotherm parameters of the persistent host cell protein contaminants. From these results, strategies can be developed to separate abundant and problematic impurities from the target antigen.

Preparation and Purification of ß-1,3-glucan-Linked Candida glabrata Cell Wall Proteases by Ion-Exchange Chromatography, Gel Filtration, and MDPF-Gelatin-Zymography Assay.

Candida glabrata is an opportunistic pathogen that may cause serious infections in an immunocompromised host. C. glabrata cell wall proteases directly interact with host cells and affect yeast virulence and host immune responses. This protocol describes methods to purify ß-1,3-glucan-bonded cell wall proteases from C. glabrata. These cell wall proteases are detached from the cell wall glucan network by lyticase treatment, which hydrolyzes ß-1,3-glucan bonds specifically without rupturing cells. The cell wall supernatant is further fractioned by centrifugal devices with cut-offs of 10 and 50 kDa, ion-exchange filtration (charge), and gel filtration (size exclusion). The enzymatic activity of C. glabrata proteases is verified with MDPF-gelatin zymography and the degradation of gelatin is visualized by loss of gelatin fluorescence. With this procedure, the enzymatic activities of the fractions are kept intact, differing from methods used in previous studies with trypsin digestion of the yeast cell wall. The protein bands may be eventually located from a parallel silver-stained gel and identified with LC-MS/MS spectrometry. The advantage of this methodology is that it allows further host protein degradation assays; the protocol is also suitable for studying other Candida yeast species. Key features • Uses basic materials and laboratory equipment, enabling low-cost studies. • Facilitates the selection and identification of proteases with certain molecular weights. • Enables further functional studies with host proteins, such as structural or immune response-related, or enzymes and candidate protease inhibitors (e.g., from natural substances). • This protocol has been optimized for C. glabrata but may be applied with modifications to other Candida species.

Efficient experimental method for purifying allergens from aqueous extracts.

Studying the molecular and immunological basis of allergic diseases often requires purified native allergens. The methodologies for protein purification are usually difficult and may not be completely successful. The objective of this work was to describe a methodology to purify allergens from their natural source, while maintaining their native form. The purification strategy consists of a three-step protocol and was used for purifying five specific allergens, Ole e 1, Amb a 1, Alt a 1, Bet v 1 and Cup a 1. Total proteins were extracted in PBS (pH 7.2). Then, the target allergens were pre-purified and enriched by salting-out using increasing concentrations of ammonium sulfate. The allergens were further purified by anion exchange chromatography. Purification of Amb a 1 required an extra step of cation exchange chromatography. The detection of the allergens in the fractions obtained were screened by SDS-PAGE, and Western blot when needed. Further characterization of purified Amb a 1 was performed by mass spectrometry. Ole e 1, Alt a 1, Bet v 1 and Cup a 1 were obtained at > 90 % purity. Amb a 1 was obtained at > 85 % purity. Overall, we propose an easy-to-perform purification approach that allows obtaining highly pure allergens. Since it does not involve neither chaotropic nor organic reagents, we anticipate that the structural and biological functions of the purified molecule remain intact. This method provides a basis for native allergen purification that can be tailored according to specific needs.

Online Mixed-Bed Ion Exchange Chromatography for Native Top-Down Proteomics of Complex Mixtures.

Native top-down mass spectrometry (nTDMS) allows characterization of protein structure and noncovalent interactions with simultaneous sequence mapping and proteoform characterization. The majority of nTDMS studies utilize purified recombinant proteins, with significant challenges hindering application to endogenous systems. To perform native top-down proteomics (nTDP), where endogenous proteins from complex biological systems are analyzed by nTDMS, it is essential to separate proteins under nondenaturing conditions. However, it remains difficult to achieve high resolution with MS-compatible online chromatography while preserving protein tertiary structure and noncovalent interactions. Herein, we report the use of online mixed-bed ion exchange chromatography (IEC) to enable separation of endogenous proteins from complex mixtures under nondenaturing conditions, preserving noncovalent interactions for nTDP analysis. We have successfully detected large proteins (>146 kDa) and identified endogenous metal-binding and oligomeric protein complexes in human heart tissue lysate. The use of a mixed-bed stationary phase allowed retention and elution of proteins over a wide range of isoelectric points without altering the sample or mobile phase pH. Overall, our method provides a simple online IEC-MS platform that can effectively separate proteins from complex mixtures under nondenaturing conditions and preserve higher-order structure for nTDP applications.

Valorization of agro-industrial residues using Aspergillus heteromorphus URM0269 for protease production: Characterization and purification.

This study aimed to produce, characterize and purify a protease from Aspergillus heteromorphus URM0269. After production by solid fermentation of wheat bran performed according to a central composite design, protease was characterized in terms of biochemical, kinetic, and thermodynamic parameters for further purification by chromatography. Proteolytic activity achieved a maximum value of 57.43 U/mL using 7.8 g of wheat bran with 40 % moisture. Protease displayed high stability in the pH and temperature ranges of 5.0-10.0 and 20-30 °C, respectively, and acted optimally at pH 7.0 and 50 °C. The enzyme, characterized as a serine protease, followed Michaelis-Menten kinetics with a maximum reaction rate of 140.0 U/mL and Michaelis constant of 11.6 mg/mL. Thermodynamic activation parameters, namely activation Gibbs free energy (69.79 kJ/mol), enthalpy (5.86 kJ/mol), and entropy (-214.39 J/mol.K) of the hydrolysis reaction, corroborated with kinetic modeling showing high affinity for azocasein. However, thermodynamic parameters suggested a reversible mechanism of unfolding. Purification by chromatography yielded a protease purification factor of 7.2, and SDS-PAGE revealed one protein band with a molecular mass of 14.7 kDa. Circular dichroism demonstrated a secondary structure made up of 45.6 % α-helices. These results show the great potential of this protease for future use in the industrial area.

A Single-Step Method for Harvesting Influenza Viral Particles from MDCK Cell Culture Supernatant with High Yield and Effective Impurity Removal.

Influenza vaccines, which are recommended by the World Health Organization (WHO), are the most effective preventive measure against influenza virus infection. Madin-Darby canine kidney (MDCK) cell culture is an emerging technology used to produce influenza vaccines. One challenge when purifying influenza vaccines using this cell culture system is to efficiently remove impurities, especially host cell double-stranded DNA (dsDNA) and host cell proteins (HCPs), for safety assurance. In this study, we optimized ion-exchange chromatography methods to harvest influenza viruses from an MDCK cell culture broth, the first step in influenza vaccine purification. Bind/elute was chosen as the mode of operation for simplicity. The anion-exchange Q chromatography method was able to efficiently remove dsDNA and HCPs, but the recovery rate for influenza viruses was low. However, the cation-exchange SP process was able to simultaneously achieve high dsDNA and HCP removal and high influenza virus recovery. For the SP process to work, the clarified cell culture broth needed to be diluted to reduce its ionic strength, and the optimal dilution rate was determined to be 1:2 with purified water. The SP process yielded a virus recovery rate exceeding 90%, as measured using a hemagglutination units (HAUs) assay, with removal efficiencies over 97% for HCPs and over 99% for dsDNA. Furthermore, the general applicability of the SP chromatography method was demonstrated with seven strains of influenza viruses recommended for seasonal influenza vaccine production, including H1N1, H3N2, B (Victoria), and B (Yamagata) strains, indicating that the SP process could be utilized as a platform process. The SP process developed in this study showed four advantages: (1) simple operation, (2) a high recovery rate for influenza viruses, (3) a high removal rate for major impurities, and (4) general applicability.

Pore-blocking steric mass-action model for adsorption of bioparticles.

The steric mass-action (SMA) model has been widely reported to describe the adsorption of proteins in different types of chromatographic adsorbents. Here in the present work, a pore-blocking steric mass-action model (PB-SMA) was developed for the adsorption of large-size bioparticles, which usually exhibit the unique pore-blocking characteristic on the adsorbent and thus lead to a fraction of ligands in the deep channels physically inaccessible to bioparticles adsorption, instead of being shielded due to steric hindrance by adsorbed bioparticles. This unique phenomenon was taken into account by introducing an additional parameter, Lin, which is defined as the inaccessible ligand densities in the physically blocked pore area, into the PB-SMA model. This fraction of ligand densities (Lin) will be deducted from the total ligand (Lt) for model development, thus the steric factor (σ) in the proposed PB-SMA will reflect the steric shielding effect on binding sites by adsorbed bioparticles more accurately than the conventional SMA model, which assumes that all ligands on the adsorbent have the same accessibility to the bioparticles. Based on a series of model assumptions, a PB-SMA model was firstly developed for inactivated foot-and-mouth disease virus (iFMDV) adsorption on immobilized metal affinity chromatography (IMAC) adsorbents. Model parameters for static adsorption including equilibrium constant (K), characteristic number of binding sites (n), and steric factor (σ) were determined. Compared with those derived from the conventional SMA model, the σ values derived from the PB-SMA model were dozens of times smaller and much closer to the theoretical maximum number of ligands shielded by a single adsorbed iFMDV, indicating the modified model was more accurate for bioparticles adsorption. The applicability of the PB-SMA model was further validated by the adsorption of hepatitis B surface antigen virus-like particles (HBsAg VLPs) on an ion exchange adsorbent with reasonably improved accuracy. Thus, it is considered that the PB-SMA model would be more accurate in describing the adsorption of bioparticles on different types of chromatographic adsorbents.

Liquid phase detection in the miniature scale. Microfluidic and capillary scale measurement and separation systems. A tutorial review.

Microfluidic and capillary devices are increasingly being used in analytical applications while their overall size keeps decreasing. Detection sensitivity for these microdevices gains more importance as device sizes and consequently, sample volumes, decrease. This paper reviews optical, electrochemical, electrical, and mass spectrometric detection methods that are applicable to capillary scale and microfluidic devices, with brief introduction to the principles in each case. Much of this is considered in the context of separations. We do consider theoretical aspects of separations by open tubular liquid chromatography, arguably the most potentially fertile area of separations that has been left fallow largely because of lack of scale-appropriate detection methods. We also examine the theoretical basis of zone electrophoretic separations. Optical detection methods discussed include UV/Vis absorbance, fluorescence, chemiluminescence and refractometry. Amperometry is essentially the only electrochemical detection method used in microsystems. Suppressed conductance and especially contactless conductivity (admittance) detection are in wide use for the detection of ionic analytes. Microfluidic devices, integrated to various mass spectrometers, including ESI-MS, APCI-MS, and MALDI-MS are discussed. We consider the advantages and disadvantages of each detection method and compare the best reported limits of detection in as uniform a format as the available information allows. While this review pays more attention to recent developments, our primary focus has been on the novelty and ingenuity of the approach, regardless of when it was first proposed, as long as it can be potentially relevant to miniature platforms.

Simultaneous separation and detection of monochloramine, nitrite, and nitrate by step-gradient mixed-mode ion chromatography: Translation from benchtop to portable ion chromatograph.

BACKGROUND: Nitrite (NO2-) and nitrate (NO3-) can be produced in the distribution systems of chloraminated drinking water due to the nitrification of ammonia. The most applied inorganic chloramine for this purpose, namely monochloramine (NH2Cl), is also released into aquatic environments from water treatment plants' effluent and within industrial waste streams. Within the treatment process, the continuous monitoring of disinfectant levels is necessary to limit the harmful disinfectant by-product (DBP) formation. Currently, NH2Cl can interfere with nutrient analysis in water samples, and there are no analytical techniques available for the simultaneous analysis of NH2Cl, NO2-, and NO3-. RESULTS: A green analytical method based on mixed-mode ion chromatography, specifically ion exchange and ion exclusion modes, was developed for the simultaneous separation and detection of NH2Cl, NO2-, and NO3-. The separation was achieved using a Dionex IonPac AG15 column guard column and a step gradient elution involving deionized water and 120.0 mM NaCl. The method was developed using a benchtop HPLC with a custom-made multi-wavelength UV absorbance detector with a 50-mm flow cell to enable the sensitive detection of NH2Cl, NO2-, and NO3- at 240 nm, 220 nm, and 215 nm, respectively. The developed method was then transferred to a portable ion chromatography (IC) system, the Aquamonitrix analyser. The total run time was less than 10 min for both systems. The benchtop HPLC method had a limit of detection (LOD) of 0.07 µg mL-1 as Cl2 for NH2Cl, 0.01 µg mL-1 for NO2-, and 0.03 µg mL-1 for NO3-. The LODs obtained using the portable Aquamonitrix analyser were found to be 0.36 µg mL-1 as Cl2, 0.02 µg mL-1, and 0.11 µg mL-1 for NH2Cl, NO2-, and NO3-, respectively. Excellent linearity (r ≥ 0.9999) was achieved using the portable analyser over the studied concentration ranges. The developed system was applied to the analysis of spiked municipal drinking water samples and showed excellent repeatability for the three analytes at three different concentration levels (RSD of triplicate recovery experiments ≤ 1.9 %). Moreover, the variation in retention time was negligible for the three target analytes with RSD ≤ 0.8 % over 12 runs. SIGNIFICANCE: We are reporting the first ion chromatographic method for the simultaneous separation and detection of NH2Cl, NO2-, and NO3- in water samples. The monitoring of NH2Cl, NO2-, and NO3- is critical for the determination of disinfectant dosing, water quality, and nitrification status. The developed method can be applied using a benchtop HPLC or via the portable automated IC system to monitor for the three target compounds analysis in water treatment plants.

Profiling Protein-Protein Interactions in the Human Brain by Refined Cofractionation Mass Spectrometry.

Proteins usually execute their biological functions through interactions with other proteins and by forming macromolecular complexes, but global profiling of protein complexes directly from human tissue samples has been limited. In this study, we utilized cofractionation mass spectrometry (CF-MS) to map protein complexes within the postmortem human brain with experimental replicates. First, we used concatenated anion and cation Ion Exchange Chromatography (IEX) to separate native protein complexes in 192 fractions and then proceeded with Data-Independent Acquisition (DIA) mass spectrometry to analyze the proteins in each fraction, quantifying a total of 4,804 proteins with 3,260 overlapping in both replicates. We improved the DIA's quantitative accuracy by implementing a constant amount of bovine serum albumin (BSA) in each fraction as an internal standard. Next, advanced computational pipelines, which integrate both a database-based complex analysis and an unbiased protein-protein interaction (PPI) search, were applied to identify protein complexes and construct protein-protein interaction networks in the human brain. Our study led to the identification of 486 protein complexes and 10054 binary protein-protein interactions, which represents the first global profiling of human brain PPIs using CF-MS. Overall, this study offers a resource and tool for a wide range of human brain research, including the identification of disease-specific protein complexes in the future.

Vanadium Accumulation and Reduction by Vanadium-Accumulating Bacteria Isolated from the Intestinal Contents of Ciona robusta.

The sea squirt Ciona robusta (formerly Ciona intestinalis type A) has been the subject of many interdisciplinary studies. Known as a vanadium-rich ascidian, C. robusta is an ideal model for exploring microbes associated with the ascidian and the roles of these microbes in vanadium accumulation and reduction. In this study, we discovered two bacterial strains that accumulate large amounts of vanadium, CD2-88 and CD2-102, which belong to the genera Pseudoalteromonas and Vibrio, respectively. The growth medium composition impacted vanadium uptake. Furthermore, pH was also an important factor in the accumulation and localization of vanadium. Most of the vanadium(V) accumulated by these bacteria was converted to less toxic vanadium(IV). Our results provide insights into vanadium accumulation and reduction by bacteria isolated from the ascidian C. robusta to further study the relations between ascidians and microbes and their possible applications for bioremediation or biomineralization.

Mass spectrometry study on SARS-CoV-2 recombinant vaccine with comprehensive separation techniques to characterize complex heterogeneity.

SARS-CoV-2, the causative agent of COVID-19, has imposed a major public health threat, which needs effective therapeutics and vaccination strategies. Several potential candidate vaccines being rapidly developed are in clinical evaluation and recombinant vaccine has gained much attention thanks to its potential for greater response predictability, improved efficacy, rapid development and reduced side effects. Recombinant vaccines are designed and manufactured using bacterial, yeast cells or mammalian cells. A small piece of DNA is taken from the virus or bacterium against which we want to protect and inserted into the manufacturing cells. Due to the extremely complex heterogeneity of SARS-CoV-2 recombinant vaccine, single technology platform cannot achieve thorough and accurate characterization of such difficult proteins so integrating comprehensive technologies is essential. This study illustrates an innovative workflow employing multiple separation techniques tandem high-resolution mass spectrometry for comprehensive and in-depth characterization of SARS-CoV-2 recombinant vaccine, including ultra-high performance liquid chromatography (UHPLC), ion exchange chromatography (IEX) and imaged capillary isoelectric focusing (icIEF). The integrated methodology focuses on the importance of cutting-edge icIEF-MS online coupling and icIEF fractionation applied to revealing the heterogeneity secret of SARS-CoV-2 recombinant vaccine.

Standardized approach for accurate and reliable model development of ion-exchange chromatography based on parameter-by-parameter method and consideration of extra-column effects.

Developing an accurate and reliable model for chromatographic separation that meets regulatory requirements and ensures consistency in model development remains challenging. In order to address this challenge, a standardized approach was proposed in this study with ion-exchange chromatography (IEC). The approach includes the following steps: liquid flow identification, system and column-specific parameters determination and validation, multi-component system identification, protein amount validation, steric mass action parameters determination and evaluation, and validation of the calibrated model's generalization ability. The parameter-by-parameter (PbP) calibration method and the consideration of extra-column effects were integrated to enhance the accuracy of the developed models. The experiments designed for implementing the PbP method (five gradient experiments for model calibration and one stepwise experiment for model validation) not only streamline the experimental workload but also ensure the extrapolation abilities of the model. The effectiveness of the standardized approach is successfully validated through an application about the IEC separation of industrial antibody variants, and satisfactory results were observed with R2 ≈ 0.9 for the majority of calibration and validation experiments. The standardized approach proposed in this work contributes significantly to improve the accuracy and reliability of the developed IEC models. Models developed using this standardized approach are ready to be applied to a broader range of industrial separation systems, and are likely find further applications in model-assisted decision-making of process development.

Development of a method for the simultaneous determination of ionic nutrients in hydroponic solutions using cation-/anion-exchange chromatography with a neutral eluent.

Nutrient availability in hydroponic solutions must be accurately monitored to maintain crop productivity; however, few cost-effective, accurate, real-time, and long-term monitoring technologies have been developed. In this study, we describe the development and application of cation-/anion-exchange chromatography with a neutral eluent (20-mmol/L sodium formate, pH 7.87) for the simultaneous separation (within 50 min) of ionic nutrients, including K+, NH4+, NO2-, NO3-, and phosphate ion, in a hydroponic fertilizer solution. Using the neutral eluent avoided degradation of the separation column during precipitation of metal ion species, such as hydroxides, with an alkaline eluent and oxidation of NO2- to NO3- with an acidic eluent. The suitability of the current method for monitoring ionic components in a hydroponic fertilizer solution was confirmed. Based on our data, we propose a controlled fertilizer strategy to optimize fertilizer consumption and reduce the chemical load of drained fertilizer solutions.

Biochemical evaluation and ligand binding studies on glycerophosphodiester phosphodiesterase from Staphylococcus aureus using STD-NMR spectroscopy and molecular docking analysis.

Glycerophosphodiester phosphodiesterase (GDPD) is a highly conserved enzyme in both prokaryotic and eukaryotic organisms. It catalyses the hydrolysis of various glycerophosphodiesters into glycerol-3-phosphate and corresponding alcohols, which serve as building blocks in several biosynthetic pathways. This enzyme is a well-known virulence factor in many pathogenic bacteria, including Staphylococcus aureus, and is thus considered a potential drug target. In this study, competent E. coli BL21(DE3)pLysS expression cells were used to express the GDPD enzyme from vancomycin-resistant Staphylococcus aureus (VRSA), which was then purified using size exclusion and anion exchange chromatography. The hydrolytic activity of GDPD was evaluated on the non-physiological substrate bis(p-nitrophenyl) phosphate (BpNPP), which indicated functional activity of the enzyme. 79 drugs were evaluated for their inhibitory potential against GDPD enzyme by the colorimetric assay. Out of 79 drugs, 13 drugs, including tenofovir (1), adenosine (2), clioquinol (11), bromazepam (12), lamotrigine (13), sulfadiazine (14), azathioprine (15), nicotine (16), sitagliptin PO4 (17), doxofylline (18), clindamycin phosphate (19), gentamycin sulphate (20), and ceftriaxone sodium (21) revealed varying degrees of inhibitory potential with IC50 values in the range of 400 ± 0.007-951 ± 0.016 µM. All drugs were also evaluated for their binding interactions with the target enzyme by saturation transfer difference (STD-NMR) spectroscopy. 10 drugs demonstrated STD interactions and hence, showed binding affinity with the enzyme. Exceptionally, tenofovir (1) was identified to be a better inhibitor with an IC50 value of 400 ± 0.007 µM, as compared to the standard EDTA (ethylenediaminetetraacetic acid) (IC50 = 470 ± 0.008 µM). Moreover, molecular docking studies have identified key interactions of the ligand (tenofovir) with the binding site residues of the enzyme.

Rational downstream development for adeno-associated virus full/empty capsid separation - A streamlined methodology based on high-throughput screening and mechanistic modeling.

Recombinant adeno-associated virus (AAV) has emerged as one of the most promising systems for therapeutic gene delivery and has demonstrated clinical success in a wide range of genetic disorders. However, manufacturing of high-quality AAV in large amounts still remains a challenge. A significant difficulty for downstream processing is the need to remove empty capsids that are generated in all currently utilized expression systems and that represent product-related impurities that adversely affect safety and efficacy of AAV vectors. Empty and full capsids exhibit only subtle differences in surface charge and size, making chromatography-based separations highly challenging. Here, we present a rapid methodology for the systematic process development of the crucial AAV full/empty capsid separation on ion-exchange media based on high-throughput screening and mechanistic modeling. Two of the most commonly employed serotypes, AAV8 and AAV9, are used as case studies. First, high-throughput studies in filter-plate format are performed that allow the rapid and comprehensive study of binding and elution behavior of AAV on different resins, using different buffer systems, pH, salt conditions, and solution additives. Small amounts of separated empty and full AAV capsids are generated by iodixanol gradient centrifugation that allow studying the binding and elution behavior of the two vector species separately in miniaturized format. Process conditions that result in maximum differences in elution behavior between empty and full capsids are then transferred to benchtop chromatography systems that are used to generate calibration data for the estimation of steric mass-action isotherm and mass transport parameters for process simulation. The resulting column models are employed for in-silico process development that serves to enhance understanding of separation constraints and to identify optimized conditions for the removal of empty particles. Finally, optimized separation conditions are verified experimentally. The methodology presented in this work provides a systematic framework that affords mechanistic understanding of the crucial empty/full capsid separation and accelerates the development of a scalable AAV downstream process.