Biopharmaceutical Applications Research Articles

Preparation and Properties of Glycerohydrogels Based on Silicon Tetraglycerolate, Chitosan Hydrochloride and Glucomannan

Glycerohydrogels based on silicon glycerolate, chitosan (CS) and polyvinyl alcohol (PVA) are widely studied for use in biomedical applications. In line with the general trend of replacing synthetic polymers with natural ones in such compositions, it would be of interest to replace PVA with the polysaccharide glucomannan (GM), as well as to introduce functional additives to impart the desired properties, including gelation time, to the final hydrogel. In this work, a comprehensive study of the preparation conditions and properties of glycerohydrogels based on silicon tetraglycerolate, chitosan hydrochloride (CS·HCl) and GM was carried out. Viscometry was used to assess the conformational state of CS·HCl and GM macromolecules, and their associates in solution before gelation. Gelation was studied using the vessel inversion method. The mucoadhesive and the dermoadhesive properties of the glycerohydrogels obtained were assessed using the tearing off method from the model substrates simulating mucous and dermal tissues. The conformational state of the individual polymers and their mixed associates in solution before gelation was estimated; the intrinsic viscosity and the hydrodynamic radius of the macromolecular coils were calculated. The influence of various factors (addition of ε-aminocaproic and hydrochloric acids, sodium chloride, hydroxide and tetraborate to vary the acidity and ionic strength of the medium, as well as temperature) and the molecular weight of chitosan on the gelation time was studied. The gelation time achieved was less than 2 min, which is promising in practical terms, i.e., for creating liquid plasters. Our best samples are not inferior to the commercial preparation “Metrogyl Denta”® in terms of tearing force during mucoadhesion and dermoadhesion at short gelation times. Thus, the glycerohydrogels synthesized by us and based on silicon tetraglycerolate, CS·HCl and GM could find usage in new biopharmaceutical and biomedical applications.

Glyco-engineering Using Pneumococcal Exoglycosidases: A Pilot Study on Deglycosylation of Human Alpha-1-Acid Glycoprotein

Glyco-engineering is an emerging field that focuses on modifying glycan structures to enhance or alter biological functions. Enzymatic glycan remodeling has numerous applications in biopharmaceuticals, vaccine development, and glycan-based diagnostics. Pneumococcal exoglycosidases, such as neuraminidase NanA and β-galactosidase BgaA, selectively hydrolyze terminal sugars from glycoconjugates, making them potential tools for glyco-engineering. However, their ability to modify human glycoproteins in vitro remains largely unexplored. In this study, we assessed the glyco-engineering potential of pneumococcal exoglycosidases by examining their role in deglycosylating human alpha-1-acid glycoprotein (AGP), a plasma glycoprotein involved in inflammation. Our results demonstrate that NanA removes terminal sialic acid residues from AGP, enabling subsequent galactose removal by BgaA, as confirmed by SDS-PAGE and lectin blot analysis. These findings provide experimental evidence supporting the utility of pneumococcal glycosidases for enzymatic glyco-engineering.

Therapeutic Efficacy Studies on the Monoterpenoid Hinokitiol in the Treatment of Different Types of Cancer.

Hinokitiol (HK), a monoterpenoid that naturally occurs in plants belonging to the Cupressaceae family, possesses important biological activities, including an anticancer effect. This review summarizes its anticancer potential and draws possible molecular interventions. In addition, it evaluates the biopharmaceutical, toxicological properties, and clinical application of HK to establish its viability for future advancement as a dependable anticancer medication. The assessment is based on the most recent information available from various databases. Findings demonstrate that HK possesses substantial therapeutic advantages against diverse types of cancer (colon, cervical, breast, bone, endometrial, liver, prostate, oral, and skin) through various molecular mechanisms. HK induces oxidative stress, cytotoxicity, apoptosis, cell-cycle arrest at the G and S phases, and autophagy through modulation of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), p38/ERK/MAPK, nuclear factor kappa B, and c-Jun N-terminal kinase signaling pathways. Furthermore, this compound exhibits good oral bioavailability with excellent plasma clearance. Clinical uses of HK demonstrate therapeutic advantages without any significant negative effects. A thorough study of the pertinent data suggests that HK may serve as a viable candidate for developing novel cancer therapies. Consequently, more extensive studies are necessary to evaluate its cancer treatment efficacy, safety, and possible long-term hazards.

Polynorepinephrine nanoparticles activate vascular smooth muscle alpha-1 adrenergic receptors.

Biocompatible polymeric nanoparticles (NPs) as carriers for therapeutic agents with multifunctional activities have received unprecedented attention for a variety of bio-pharmaceutical applications. We describe the synthesis, the fluorescence properties, the bio-compatible nature and the alpha-1 adrenergic receptor bio-activity of engineered quantum dot-like polynorepinephrine (PNE) NPs. The spherical PNE NPs, which are internalized in smooth muscle cells via a receptor-selective mechanism, activate alpha-1-adrenoceptors in intact mouse aorta and aorta-derived cultured smooth muscle cells, leading to the activation of calcium signaling/contraction and stimulation of mitogen-activated protein kinase (MAPK), thereby displaying receptor-triggering biological activity, possibly acting both extracellularly and intracellularly. Our data indicate that NPs generated by the polymerization of pharmacologically active compounds like norepinephrine can retain receptor-selective biological activity coupled to inherent fluorescence.

Application of integrated full-membrane platform in antibody purification

AbstractAntibody products are promising therapeutic candidates for various diseases, such as cancers and autoimmune disorders. Owing to the improvement of cell culture technology in recent years, the production titers of antibodies can reach up to 10 g/L. With such high titers in upstream production, advancements in the development of downstream separation and purification techniques are highly desired. To ensure a balance between handling the increased product supply and maintaining the high purity required for biopharmaceutical applications, membrane chromatography is an efficient and cost-effective technique that can be used to separate and purify proteins and other biological components. In this study, we developed a full-membrane platform that uses affinity membrane chromatography to capture antibodies, which is followed by a two-step flow-through polishing process involving anion exchange membrane chromatography and hydrophobic interaction membrane chromatography. We tested five molecules using this platform, and the outcomes of membrane chromatography were comparable to those of column chromatography in terms of yield and product quality. Moreover, full-membrane chromatography might lead to a cost reduction of > 70% in chromatography media. Altogether, the full-membrane platform developed in this has potential applications in downstream processes of antibody production.

Target Bioconjugation of Protein Through Chemical, Molecular Dynamics, and Artificial Intelligence Approaches.

Covalent modification of proteins at specific, predetermined sites is essential for advancing biological and biopharmaceutical applications. Site-selective labeling techniques for protein modification allow us to effectively track biological function, intracellular dynamics, and localization. Despite numerous reports on modifying target proteins with functional chemical probes, unique organic reactions that achieve site-selective integration without compromising native functional properties remain a significant challenge. In this review, we delve into site-selective protein modification using synthetic probes, highlighting both chemical and computational methodologies for chemo- and regioselective modifications of naturally occurring amino acids, as well as proximity-driven protein-selective chemical modifications. We also underline recent traceless affinity labeling strategies that involve exchange/cleavage reactions and catalyst tethering modifications. The rapid development of computational infrastructure and methods has made the bioconjugation of proteins more accessible, enabling precise predictions of structural changes due to protein modifications. Hence, we discuss bioconjugational computational approaches, including molecular dynamics and artificial intelligence, underscoring their potential applications in enhancing our understanding of cellular biology and addressing current challenges in the field.

Stimuli-responsive hydrogels for skin wound healing and regeneration

AbstractSkin wounds are not only an aesthetic concern but also pose great risks to quality of life and general health. As the most promising biomaterial, hydrogels are three-dimensional polymeric networks and have attracted intense research attention. Hydrogels have developed a diverse range of biomedical and biopharmaceutical applications, owing to their large water content, biocompatibility, tunable mechanical properties, and stimuli-responsiveness. Stimuli-responsive hydrogels are smart materials which exhibit gelation, structural, degradation, performance and function changes when treated with external stimulations. Using these hydrogels to prepare wound dressing is a rapidly growing research area and has exhibited encouraging healing outcomes in small animal models, especially for the treatment of chronic wounds, diabetic wounds, and persistent skin inflammations. The present work gives a detailed and critical analysis on the design strategies, gelation mechanisms, materials selection, stimuli-responsiveness, hydrogel degradation, drug release profiles, and treatment outcomes of wound dressings prepared by the hydrogels with sensitivity to temperature, pH, reactive oxygen species (ROS), glucose, enzymes, and lights. We summarize, analyze, and critically evaluate the most recent publications in this area to explain, compare, and assess why and how various synthetic and bio-polymers are utilized by materials scientists to develop the next generation of skin wound dressing and regeneration. Graphical abstract

COP II-mediated ER-to-Golgi transport is a bottleneck for IgNAR-Fc production in the Chinese hamster ovary cell expression system

The novel heavy-chain antibody known as immunoglobulin new antigen receptor (IgNAR) is derived from cartilaginous fishes such as sharks. IgNAR, which binds to antigens with the high specificity and affinity of a conventional IgG antibody and exhibits high resistance to denaturation, has potential as a next-generation antibody in biopharmaceutical and biotechnological applications. High-level expression of recombinant IgNAR in animal cells has been challenging. In our previous study, IgNAR was expressed as a fusion protein with a human IgG Fc region (IgNAR-Fc) in Chinese hamster ovary (CHO) cells, but did not meet the production level required for further research and application. In this study, we sought to identify the production bottleneck in CHO cells as a first step toward achieving abundant production of IgNAR. Using an established IgG high-production CHO cell line as a comparator, we found that the amounts of intracellular dimeric IgNAR-Fc produced in CHO cells were similar to those of intracellular dimeric IgG. Furthermore, the majority of intracellular IgNAR-Fc was retained in the endoplasmic reticulum (ER) and strongly colocalized to ERGIC-53, the cargo receptor for coat protein complex II (COP II)-coated vesicles. These findings suggest that COP II-mediated ER-to-Golgi transport may represent a bottleneck for IgNAR-Fc production in the CHO cell expression system.

Unrivalled Insight into Possible Biopharmaceutical Application of Justicia vahlii Roth. (Acanthaceae): Chemodiversity, In Vitro Bioactivities, and Computational Analysis.

Justicia vahliiRoth. is an important wild medicinal food plant traditionally used for treating inflammation and various common ailments. This study investigated the chemical composition, antioxidant, enzyme inhibition and toxicity profiles of n-hexane (nHEJv) and chloroform (CEJv) extracts of J. vahlii. Moreover, the effect of the extracts was evaluated on CCl4 induced liver injury. The total phenolic and flavonoid contents were present in both extracts in significant amount. The UPLC-Q-TOF-MS and GC-MS profiling of CEJv tentatively identified several important phytocompounds. The CEJv extract was comparatively more active for antioxidant activity and α-amylase inhibition, whereas the nHEJv extract presented higher inhibition potential against urease, tyrosinase, and α-glucosidase enzymes. Similarly, the in-silicostudy of four major compounds, i. e., 1-acetoxypinoresinol, 3-hydroxysebacic acid, nortrachelogenin, and viscidulin-III have shown a good docking score against the clinically significant enzymes. The acute oral toxicity and brine shrimp lethality assaysrevealed the extracts as non-toxic. The CCl4 treated animals showed a geared depletion of various antioxidant enzymes which were significantly reversed with the treatment of the extracts. Overall, the study's findings revealed J. vahliiwith antioxidant mediated hepatoprotective and enzyme inhibition potential and warrant further research on isolation of the bioactive compounds.

Small scale model for predicting transportation-induced particle formation in biotherapeutics

Understanding protein adsorption and aggregation at the air-liquid interfaces of protein solutions is an important open challenge in biopharmaceutical, medical, and biotechnological applications, among others. Proteins, being amphiphilic, adsorb at the surface, partially unfold, and form a viscoelastic film through non-covalent interactions. Mechanical agitation of the surface can break this film up, releasing insoluble protein particles into the solution. These aggregates are usually highly undesirable and even toxic in cases, such as for biopharmaceutical application. Therefore, it is imperative to be able to predict the behavior of such solutions undergoing surface agitation during handling, usually transport or mixing. We apply the findings on the viscoelastic protein film, formed at the air-liquid interface, to the prediction of surface mediated aggregation in selected protein solutions of direct biopharmaceutical relevance. Our broad study of Brewster angle microscopy and aggregation monitoring across multiple size ranges by micro-flow imaging, light scattering, and size exclusion chromatography shows that formation of protein particles is driven by the adsorption rate as compared to the rate of surface turnover and that surface film dynamics in the quiescent phase directly affect aggregation. We demonstrate how these learnings can be directly applied to the design of a novel small scale biopharmaceutical stability study, simulating relevant transport conditions. More generally, we show the impact of adsorption dynamics at the air-liquid interface on the stability of a distinct protein solution, as a general contribution to understanding different colloidal and biological interfacial systems.

Role of Lichen Extracts as a Potential Source of Natural Fungicides

Plant pathogens and the diseases they cause are major to humanity. Each year, we lose more than thirty percent of crops worldwide to bacterial and fungal diseases. Lichen is a composite organism made up of two unrelated species: algae and fungi. These two components live together and behave as a single organism. When two organisms live together in this way, they provide mutual benefits to one another. They are referred to as symbionts. Lichens produce many rare secondary products not found in other plants. The uniqueness of many aromatic products sparked early chemical interest in lichen compounds. Lichens are an underutilized source of industrially important as it proven many biological activities, and yet there is much more to study about their efficacy. Lichen bioactive metabolites showed promising use in biopharmaceutical applications and for the preparation of new formulations or innovations to support human life. This review aims to summarize the research and development trends from the past and present in the bioactive fungicides against plant pathogens.

Evaluation of a green synthesized biopolymer polymethyl methacrylate grafted Moringa gum amphiphilic graft copolymer (MOG-g-PMMA) with polymeric-surfactant like properties for biopharmaceutical applications

Evaluation of a green synthesized biopolymer polymethyl methacrylate grafted Moringa gum amphiphilic graft copolymer (MOG-g-PMMA) with polymeric-surfactant like properties for biopharmaceutical applications

Machine Learning for Deconvolution and Segmentation of Hyperspectral Imaging Data from Biopharmaceutical Resins.

Biopharmaceutical resins are pivotal inert matrices used across industry and academia, playing crucial roles in a myriad of applications. For biopharmaceutical process research and development applications, a deep understanding of the physical and chemical properties of the resin itself is frequently required, including for drug purification, drug delivery, and immobilized biocatalysis. Nevertheless, the prevailing methodologies currently employed for elucidating these important aspects of biopharmaceutical resins are often lacking, frequently require significant sample alteration, are destructive or ionizing in nature, and may not adequately provide representative information. In this work, we propose the use of unsupervised machine learning technologies, in the form of both non-negative matrix factorization (NMF) and k-means segmentation, in conjugation with Raman hyperspectral imaging to rapidly elucidate the molecular and spatial properties of biopharmaceutical resins. Leveraging our proposed technology, we offer a new approach to comprehensively understanding important resin-based systems for application across biopharmaceuticals and beyond. Specifically, focusing herein on a representative resin widely utilized across the industry (i.e., Immobead 150P), our findings showcase the ability of our machine learning-based technology to molecularly identify and spatially resolve all chemical species present. Further, we offer a comprehensive evaluation of optimal excitation for hyperspectral imaging data collection, demonstrating results across 532, 638, and 785 nm excitation. In all cases, our proposed technology deconvoluted, both spatially and spectrally, resin and glass substrates via NMF. After NMF deconvolution, image segmentation was also successfully accomplished in all data sets via k-means clustering. To the best of our knowledge, this is the first report utilizing the combination of two unsupervised machine learning methodologies, combining NMF and k-means, for the rapid deconvolution and segmentation of biopharmaceutical resins. As such, we offer a powerful new data-rich experimentation tool for application across multidisciplinary fields for a deeper understanding of resins.

Medical Applications and Cellular Mechanisms of Action of Carboxymethyl Chitosan Hydrogels.

Carboxymethyl chitosan (CMCS) hydrogels have been investigated in biomedical research because of their versatile properties that make them suitable for various medical applications. Key properties that are especially valuable for biomedical use include biocompatibility, tailored solid-like mechanical characteristics, biodegradability, antibacterial activity, moisture retention, and pH stimuli-sensitive swelling. These features offer advantages such as enhanced healing, promotion of granulation tissue formation, and facilitation of neutrophil migration. As a result, CMCS hydrogels are favorable materials for applications in biopharmaceuticals, drug delivery systems, wound healing, tissue engineering, and more. Understanding the interactions between CMCS hydrogels and biological systems, with a focus on their influence on cellular behavior, is crucial for leveraging their versatility. Because of the constantly growing interest in chitosan and its derivative hydrogels in biomedical research and applications, the present review aims to provide updated insights into the potential medical applications of CMCS based on recent findings. Additionally, we comprehensively elucidated the cellular mechanisms underlying the actions of these hydrogels in medical settings. In summary, this paper recapitulates valuable data gathered from the current literature, offering perspectives for further development and utilization of carboxymethyl hydrogels in various medical contexts.

2PP-Hydrogel Covered Electrodes to Compensate for Media Effects in the Determination of Biomass in a Capillary Wave Micro Bioreactor.

Microbioreactors increase information output in biopharmaceutical screening applications because they can be operated in parallel without consuming large quantities of the pharmaceutical formulations being tested. A capillary wave microbioreactor (cwMBR) has recently been reported, allowing cost-efficient parallelization in an array that can be activated for mixing as a whole. Although impedance spectroscopy can directly distinguish between dead and viable cells, the monitoring of cells in suspension within bioreactors is challenging because the signal is influenced by the potentially varying properties of the culture medium. In order to address this challenge, an impedance sensor consisting of two sets of microelectrodes in a cwMBR is presented. Only one set of electrodes was covered by a two-photon cross-linked hydrogel to become insensitive to the influence of cells while remaining sensitive to the culture medium. With this impedance sensor, the biomass of Saccharomyces cerevisiae could be measured in a range from 1 to 20 g L-1. In addition, the sensor can compensate for a change in the conductivity of the suspension of 5 to 15 mS cm-1. Moreover, the two-photon cross-linking of hydroxyethyl starch methacrylate hydrogel, which has been studied in detail, recommends itself for even much broader sensing applications in miniaturized bioreactors and biosensors.

Eco-benevolent synthesis of ZnO-NPs and ZnO-MFs from Inula oculus-christi L. (Asteraceae) with effective antioxidant, antimicrobial, DNA cleavage, and decolorization efficiencies.

As a result of the changes occurring globally in recent years, millions of people are facing challenging and even life-threatening diseases such as cancer and the COVID-19 pandemic, among others. This phenomenon has spurred researchers towards developing and implementing innovative and environmentally friendly scientific methods, merging disciplines with significant technological potential, such as nanotechnology with medicinal plants. Therefore, the focus of this research is to synthesize zinc nanoparticles (ZnO-NPs) and microflowers (ZnO-MFs) using extracts of the medicinal plant I. oculus christi prepared in n-hexane and methanol as new bioreduction and capping agents through a simple and environmentally friendly chemical approach. Optical, thermal, and morphological structural analyses of ZnO-NPs and ZnO-MFs were conducted using Ultraviolet-Visible (UV-Vis) spectroscopy, Fourier Transform Infrared (FT-IR) spectroscopy, Thermogravimetric Analysis (TGA), and Field Emission Scanning Electron Microscopy (FE-SEM). Metabolic profiles of extracts from different plant parts were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS) and supported by visualization of contents through Principal Component Analysis (PCA), hierarchical cluster analysis heatmaps, and Pearson correlation graphs. Interestingly, ZnO-NPs and ZnO-MFs exhibited strong antioxidant properties and demonstrated particularly potent antimicrobial activity against Micrococcus luteus NRRL B-4375, Escherichia coli ATCC 25922, and Candida albicans ATCC 10231 strains compared to standard antibiotics. Furthermore, ZnO-NPs and ZnO-MFs showed excellent plasmid DNA-cleavage activity of pBR322 with increasing doses. The photocatalytic performance of the synthesized ZnO-NPs and ZnO-MFs was evaluated for methylene blue (MB), congo red (CR), and safranin-O(SO) dyes, demonstrating remarkable color removal efficiency. Overall, the results provide a promising avenue for the green synthesis of ZnO-NPs and ZnO-MFs using I. oculus-christi L. inflorescence and pappus extracts, potentially revolutionizing biopharmaceutical and catalytic applications in these fields.

Deep Learning-Based Self-Adaptive Evolution of Enzymes

AbstractBiocatalysis has been widely used to prepare drug leads and intermediates. Enzymatic synthesis has advantages, mainly in terms of strict chirality and regional selectivity compared with chemical methods. However, the enzymatic properties of wild-type enzymes may or may not meet the requirements for biopharmaceutical applications. Therefore, protein engineering is required to improve their catalytic activities. Thanks to advances in algorithmic models and the accumulation of immense biological data, artificial intelligence can provide novel approaches for the functional evolution of enzymes. Deep learning has the advantage of learning functions that can predict the properties of previously unknown protein sequences. Deep learning-based computational algorithms can intelligently navigate the sequence space and reduce the screening burden during evolution. Thus, intelligent computational design combined with laboratory evolution is a powerful and potentially versatile strategy for developing enzymes with novel functions. Herein, we introduce and summarize deep-learning-assisted enzyme functional adaptive evolution strategies based on recent studies on the application of deep learning in enzyme design and evolution. Altogether, with the developments of technology and the accumulation of data for the characterization of enzyme functions, artificial intelligence may become a powerful tool for the design and evolution of intelligent enzymes in the future.

Activity-based metaproteomics driven discovery and enzymological characterization of potential α-galactosidases in the mouse gut microbiome

The gut microbiota offers an extensive resource of enzymes, but many remain uncharacterized. To distinguish the activities of similar annotated proteins and mine the potentially applicable ones in the microbiome, we applied an effective Activity-Based Metaproteomics (ABMP) strategy using a specific activity-based probe (ABP) to screen the entire gut microbiome for directly discovering active enzymes and their potential applications, not for exploring host-microbiome interactions. By using an activity-based cyclophellitol aziridine probe specific to α-galactosidases (AGAL), we successfully identified and characterized several gut microbiota enzymes possessing AGAL activities. Cryo-electron microscopy analysis of a newly characterized enzyme (AGLA5) revealed the covalent binding conformations between the AGAL5 active site and the cyclophellitol aziridine ABP, which could provide insights into the enzyme’s catalytic mechanism. The four newly characterized AGALs have diverse potential activities, including raffinose family oligosaccharides (RFOs) hydrolysis and enzymatic blood group transformation. Collectively, we present a ABMP platform that facilitates gut microbiota AGALs discovery, biochemical activity annotations and potential industrial or biopharmaceutical applications.

A Review of the Latest Spectroscopic Research in Pharmaceutical and Biopharmaceutical Applications

Spectroscopic analytical techniques are pivotal in the pharmaceutical and biopharmaceutical industries, facilitating the classification and quantification of processes and products. This review highlights very recent advancements in several key spectroscopic methods, including atomic, vibrational, molecular, electronic, and diffraction techniques. The applications of these analytical techniques in drug development, process monitoring, and quality control are discussed, showcasing their integral roles in advancing pharmaceutical sciences.

DiaPASEF Enables High-Throughput Proteomic Analysis of Host Cell Proteins for Biopharmaceutical Process Development.

Monitoring and quantifying host cell proteins (HCPs) in biotherapeutic production processes is crucial to ensure product quality, stability, and safety. Liquid chromatography-mass spectrometry (LC-MS) analysis has emerged as an important tool for identifying and quantifying individual HCPs. However, LC-MS-based approaches face challenges due to the wide dynamic range between HCPs and the therapeutic protein as well as laborious sample preparation and long instrument time. To address these limitations, we evaluated the application of parallel accumulation-serial fragmentation combined with data-independent acquisition (diaPASEF) to HCP analysis for biopharmaceutical process development applications. We evaluated different library generation strategies and LC methods, demonstrating the suitability of these workflows for various HCP analysis needs, such as in-depth characterization and high-throughput analysis of process intermediates. Remarkably, the diaPASEF approach enabled the quantification of hundreds of HCPs that were undetectable by a standard data-dependent acquisition mode while considerably improving sample requirement, throughput, coverage, quantitative precision, and data completeness.