Free Protein Research Articles

Polyphenol autoxidation and prooxidative activity induce protein oxidation and protein-polyphenol adduct formation in model systems

Polyphenols are well-known for their antioxidant properties, but their prooxidative activity remain less understood. This study quantitatively examined the formation of hydrogen peroxide (H2O2) during the autooxidation of nine different polyphenols in model systems, investigating how it impacts protein oxidation and protein-polyphenol covalent adduct formation. Polyphenols (4 mM) generated H2O2 in the range of 0.2–242 μM, depending on type of polyphenol, incubation time, temperature, and pH, but no clear relationship between polyphenol structure and H2O2 production was observed. The presence of free amino acids and proteins (bovine serum albumin and β-lactoglobulin) inhibited H2O2 formation, with Cys completely scavenging H2O2. Met was highly susceptible to oxidation with a 25–75 % loss, forming methionine sulfoxide through a two-electron oxidation pathway. Trp and Tyr were oxidized to produce dioxindolyl-L-alanine, kynurenine, 3,4-dihydroxyphenylalanine, N′-formylkynurenine, and 5-hydroxytryptophan in the nmol/mol-mmol/mol amino acid range. Furthermore, autoxidation of polyphenols resulted in >177 distinct amino acid/protein-polyphenol adducts as identified using LC-Orbitrap-MS/MS analysis.

Silica Nanoparticle-Protein Aggregation and Protein Corona Formation Investigated with Scattering Techniques.

Protein-nanoparticle interactions and the resulting corona formation play crucial roles in the behavior and functionality of nanoparticles in biological environments. In this study, we present a comprehensive analysis of protein corona formation with superfolder green fluorescent protein (sfGFP) and bovine serum albumin in silica nanoparticle dispersions using small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS). For the first time, we subtracted the scattering of individual proteins in solution and individual nanoparticles from the protein-nanoparticle complexes. This approach effectively isolated the contributions of specific components within the corona. Our form factor analysis revealed consistent core-shell sphere thicknesses but varied attractive interaction strengths of the nanoparticle complexes, influenced by the protein corona and the surface properties of silica and aminated silica nanoparticles. Interestingly, fractal analysis of nanoparticles showed a transition from surface to mass fractals for sfGFP samples at high protein:nanoparticle molar ratios of over 264,000:1. DLS analysis highlighted aggregation behaviors, including the increasing size of protein-nanoparticle complexes and significant aggregation of both free proteins and complexes at ∼264,000 molar ratio. Large polydispersity and heterogeneous protein aggregation were observed at these high molar ratios. Both SAXS and DLS revealed transitions and changes in protein-nanoparticle interactions at molar ratios of 4000 to 44,000, consistent with corona formation, while pronounced aggregation was observed at a molar ratio of ∼264,000. These findings advance our understanding of the structural complexities in protein-nanoparticle association and suggest further avenues for refining characterization techniques in protein corona research.

Visualizing Tomato Spotted Wilt Virus Protein Localization: Cross-Kingdom Comparisons of Protein-Protein Interactions.

Tomato spotted wilt virus (TSWV) is an orthotospovirus that infects both plants and insect vectors, and understanding its protein localization and interactions is crucial for unraveling the infection cycle and host-virus interactions. We investigated and compared the localization of TSWV proteins. The localization between plant and insect cells was overall consistent, indicating a similar mechanism is utilized by the virus in both types of cells. However, a change in localization over time was associated with the viral proteins that did not contain signal peptides and transmembrane domains such as N, NSs, and NSm, which only occurred in the plant cells and not in the insect cells. We also tested the localization of the proteins during an active plant infection using free red fluorescent protein (RFP) as a marker to highlight the nucleus and cytoplasm. Voids in the cytoplasm were shown only during infection, and N, NSs, NSm, and to a lesser extent GN and GC were surrounding these areas, suggesting it may be a site of replication or morphogenesis. Furthermore, we tested the interactions of viral proteins using both bimolecular fluorescence complementation (BiFC) and membrane-based yeast two-hybrid (MbY2H) assays. These revealed self-interactions of NSm, N, GN, GC, and NSs. We also identified interactions between different TSWV proteins, indicating their possible roles, such as between NSs and GC and N and GC, which may be necessary during the replication and assembly processes, respectively. This research expands our knowledge of TSWV infection and elaborates on the intricate relationships between viral proteins, cellular dynamics, and host responses. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Diverse single-stranded nucleic acid binding proteins enable both stable protection and rapid exchange required for biological function

Abstract Single-stranded nucleic acid (ssNA) binding proteins must both stably protect ssNA transiently exposed during replication and other NA transactions, and also rapidly reorganize and dissociate to allow further NA processing. How these seemingly opposing functions can coexist has been recently elucidated by optical tweezers (OT) experiments that isolate and manipulate single long ssNA molecules to measure conformation in real time. The effective length of an ssNA substrate held at fixed tension is altered upon protein binding, enabling quantification of both the structure and kinetics of protein–NA interactions. When proteins exhibit multiple binding states, however, OT measurements may produce difficult to analyze signals including non-monotonic response to free protein concentration and convolution of multiple fundamental rates. In this review we compare single-molecule experiments with three proteins of vastly different structure and origin that exhibit similar ssNA interactions. These results are consistent with a general model in which protein oligomers containing multiple binding interfaces switch conformations to adjust protein:NA stoichiometry. These characteristics allow a finite number of proteins to protect long ssNA regions by maximizing protein–ssNA contacts while also providing a pathway with reduced energetic barriers to reorganization and eventual protein displacement when these ssNA regions are diminished.

Development of high-performance inducible and secretory expression vector and host system for enhanced recombinant protein production

The production of lipopolysaccharide (LPS)-free recombinant proteins from culture supernatants is of great interest to biomedical research and industry. Due to the LPS-free cell wall structure and the well-defined secretion factor B (SecB)-dependent secretion pathway, Gram-positive bacteria are a superior alternative to Escherichia coli expression systems. However, the lack of inducible expression systems for high yields has been a bottleneck. To address this, we developed the pKS81 plasmid, featuring the uhpT (glucose-6-phosphate [G6P] transporter) promoter for high expression of recombinant proteins induced by extracellular G6P via a three-component hexose phosphate transport regulatory system (HptARS), the N-terminal SecB-dependent signal peptide sequence for recombinant protein secretion, and the C-terminal 8 × histidine tag for purification by nickel affinity chromatography. We also generated an expression host strain, Staphylococcus aureus LAC9, lacks the uhpT gene and harmful superantigen and leukotoxin genes, allowing for constitutive HptARS activation by extracellular G6P and increased safety, respectively. Using the pKS81 plasmid, we successfully achieved high yields of prokaryotic (staphylococcal leukotoxin E) and eukaryotic (human annexin A2 protein tagged with mouse IgG1) recombinant proteins, up to 900 mg/L. Our newly established inducible and secretory expression system provides for efficient production and easy purification of LPS-free recombinant proteins, making it valuable for biomedical research and industrial applications.

Apocynin, a Selective NADPH Oxidase (Nox2) Inhibitor, Ameliorates Behavioural and Learning Deficits in the Fragile X Syndrome Mouse Model.

Background/Objectives: Fragile X Syndrome (FXS) is associated with intellectual disability, hyperactivity, social anxiety and signs of autism. Hyperactivation of NADPH oxidase has been previously described in the brain of the male Fmr1-KO mouse. This work aims to demonstrate the efficacy of Apocynin, a specific NADPH oxidase inhibitor, in treating Fragile X mouse hallmarks. Methods: Free radicals, lipid and protein oxidation markers and behavioural and learning paradigms were measured after chronic treatment with orally administered vehicle, 10 mg/kg/day or 30 mg/kg/day of Apocynin. Results: The results revealed a reduction in testis weight, an increase in peritoneal fat, and no variation in body weight after chronic treatment. Furthermore, a reduction in hyperactivity was detected in Apocynin-treated male Fmr1-KO mice. Additionally, the higher dose of 30 mg/kg/day also improves behaviour and learning in the male Fmr1-KO mice, normalising free radical production and oxidative parameters. Moreover, a reduction in phospho-EKR1 and P47-Phox protein signals was observed in specific brain areas. Conclusions: Thus, chronic treatment with Apocynin could lead to a new therapeutic option for the Fragile X Syndrome.

Consistent theoretical description of nuclear spin long-lived states decay under conditions of reversible ligand-protein binding.

Determining the stability constant of the complex formed by an organic ligand with a protein is the first stage in the screening of new drugs. Nuclear spin long-lived states, in particular the singlet state, can be used to study the reversible binding of ligands to proteins. In a complex with a protein, the spins of the ligand interact with the spins of the protein, the system of protein and ligand nuclei can relax by a dipole-dipole mechanism, and the lifetime of the singlet state is strongly reduced. In this theoretical study, a system of encounter theory equations with the condition of fast relaxation in free protein was solved to determine the lifetime of the LLS in the presence of protein. It was shown that in the limit of fast chemical exchange, the relaxation of the LLS of the ligand nuclei due to dipole interaction with the protein nuclei is reduced to relaxation by the mechanism of dipole interaction with one proton of the protein, which is located at some effective distance from the ligand nuclei. Numerical calculations were made to test the applicability of the approximations used to process the experimental lifetime dependencies on the ligand concentration and external field, and it was shown that these approximations coincide with the limit of fast exchange in strong and weak magnetic fields, but not in the medium field. An analytical expression for the lifetime of the singlet state of ligand nuclei in an arbitrary magnetic field in the absence of protein was obtained.

A finger that gets in the way: When binding isn’t just about the bound state

In this issue of Structure, Viennet etal.1 report a study characterizing the DNA binding by a three-zinc-finger fragment from the transcription factor BCL11A, with the unusual feature that an interfinger interaction in the free protein is disrupted during binding, which provides a positive entropic contribution that enhances the affinity.

Enhanced proteomic profiling of human plasma-derived extracellular vesicles through charge-based fractionation to advance biomarker discovery potential.

The study introduces a charge-based fractionation method for fractionating plasma-derived extracellular vesicles (EVs) into sub-populations aimed at the improved purification from free plasma proteins to enhance the diagnostic potential of EV sub-populations for specific pathophysiological states. Here, we present a novel approach for EV fractionation that leverages EVs' inherent surface charges, differentiating them from other plasma components and, thus, reducing the sample complexity and increasing the purity of EVs. The developed method was optimized and thoroughly evaluated using proteomic analysis, transmission electron microscopy, nanoparticle tracking, and western blotting of isolated EVs from healthy donors. Subsequently, we pilot-tested the developed technique for its applicability to real-world specimens using a small set of clinical prostate cancer samples and matched controls. The presented technique demonstrates the effective isolation and fractionation of EV sub-populations based on their surface charge, which may potentially help enhance EV-based diagnostics, biomarker discovery, and basic biology research. The method is designed to be straightforward, scalable, easy-to-use, and it does not require specialized skills or equipment.

Hepatic Vein Thrombosis Secondary to Protein C and S Deficiency

Hepatic Venous Outflow Tract Obstruction is obstruction of the hepatic venous outflow tract can be primary due to obstruction by thrombosis or secondary due to compression of hepatic vein, inferior vena cava or both. Causes being prothrombotic inherited or acquired. Classical clinical triad being abdominal pain, ascites, and tender hepatomegaly.Ultrasound, Computed Tomography or Magnetic Resonance Imaging of abdomen are required for confirmation.Long-term anticoagulation with vitamin K antagonists should be started. 24year female with abdominal distention and pain, having pallor, fluid distended abdomen. CT abdomen revealed an hypodense focal lesion in right lobe, ascites with Hepatic Vein Thrombosis. Haematology Low Functional antithrombin activity of 29 units, low Free Protein S Antigen of 42.8 units, very low Protein C functional activity of 16.5 units was observed. Frequency of prothrombotic hepatic venous outflow tract obstruction in India is more compared to west. So considered in all cases with acute, chronic liver disease with complications.

Green Tea Epigallocatechin 3-Gallate Reduced Platelet Aggregation and Improved Anticoagulant Proteins in Patients with Transfusion-Dependent β-Thalassemia: A Randomized Placebo-Controlled Clinical Trial.

Patients with transfusion-dependent β-thalassemia (TDT) with iron overload have been linked to hypercoagulability and increased platelet (PLT) activation that causes thrombosis. Green tea extract (GTE) rich in epigallocatechin-3-gallate (EGCG) exerts iron-chelating and antithrombotic properties. The study aimed to assess the effects of GTE treatment on plasma coagulation state and PLT function in vitro and in patients with TDT. The subjects consumed a placebo or GTE tablets (50 mg and 2 × 50 mg EGCG equivalent) every day for two months. Blood was then collected from the treated patients for analyses of PLT numbers, agonist-induced PLT aggregation, and anti-coagulation proteins. In our findings indicate that the in vitro treatment of GTE (at least 1 mg EGCG equivalent) inhibited PLT aggregation in patients who were healthy and with thalassemia platelet-rich plasma (PRP), which was significant in the healthy PRP. Consistently, GTE treatment inhibited the PLT aggregation that had been ex vivo generated by collagen or ADP. In addition, consumption of GTE tablets greatly inhibited PLT aggregation and increased the plasma levels of proteins C and S, as well as the free protein S concentrations depending upon the time course, but not the GTE dosage. Moreover, plasma ferritin levels decreased in both green tea tablet groups in a time-dependent manner (p < 0.05 in the second month). In conclusion, EGCG-rich GTE diminished PLT aggregation in patients who were healthy and patients with thalassemia plasma. It also improved PLT aggregation and hypercoagulability in patients with TDT by increasing the antithrombotic activity of protein C and protein S. This would suggest an adjuvant of GTE could reduce the risk of thrombosis associated with iron overload.

Exploration on antifreeze potential of thawed drip enzymatic hydrolysates on myofibrillar proteins in pork patties during freeze-thaw cycles

This study explored using small molecular weight hydrolysates from enzymolyzed thawed drip as cryoprotectants to preserve myofibrillar protein quality in pork patties during freeze-thaw cycles. Hydrolysates were added at 0.36 %, 0.72 %, and 1.4 % concentrations, compared to a control with deionized water and a positive control with sorbitol and sucrose. Results indicated that thawed drip hydrolysates significantly reduced thawing loss and cooking loss. Moreover, the color deterioration during the 3rd and 6th freeze-thaw cycles was delayed. Myofibrillar protein denaturation and oxidation in the experimental groups were inhibited, shown by decreased surface hydrophobicity, reduced carbonyl groups and protein surface roughness, and increased free sulfhydryl groups, α-helix content, and protein particle height. The highest hydrolysate concentration (1.4 %) provided the most benefits, performing comparably to the positive control. Correlation analysis confirmed that hydrolysates enhanced both myofibrillar protein and pork quality, offering a promising approach to improve meat resilience against freeze-thaw conditions.

Surface properties and biofilm formation on resins for subtractively and additively manufactured fixed dental prostheses aged in artificial saliva: Effect of material type and surface finishing

Statement of problemAdditive manufacturing (AM) and subtractive manufacturing (SM) have been widely used for fabricating resin-based fixed dental prostheses. However, studies on the effects of material type (AM or SM resin) and surface finishing (polishing or glazing) on the surface properties and biofilm formation are lacking. PurposeThe purpose of this in vitro study was to investigate the effects of material type and surface finishing on the surface roughness, wettability, protein adsorption, and microbial adhesion of the AM and SM resins marketed for fixed restorations under artificial saliva-aged conditions. Material and methodsDisk-shaped specimens (∅10×2 mm) were fabricated using 3 types of resins: AM composite resin with fillers (AMC), AM resin without fillers (AMU), and SM composite resin with fillers (SMC). Each resin group was divided into 2 subgroups based on surface finishing: polished (P) and glazed (G). Therefore, 3 polished surface groups (AMCP, AMUP, and SMCP) and 3 glazed surface groups (AMCG, AMUG, and SMCG) were prepared. Specimens were then categorized according to aging condition in artificial saliva. Surface roughness (Ra and Sa), contact angle, surface free energy (SFE), protein adsorption, and microbial adhesion were measured. The data were analyzed using a nonparametric factorial analysis of variances and post hoc tests with Bonferroni correction (α=.05). ResultsWhen nonaged, significant interactions between material type and surface finishing were detected for Ra, contact angle, SFE, protein adsorption, and microbial adhesion (P≤.008). AMCP showed higher Ra and microbial adhesion than AMUP and SMCP, and higher contact angle and protein adsorption than SMCP (P<.001). AMCG had lower SFE than AMUG (P=.005) and higher bacterial adhesion than SMCG (P<.001). AMC had higher Sa than AMU and SMC (P≤.006). When aged, significant interactions between material type and surface finishing were detected for Ra, Sa, protein adsorption, and microbial adhesion (P≤.026). The contact angle and SFE were significantly affected only by the material type (P≤.001), as AMC exhibited higher wettability than SMC (P≤.004). AMCP had higher Ra and microbial adhesion than AMUP and SMCP (P≤.003). AMCP had higher Sa and protein adsorption than SMCP (P≤.004). AMCG showed lower Ra and higher protein adsorption than AMUG (P≤.001). ConclusionsBoth material type and surface finishing significantly affected surface properties and biofilm formation. AMCP exhibited higher surface roughness, protein adsorption, and microbial adhesion compared with SMCP. Glazing may reduce the differences in surface-biofilm interactions between AMC and SMC.

Modulating the highland barley hordein/glutelin ratio can improve reconstituted fermented dough characteristics to promote Chinese Mantou quality

To improve the defective processing of barley fermented dough, this study constructed barley model dough using reconstituted hordein/glutelin ratios (75:25, 50:50, and 25:75) and elucidated its regulatory roles and potential mechanisms. SEM and CLSM results showed that increasing the hordein ratio improved the continuity and completion of the reconstituted gluten network compared to Control, thus allowing the gluten to stretch and elongate during fermentation. Also, LF-NMR revealed that the water distribution of the reconstituted system tended to shift from a free to a bound state, contributing to water retention during the dough hydration phase. Rheological determination indicated that the 75H-25G sample exhibited maximum tan δ (G′/G″) with optimal viscoelastic solid behavior. Although the reconstituted system is homogeneous in composition, a moderate increase in the hordein ratio can improve the lack of barley dough textural properties, which agrees with the stress-relaxation results. Further analysis revealed that an increased hordein ratio results in a gluten-free sulfhydryl content decrease, SDS extractability rate increase, and λmax redshift; this is due to the ionic and hydrogen bonding weakening as well as hydrophobic interactions enhancement within its system. Furthermore, conformational relationship analysis revealed that free sulfhydryl, intermolecular forces, and protein secondary and tertiary structures were the characteristic indicators influencing the reconstituted barley dough and Mantou. The obtained results are expected to provide new insights into the quality improvement of barley dough during fermentation; in the future, attention towards blending strategies with other cereal flours should be focused on facilitating the barley gluten network deficiencies, thus promoting the flourishing of cereal products.

Establishment of liquid biopsy procedure for the analysis of circulating cell free DNA, exosomes, RNA and proteins in colorectal cancer and adenoma patients

Liquid biopsy has an underexplored diagnostic potential in colorectal cancer (CRC). Sufficient quantity and quality of its elements (circulating cell-free DNA (ccfDNA), exosomes and exosomal RNA) are essential for accurate results. The present study aims to establish the optimal protocol for handling liquid biopsy samples. Samples were obtained by collecting peripheral blood from colorectal adenoma patients in CellSave tubes. Plasma was separated within six hours using differential centrifugation and aliquots stored at – 20/– 80 °C until further processing. Three methods for isolation of ccfDNA, and two combinations of kits for isolation of exosomes and exosomal RNA were tested. The quality and quantity of ccfDNA isolates were evaluated. Exosomes were characterised by determining size, concentration, and total and specific protein content. Expression of chosen microRNAs, miR-19a-3p and miR-92-3p, which have been implicated in CRC progression, were determined. The vacuum-column-based kit showed the highest quantities of isolated ccfDNA (P-value < 0.001). Kits for exosome isolation significantly differed in size (P-value = 0.016), concentration (P-value = 0.016) and protein content (P-value = 0.016). There was no significant difference in expressions of miR-19a-3p (P-value = 0.219) and miR-92a-3p (P-value = 0.094) between the two isolation kits. The new, adapted protocol described, enables simultaneous analysis of multiple elements when investigating potential biomarkers of CRC.

Small Molecule Kinetic Stabilizers Reduce Amyloidogenicity of Free Light Chain Proteins of Diverse Sequences in λ Light Chain Amyloidosis

Small Molecule Kinetic Stabilizers Reduce Amyloidogenicity of Free Light Chain Proteins of Diverse Sequences in λ Light Chain Amyloidosis

In Situ Transformable Fibrillar Clusters Disrupt Intracellular Copper Metabolic Homeostasis by Comprehensive Blockage of Cuprous Ions Efflux.

Dysregulation of copper metabolism is intricately associated with the occurrence and therapeutic management of colorectal cancer. Previous studies have attempted to induce cuproptosis by delivering lethal doses of copper ions into tumor cells, often with systemic safety risks. In vivo, transformable peptide is modular and designed for various tumor-related proteins, which can affect protein function and distribution. Here, a fibrillar transformation peptidic (FTP) nanoparticle is synthesized, which can bind ATP7B membrane proteins (cuprous ions transporter) and transform into nanofibrils/ATP7B clusters, inducing "copper-free cuproptosis" in vivo. Without adding exogenous copper ions, the spherical FTP nanoparticles bound the high distribution regions of ATP7B membrane proteins, transforming into fibrillar networks in situ with prolonged retention. The cage-like fibrillar network would further capture unbound or newly generated free ATP7B membrane proteins, thereby significantly and consistently preventing cuprous ions efflux. The FTP nanoparticles would not undergo in situ fibrillar transformation on the low expression region of ATP7B membrane proteins but enter the cell for safe degradation, which exhibited high specificity and safety in vivo. By disrupting intracellular copper homeostasis, the transformable fibrillar clusters displayed a long-term anti-tumor effect on subcutaneous transplantation and liver metastatic CRC models.

Measurement of α-synuclein as protein cargo in plasma extracellular vesicles

Extracellular vesicles (EVs) are released by all cells and hold great promise as a class of biomarkers. This promise has led to increased interest in measuring EV proteins from both total EVs as well as brain-derived EVs in plasma. However, measuring cargo proteins in EVs has been challenging because EVs are present at low levels, and EV isolation methods are imperfect at separating EVs from free proteins. Thus, knowing whether a protein measured after EV isolation is truly inside EVs is difficult. In this study, we developed methods to measure whether a protein is inside EVs and quantify the ratio of a protein in EVs relative to total plasma. To achieve this, we combined a high-yield size-exclusion chromatography protocol with an optimized protease protection assay and Single Molecule Array (Simoa) digital enzyme-linked immunoassays (ELISAs) for ultrasensitive measurement of proteins inside EVs. We applied these methods to analyze α-synuclein and confirmed that a small fraction of the total plasma α-synuclein is inside EVs. Additionally, we developed a highly sensitive Simoa assay for phosphorylated α-synuclein (phosphorylated at the Ser129 residue). We found enrichment in the phosphorylated α-synuclein to total α-synuclein ratio inside EVs relative to outside EVs. Finally, we applied the methods we developed to measure total and phosphorylated α-synuclein inside EVs from Parkinson's disease and Lewy body dementia patient samples. This work provides a framework for determining the levels of proteins in EVs and represents an important step in the development of EV diagnostics for diseases of the brain, as well as other organs.

Detection and optimization of microbial expression systems for extracellular production and purification of Ca2+-responsive phase-changing annexin fusions

Previously, we identified the human annexin A1 as a purification tag for column-free purification with gentler calcium-responsive precipitation. In this work, we used the annexin A1 tagged green fluorescent protein constructs for detecting extracellular production in Escherichia coli, Bacillus subtilis, and Pichia pastoris, and identified that the leaderless fusion protein was transported extracellularly in E. coli with supply of additives including Triton X-100. The coexpressed enzymes, culture compositions, and induction conditions in E. coli extracellular expression systems were optimized. With coexpression of phospholipase C from Bacillus cereus and addition of 0.2 % Triton X-100 after induction for 60 h at 28 °C, the annexin A1 tagged green fluorescent protein and 5-aminolevulinate dehydratase from E. coli were overexpressed and purified from lysogeny broth by precipitation with 20 mM Ca2+ and redissolution with 25 mM EDTA with the acceptable protein purities and recoveries. The silica binding peptide was fused to the annexin A1 tagged fluorescent protein fusion for successive affinity precipitation and purification. With incubation of the specific protease, the released tag-free protein displayed higher purity via on-resin cleavage than that through cleavage of the free fusion protein. The tandem tag is applicable for two-step purification of small or large amounts of other fusion proteins in the culture and recovery of tag-free proteins at low cost.

Analytical Challenges in Novel Pentavalent Meningococcal Conjugate Vaccine (A, C, Y, W, X).

Multivalent meningococcal conjugate vaccines are a significant focus for the scientific community in light of the WHO's mission to defeat meningitidis by 2030. Well-known meningococcal vaccines such as MenAfriVac, Nimenrix, Menveo, and MenQuadfi are licensed in various parts of the world and have been successful. Recently, the World Health Organization (WHO) qualified MenFive (meningococcal A, C, Y, W, and X) conjugate vaccine, further enhancing the battery of vaccines against meningitis. The antigenic nature of the current and new serogroups, the selection of carrier proteins, and the optimal formulation of these biomolecules are pivotal parameters for determining whether a biological preparation qualifies as a vaccine candidate. Creating appropriate quality control analytical tools for a complex biological formulation is challenging. A scoping review aims to identify the main challenges and gaps in analyzing multivalent vaccines, especially in the case of novel serogroups, such as X, as the limited literature addresses these analytical challenges. In summary, the similarities in polysaccharide backbones between meningococcal serogroups (C, Y, W sharing a sialic acid backbone and A, X sharing a phosphorous backbone) along with various conjugation chemistries (such as CNBr activation, reductive amination, CDAP, CPIP, thioether bond formation, N-hydroxy succinimide activation, and carbodiimide-mediated coupling) resulting into a wide variety of polysaccharide -protein conjugates. The challenge in analyzing carrier proteins used in conjugation (such as diphtheria toxoid, tetanus toxoid, CRM diphtheria protein, and recombinant CRM) is assessing their purity (whether they are monomeric or polymeric in nature as well as their polydispersity). Additional analytical challenges include the impact of excipients, potential interference from serogroups, selection and establishment of standards, age-dependent behavior of biomolecules indicated by molecular size distributions, and process-driven variations. This article explains the analytical insights gained (polysaccharide content, free saccharide, free proteins, MSD) during the development of the MenFive vaccine and highlights the crucial gaps and challenges in testing.