Search Results (7)

Background/Objectives: Multi-dimensional liquid chromatography coupled with mass spectrometry (mD-LC-MS) has emerged as a powerful technique for the in-depth characterization of biopharmaceuticals by assessing chromatographically resolved product variants in a streamlined and semi-automated manner. The study aims to demystify and enhance the accessibility to this powerful but inherently complex technique by detailing a robust and user-friendly instrument platform, allowing analysts to switch seamlessly between intact, subunit, and peptide mapping workflows. Methods: Starting from a commercially available Two-Dimensional Liquid Chromatography (2D-LC) system, we introduce specific hardware and software extensions leading to two versatile mD-LC-MS setups, in slightly different configurations. The technique’s efficacy is demonstrated through a case study on a cation exchange chromatography method assessing the charge variants of a bispecific antibody, isolating peak(s) of interest, followed by online sample processing, including reduction and enzymatic digestion, and subsequently mass spectrometry analysis. Results: The accuracy and reproducibility of both mD-LC-MS setups proposed in this study were successfully tested. Despite the complex peak patterns in the first dimension, the systems were equally effective in identifying and quantifying the underlying product species. This case study highlights the routine usability of mD-LC-MS technology for the characterization of (ultra) high-performance liquid chromatography (UHPLC) of therapeutic biomolecule. Conclusions: The demonstrated reliability and accuracy underscore the practicality of mD-LC-MS for routine use in biopharmaceutical analysis. Our detailed description of the mD-LC-MS systems and insights simplify access to this advanced technology for a broader scientific community, regardless of expertise level, and lower the entry barrier for its use in various research and industrial settings. Full article

Immobilized enzyme reactors (IMERs) are critical tools for developing novel oligosaccharides based on the enzymatic catalysis of polysaccharides. In this paper, a novel glucuronan lyase from Peteryoungia rosettiformans was produced, purified, and then immobilized on a CIM^® CDI disk for cleaving glucuronan. The results showed that around 63.6% of glycuronan lyases (800.9 μg) were immobilized on the disk. The V_max values of immobilized glucuronan lyases did not significantly change (56.9 ± 4.7 μM∙min⁻¹), while the K_m values (0.310 ± 0.075 g∙L⁻¹) increased by 2.5 times. It is worth noting that immobilized glucuronan lyases overcame the catalytic inhibition of free enzymes observed under high glucuronan concentrations (0.5–2 g∙L⁻¹). circumscribed central composite design (CCCD) and response surface methodology (RSM) showed that glucuronan concentration, flow rate, and reaction time significantly affected the yield of oligoglucuronans. The degree of polymerization (DP) of degraded glucuronan ranged from DP 2–8 according to the results obtained by high performance anion exchange chromatography coupled with a pulsed amperometric detector (HPAEC-PAD). The IMER retained 50.9% activity after running 2373 column volumes of glucuronan. Finally, this glucuronan lyase reactor was tentatively connected to an immobilized laccase reactor to depolymerize, and gallic acid (GA) was added to glucuronan. Approximately 8.5 mg of GA was added onto 1 g of initial glucuronan, and the GA–oligoglucuronan conjugates showed notable antioxidant activity. Full article

The chemical synthesis of heterocycles typically requires elevated temperature and acid or base addition to form the desired product. Moreover, these reactions often involve hazardous reagents, which is why biocatalytic routes for heterocycle formation have gained increasing attention. In recent years, several enzymes belonging to the amidohydrolase superfamily have been identified to generate heterocycles via cyclocondensation reactions. Of particular interest is the amidohydrolase MxcM, which catalyzes the formation of an imidazoline moiety in the biosynthesis of the anti-inflammatory natural product pseudochelin A. In this study, we present a concept for the immobilization of this enzyme using a fused hexahistidine tag for fixation onto a solid, porous carrier. Notably, the immobilization improves the enzyme’s tolerance to organic solvents. The immobilized MxcM exhibits a residual activity of 169% in the polar solvent acetonitrile compared to the free enzyme, and the storage stability in the presence of 20 vol% acetonitrile was ameliorated. In addition, an immobilized enzyme reactor (IMER) was designed that can be operated under flow conditions. The MxcM-IMER retains its biocatalytic activity and mechanic stability over the tested operation time. These results provide important insights for the integration of heterocycle-forming amidohydrolases in chemical processes. Full article

Xanthine oxidase (XO) is an enzyme involved in the oxidative process of hypoxanthine and xanthine to uric acid (UA). This process also produces reactive oxygen species (ROS) as byproducts. Both UA and ROS are dangerous for human health, and some health conditions trigger upregulation of XO activity, which results in many diseases (cancer, atherosclerosis, hepatitis, gout, and others) given the worsened scenario of ROS and UA overproduction. So, XO became an attractive target to produce and discover novel selective drugs based on febuxostat, the most recent XO inhibitor out of only two approved by FDA. Under this context, high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) have been successfully applied to rapidly and easily screen for bioactive compounds, isolated or in complex natural matrixes, that act as enzyme inhibitors through the use of an immobilized enzyme reactor (IMER). This article’s goal is to present advances comprising febuxostat-based XO inhibitors as a new trend, bifunctional moieties capable of inhibiting XO and modulating ROS activity, and in-flow techniques employing an IMER in HPLC and CE to screen for synthetic and natural compounds that act as XO inhibitors. Full article

Given the strong interdisciplinary nature of microfluidic immobilized enzyme reactor (μ-IMER) technology, several branches of science contribute to its successful implementation. A combination of physical, chemical knowledge and engineering skills is often required. The development and application of μ-IMERs in the proteomic community are experiencing increasing importance due to their attractive features of enzyme reusability, shorter digestion times, the ability to handle minute volumes of sample and the prospect of on-line integration into analytical workflows. The aim of this review is to give an account of the current (2017–2021) trends regarding the preparation of microdevices, immobilization strategies, and IMER configurations. The different aspects of microfabrication (designs, fabrication technologies and detectors) and enzyme immobilization (empty and packed channels, and monolithic supports) are surveyed focusing on μ-IMERs developed for proteomic analysis. Based on the advantages and limitations of the published approaches and the different applications, a probable perspective is given. Full article

There is continuous effort towards developing monolithic materials as solid supports for the separation, enrichment, and digestion of glycoproteins. The intention of this review is to discuss and summarize work reported in this area during the period 2015–2021 as a follow-up to our prior review. Reports from the past three decades have already proven the advantages of monolithic materials, such as the ease with which they can be prepared and functionalized, their high permeability and low resistance to mass transfer, and their stability over a wide range of pH. Recent works on glycoprotein analysis introduce different strategies in using monolithic materials specifically in separation, enrichment, and identification of glycoproteins, glycopeptides, and free glycans. A majority of these are focused on boronic acid affinity-based technique and others on lectin affinity and HILIC-based techniques. There are also newly developed ligands that utilize different interactions with glycans, such as encapsulation into β-cyclodextrin vesicles, CH- or OH-π interactions with fullerenes, immunoaffinity with monoclonal antibodies, H-bonding interactions with metallophthalocyanines, coordination interactions with cobalt phthalocyanine tetracarboxylic acid, and hydrophilic interaction with cyclodextrin molecular tubes, zwitterionic iminodiacetic acid, and boric acid. Recent strategies for developing on-line, multidimensional systems use immobilized monolithic enzyme reactors (IMERs) for high-throughput glycoprotein analysis. These works serve as contributions to better understand glycan structure-function relationship, as glycoproteins are now widely accepted disease biomarkers. Full article

The goal of this paper was to develop an in-line immobilized enzyme reactor (IMER) integrated into a capillary electrophoresis platform. In our research, we created the IMER by adsorbing trypsin onto the inner surface of a capillary in a short section. Enzyme immobilization was possible due to the electrostatic attraction between the oppositely charged fused silica capillary surface and trypsin. The reactor was formed by simply injecting and removing trypsin solution from the capillary inlet (~1–2 cms). We investigated the factors affecting the efficiency of the reactor. The main advantages of the proposed method are the fast, cheap, and easy formation of an IMER with in-line protein digestion capability. Human tear samples were used to test the efficiency of the digestion in the microreactor. Full article