Search Results (104)

Dietary patterns have been identified as one of the most important modifiable risk factors for several non-communicable diseases, inextricably linked to the health span of older people. Poor dietary choices may act as triggers for immune responses such as aggravated inflammatory reactions and oxidative stress contributing to the pathophysiology of several ageing hallmarks. Novel dietary interventions are being explored to restore gut microbiota balance and promote overall health in ageing populations. Probiotics and, most recently, postbiotics, which are products of probiotic fermentation, have been reported to modulate different signalling biomolecules involved in immunity, metabolism, inflammation, and oxidation pathways. This review presents evidence-based literature on the effects of postbiotics in promoting healthy ageing and mitigating various age-related diseases. The development of postbiotic-based therapeutics and diet-based interventions within a personalised microbiota-targeted approach is proposed as a possible direction for improving health in the elderly population. Despite growing evidence, the data regarding their exact mechanistic pathways for antioxidant and immunomodulating activities remain largely unexplored. Expanding our understanding of the mechanistic and chemical determinants of postbiotics could contribute to disease management approaches, as well as the development of and optimisation of biotherapeutics. Full article

Maintaining a balanced skin microbiota is essential for skin health, whereas disruptions in skin microbiota composition, known as dysbiosis, can contribute to the onset and progression of various skin disorders. Microbiota dysbiosis has been associated with several inflammatory skin conditions, including atopic dermatitis, seborrheic dermatitis, acne, psoriasis, and rosacea. Recent advances in high-throughput sequencing and metagenomic analyses have provided a deeper understanding of the skin microbial communities in both health and disease. These discoveries are now being translated into novel therapeutic approaches aimed at restoring microbial balance and promoting skin health through microbiome-based interventions. Unlike conventional therapies that often disrupt the microbiota and lead to side effects or resistance, microbiome-based products offer a more targeted strategy for preventing and managing inflammatory skin diseases. These products, which include probiotics, prebiotics, postbiotics, and live biotherapeutic agents, are designed to modulate the skin ecosystem by enhancing beneficial microbial populations, suppressing pathogenic strains, and enhancing immune tolerance. As a result, they represent a promising class of products with the potential to prevent, manage, and even reverse inflammatory skin conditions. However, realizing the full therapeutic potential of microbiome-based strategies in dermatology will require continued research, robust clinical validation, and clear regulatory frameworks. Full article

Recombinantly produced monoclonal antibodies (mabs) belong to the fastest growing class of biotherapeutics. In humans, antibodies are classified into five different classes: IgA, IgD, IgE, IgG and IgM. Most of the therapeutic mabs used in the clinic belong to the IgG class, albeit other antibody classes, e.g., IgM, have been evaluated in clinical stages. Antibodies are composed of heavy chains paired with a light chain. In IgM and IgA, an additional chain, the J-chain, is present. Two types of light chains exist in humans: the κ-light chain and the λ-light chain. The κ-light chain predominates in humans and is used in the vast majority of therapeutic IgG. The reason for the preference of the κ-light chain in humans is not known. Our study investigates whether light-chain selection influences the productivity of the clinically validated mabs adalimumab and trastuzumab. Both mabs were expressed as IgG and IgM with a κ- or a λ-light chain in HEK293 cells. Besides comparing the expression levels of the different mabs, we also evaluated whether the passage number of the cell line has an impact on product yield. In addition, the expressions of adalimumab, trastuzumab, an anti-CD38 and an anti-PD-L1-antibody were analyzed in HEK293 and CHO cells when both the κ- and λ-light chains are present. In summary, IgG outperformed IgM variants in expression efficacy, while light-chain selection had minimal impact on the overall expression levels. The yields of all mab variants were higher in fresh cells, despite cell cultures with a high cell passage number having higher cell densities and cell numbers at the time of harvest. The incorporation of a particular light chain occurred at similar rates in HEK293 and CHO cells. Full article

Insect cell lines are a cornerstone of recombinant protein production, providing a versatile platform for biopharmaceutical and research applications. In the early 20th century, scientists first attempted to culture insect cells in vitro, developing continuous cell lines to produce the first insect cell-derived recombinant protein, IFN-β. Initial successes, along with advancements in the use of insect cells for recombinant protein manufacturing, primarily relied on baculovirus expression vector systems (BEVSs), which enable heterologous gene expression in infected cells. Today, growing attention is focused on baculovirus-free systems based on the transfection of insect cells with plasmid DNA. This approach simplifies the final product purification process and facilitates the development of stable monoclonal cell lines that produce recombinant proteins or protein complexes, particularly virus-like particles (VLPs). Thanks to advancements in genetic engineering and the application of adaptive laboratory evolution (ALE) methods, significant strides have been made in overcoming many limitations associated with insect cell BEVSs, ultimately enhancing the reliability, yield, and quality of the biomanufacturing process. Our manuscript discusses the history of developing insect cell lines, presents various recombinant protein production systems utilizing these cells, and summarizes modifications aimed at improving insect cell lines for recombinant protein biomanufacturing. Finally, we explore their implications in pharmaceutical production, particularly on Nuvaxovid^®/Covovax, which is the latest approved vaccine developed using insect cell BEVSs for protection against SARS-CoV-2. Full article

The vaginal microbiome can be perturbed by various intrinsic and extrinsic factors, resulting in a state of dysbiosis that decreases the abundance of commensal lactobacilli and often leads to pathological conditions such as bacterial vaginosis, yeast infections, sexually transmitted infections, and other vaginal disorders. This narrative review explores the molecular and pathophysiological mechanisms of several microbial diseases associated with the dysbiosis of the vaginal microbiome, as well as the efficacy of therapeutic tools for these conditions, such as antibiotic treatment and the use of live biotherapeutic products. A non-systematic, narrative approach was employed. Searches and data extraction were performed using the PubMed and Scopus databases between January and February 2025. All reviewed studies reported vaginal microbiome dysbiosis, with microbial pathogens inducing a specific immune response in the host. Current treatments for vaginal microbiota dysbiosis-related pathologies often result in high relapse and recurrence rates, suggesting microbial resistance and the need for alternative therapeutic strategies. In turn, live biotherapeutic products have demonstrated beneficial effects, restoring microbial balance in dysbiotic conditions. While these findings suggest promising potential for live biotherapeutic products, further rigorous clinical studies are necessary to gain a deeper understanding of the female genital tract ecosystem and to identify novel biomarkers along with their associated health implications. Moreover, the development of new diagnostic and management strategies will facilitate personalized therapeutic approaches. Ultimately, a comprehensive perspective on vaginal care is pivotal, taking into account both microbial and immune dynamics to enhance women’s health outcomes. Full article

Glycosaminoglycans (GAGs) are key ligands for proteins involved in physiological and pathological processes. Specific GAG-binding patterns are rarely identified, with the heparin pentasaccharide as an Antithrombin-III ligand being the best characterized. Generating glycan-specific antibodies is difficult due to their size, pattern dispersion, and flexibility. Single-domain variable new antigen receptors (VNAR nanobodies) from nurse sharks are highly soluble, stable, and versatile. Their unique properties suggest advantages over conventional antibodies, particularly for challenging biotherapeutic targets. Here we have used VNAR semi-synthetic phage libraries to select high-affinity fondaparinux-binding VNARs that did not show cross-reactivity with other GAG species. Competition ELISA and surface plasmon resonance identified a single fondaparinux-selective VNAR clone. This VNAR exhibited an extraordinarily stable protein fold: the beta-strands are stabilized by a robust hydrophobic network, as revealed by heteronuclear NMR. Docking fondaparinux to the VNAR structure revealed a large contact surface area between the CDR3 loop of the antibody and the glycan. Fusing the VNAR with a human Fc domain resulted in a stable product with a high affinity for fondaparinux (Kd = 9.3 × 10⁻⁸ M) that could efficiently discriminate between fondaparinux and other glycosaminoglycans. This novel glycan-targeting screening technology represents a promising therapeutic strategy for addressing GAG-related diseases. Full article

Background/Objectives: The stress testing of biotherapeutic products is a critical component of drug development, enabling the assessment of stability, biosimilarity, and degradation pathways. Subjecting biosimilar monoclonal antibodies to controlled stress conditions yields essential insights into their structural and functional integrity, informing formulation optimization and mitigating risks before clinical trials. In this study, biosimilar products were comprehensively characterized and compared with originator products under forced degradation. The aim was to expose the products to different stress conditions such as oxidative, pH, thermal, freeze/thaw, and agitation. The products were then tested at defined time points using validated analytical methods. Methods: This study employed size-exclusion chromatography to detect aggregated forms. Isoelectric focusing characterized protein charge variants (e.g., acidic/basic isoforms) from post-translational modifications, while capillary electrophoresis quantified product-related impurities (aggregates and fragments). In addition, a complement assay was used to determine the efficacy and potency under specific stress conditions. Results: Our findings showed that biosimilar and originator products exhibited similar degradation profiles. The biosimilar monoclonal antibody was found to be analytically similar to the originator product in terms of critical parameters related to efficacy and safety under various stress conditions such as aggregation profile, biological activity, and charge variant distribution. Conclusions: Forced degradation studies facilitated the comprehensive and well-validated characterization of the structure and biological activity of biosimilar monoclonal antibody products. Full article

Saccharomyces cerevisiae var. boulardii is a probiotic yeast widely recognized for its ability to enhance gut health and modulate a host’s microbiome. However, there are limited data on its large-scale cultivation in stirred tank bioreactors and subsequent downstream processing into a functional probiotic product. Different recipe formulations were evaluated and the recipe with the highest biomass yield and lowest process time was selected. Once the optimised batch was validated in the replicate batches, the statistical analysis indicated a high level of reproducibility, with low variability across key performance indicators such as biomass concentration (unit), CFU production (CFU.mL⁻¹), and substrate utilization efficiency (g.g⁻¹). The mean growth age in the bioreactor was 25.33 ± 1.16 h, with a CV of 4.56%, indicating minimal deviation between batches. Similarly, the final viable concentration exhibited a mean of 1.46 × 10⁸ CFU.mL⁻¹ with a CV of 11.68%, remaining within an acceptable range for biological processes, while the final biomass concentration had the lowest variability (CV of 3.94%) and a 95% CI of 12.134–13.266 g.L⁻¹, highlighting the accuracy and consistency of the process. Productivity indicators, including cell productivity (growth time—biomass) and YPP (biomass), maintained low CV values (3.933% and 3.389%, respectively), reinforcing process efficiency and stability. The overlapping 95% confidence intervals across batches further confirmed that no statistically significant deviations existed, ensuring minimal batch-to-batch variability, and validating the scalability and robustness of the fermentation process. These findings provide strong evidence for the feasibility of large-scale probiotic yeast production that meets industrial production standards. The final freeze-dried product retained an 81% viability post-exposure to simulated gastrointestinal conditions, meeting WHO probiotic viability standards. These findings establish a scalable, optimized process for probiotic yeast production, with potential applications in biopharmaceutical manufacturing and functional food development, as confirmed by the techno-economic evaluations performed using SuperPro Designer^®. Full article

Proteins represent a significant portion of the global therapeutics market, surpassing hundreds of billions of dollars annually. Among the various post-translational modifications, glycosylation plays a crucial role in influencing protein structure, stability, and function. This modification is especially important in biotherapeutics, where the precise characterization of glycans is vital for ensuring product efficacy and safety. Although mass spectrometry-based techniques have become essential tools for glycomic analysis due to their high sensitivity and resolution, their complexity and lengthy processing times limit their practical application. In contrast, electrochemical methods provide a rapid, cost-effective, and sensitive alternative for glycosylation assessment, enabling the real-time analysis of glycan structures on biotherapeutic proteins. These electrochemical techniques, often used in conjunction with complementary methods, offer valuable insights into the glycosylation profiles of both isolated glycoproteins and intact cells. This review examines the latest advancements in electrochemical biosensors for glycosylation analysis, highlighting their potential in enhancing the characterization of biotherapeutics and advancing the field of precision medicine. Full article

Glycosaminoglycans (GAGs) are a diverse family of ancient biomolecules that evolved over millennia as key components in the glycocalyx that surrounds all cells. GAGs have molecular recognition and cell instructive properties when attached to cell surface and extracellular matrix (ECM) proteoglycans (PGs), which act as effector molecules that regulate cellular behavior. The perception of mechanical cues which arise from perturbations in the ECM microenvironment allow the cell to undertake appropriate biosynthetic responses to maintain ECM composition and tissue function. ECM PGs substituted with GAGs provide structural support to weight-bearing tissues and an ability to withstand shear forces in some tissue contexts. This review outlines the structural complexity of GAGs and the diverse functional properties they convey to cellular and ECM PGs. PGs have important roles in cartilaginous weight-bearing tissues and fibrocartilages subject to tension and high shear forces and also have important roles in vascular and neural tissues. Specific PGs have roles in synaptic stabilization and convey specificity and plasticity in the regulation of neurophysiological responses in the CNS/PNS that control tissue function. A better understanding of GAG instructional roles over cellular behavior may be insightful for the development of GAG-based biotherapeutics designed to treat tissue dysfunction in disease processes and in novel tissue repair strategies following trauma. GAGs have a significant level of sophistication over the control of cellular behavior in many tissue contexts, which needs to be fully deciphered in order to achieve a useful therapeutic product. GAG biotherapeutics offers exciting opportunities in the modern glycomics arena. Full article

The purpose of this scoping review was to provide a comprehensive understanding of the current knowledge concerning the gut microbiome and SCFAs as emerging treatments for salt-sensitive hypertension. Relevant animal and human studies were identified via PubMed through August 2024. Twenty-four human (n = 9) and animal (n = 15) trials were included. Most human studies were observational (n = 6), aiming to compare gut microbiota differences between hypertensive and normotensive individuals. Three human studies evaluated microbiome-based interventions either via a sodium-restricted diet (n = 2) or prebiotic supplementation (n = 1). Fifteen animal trials involving either mice or rats were identified, all of which were interventional. These included dietary changes (n = 9), probiotic treatments (n = 1), postbiotic primarily bacterial metabolites (n = 4), and live biotherapeutic products (n = 4). All interventions were effective in decreasing blood pressure. Microbiome-based therapies as biologic modifiers for salt-sensitive hypertension are emerging. Substantial knowledge gaps remain, warranting further research to fully explore this promising therapeutic avenue. Full article

Saccharomyces boulardii, the only commercially available probiotic yeast, has gained attention as a recombinant live biotherapeutic product (rLBP) empowered with the expression of heterologous therapeutic proteins for treating gastrointestinal diseases. However, the genetic modification of S. boulardii intended for clinical use is hindered by regulatory and technical challenges. In this study, we developed a dihydrofolate reductase (DHFR)-based selection system as an innovative alternative to traditional auxotrophic selection strategies for engineering S. boulardii. The DHFR selection system overcame inherent resistance of the yeast to methotrexate (MTX) by incorporating sulfanilamide, a dihydrofolate synthesis inhibitor, to enhance selection efficiency. The system demonstrated robust functionality, enabling the efficient screening of high-expression clones and tunable expression of therapeutic proteins, such as cytokines and antibodies, by modulating MTX concentrations. Furthermore, the yeast’s endogenous DHFR homolog, DFR1, was shown to be a viable selection marker, providing greater host compatibility while maintaining functionality compared to DHFR. This selection system avoids reliance on foreign antibiotic selection markers and the construction of auxotrophic strains, thus simplifying engineering and allowing for a tunable protein expression. These advancements establish the DHFR/DFR1 selection system as a robust and versatile platform for developing S. boulardii-based live biotherapeutics. Full article

Background/Objectives: EPICERTIN, a biotherapeutic candidate for mucosal healing in inflammatory bowel disease (IBD) and other mucosal disorders, was subjected to an extensive long-term stability program to evaluate its molecular stability and physicochemical properties. Additionally, a forced degradation assessment was conducted to identify EPICERTIN’s degradation products under various conditions, including thermal stress, pH variations, agitation, and oxidation. Methods: The stability of EPICERTIN drug substance (DS), formulated in phosphate-buffered saline (PBS) at 1 mg/mL and stored at 5 °C and 25 °C/60% relative humidity (RH), was monitored over a 2-year period, referencing relevant regulatory guidelines. Evaluations of EPICERTIN DS over the 24-month period included assessment of purity by SDS-PAGE and size exclusion high performance liquid chromatography (SEC-HPLC), identity by electrospray ionization mass spectrometry (ESI-MS) intact mass analysis and Western blotting, and potency by GM1-binding KDEL-detection ELISA (GM1/KDEL ELISA). The forced degradation patterns were analyzed by assessing purity (using SEC-HPLC and SDS-PAGE), potency (via GM1/KDEL ELISA), and intact mass (via ESI-MS). Results: The results overall support that EPICERTIN DS remains stable for 2 years under the tested conditions. The forced degradation assessment effectively identified degradation products, particularly under conditions of high temperatures (above 40 °C for 24 h), low pH values (pH 1 and 4), and oxidation upon exposure to 2% H₂O₂. Conclusions: These findings highlight EPICERTIN’s robust long-term stability in PBS formulation, reinforcing its potential as a viable drug candidate for the treatment of IBD. Full article

Batch-to-batch reproducibility and robust quality assessment are crucial for producing cell-free biologics, such as conditioned medium (CM) derived from mesenchymal stem/stromal cells (MSCs). This study investigated the effects of freezing CM at −80 °C prior to concentration, a step that could occur in large scale pipelines, compared to freshly processed CM. Quality assessment included total protein quantification; extracellular vesicle evaluation using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and cytofluorimetry; and biochemical analysis using Raman spectroscopy. The freezing process resulted in a 34% reduction in total protein content, as confirmed for selected bioactive mediators, and significant depletion of specific particle types, particularly larger ones. Interestingly, the total particle concentration and polydispersity remained stable. Alterations in Raman spectra highlighted changes in protein, lipid, and nucleic acid content. These findings demonstrate that even routine steps like freezing can alter CM composition, likely due to temperature-induced structural changes in biological molecules. Careful consideration of pre- and intra-processing handling temperatures is critical for preserving the integrity of CM and ensuring consistent quality. This study emphasizes the importance of refining manufacturing protocols in the production of cell-free biologics. Full article

IMUNOR is an oral biotherapeutic drug that had been developed, registered, and approved in 1997 in the Czech Republic and Slovakia. IMUNOR is a dialyzable leukocyte extract (DLE) prepared from swine leukocytes. It is characterized as a mixture of small peptides with molecular weights smaller than 12 kDa and a specific portion of nucleotides. The medical uses of IMUNOR include therapeutic applications within its registered range of indications, primarily for the treatment of immunodeficiencies, allergies, and certain acute or relapsing bacterial infections in adults and children. Despite the long-term clinical application of DLE, with strong evidence of positive therapeutic effects and no serious side effects, a detailed physicochemical specification of this mixture was lacking. We developed several methods for more in-depth physicochemical characterization of IMUNOR, including a spectrophotometric method for quantification of the total protein concentration and total DNA concentration in a mixture, several chromatographic methods for identification of individual components present in significant concentrations in IMUNOR, such as HPLC methods and the Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis method, and characterization of amino acid composition of this mixture. For the investigation of the variability among different batches of IMUNOR, five to nine representative batches from a standard manufacturing process on an industrial scale were utilized. Using the analytical methods, we verified and confirmed the batch-to-batch reproducibility of the biological product IMUNOR. Full article