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Background: Peptides and peptide-based therapeutics are biomolecules that demarcate a significant chemical space to bridge small molecules with biological therapeutics, such as antibodies, recombinant proteins, and protein domains. Introduction: Cyclooligopeptides and depsipeptides, particularly cyanobacteria-derived thiazoline-based polypeptides (CTBCs), exhibit a wide array of pharmacological activities due to their unique structural features and interesting bioactions, which furnish them as promising leads for drug discovery. Methods: In the present study, we comprehensively review the natural sources, distinguishing chemistries, and pertinent bioprofiles of CTBCs. We analyze their structural peculiarities counting the mode of actions for biological portrayals which render CTBCs as indispensable sources for emergence of prospective peptide-based therapeutics. In this milieu, metal organic frameworks and their biomedical applications are also briefly discussed. To boot, the challenges, approaches, and clinical status of peptide-based therapeutics are conferred. Results: Based on these analyses, CTBCs can be appraised as ideal drug targets that have always remained a challenge for traditional small molecules, like those involved in protein- protein interactions or to be developed as potential cancer-targeting nanomaterials. Cyclization-induced reduced conformational freedom of these cyclooligopeptides contribute to improved metabolic stability and binding affinity to their molecular targets. Clinical success of several cyclic peptides provokes the large library-screening and synthesis of natural product-like cyclic peptides to address the unmet medical needs. Conclusion: CTBCs can be considered as the most promising lead compounds for drug discovery. Adopting the amalgamation of advanced biological and biopharmaceutical strategies might endure these cyclopeptides to be prospective biomolecules for futuristic therapeutic applications in the coming times.

Large surface area, rich vascularisation, well defined mucous membrane, balanced pH and relatively low enzymatic activity makes vagina a suitable site for drugs associated with women's health issues like Urinary tract infection (UTI) and vaginal infections. Therapeutic performance of intravaginal dosage forms largely depends on the properties of polymers and drugs. Chitosan (CS) because of its unique physical, chemical, pharmaceutical and biopharmaceutical properties have received a great deal of attention as an essential component in vaginal drug delivery systems. Further the presence of free amino and hydroxyl groups on the chitosan skeleton allows easy derivatization under mild conditions to meet specific application requirements. Moreover, CS-based nanopharmaceuticals like nanoparticles, nanofiber, nanogel, nanofilm, liposomes and micelles are widely studied to improve therapeutic performance of vaginal formulations. However, susceptibility of CS to the acidic pH of vagina, poor loading of hydrophobic drugs, rapid mucosal turn over are the key issues need to be addressed for successful outcomes. In this review, we have discussed the application of CS and CS derivatives in vaginal drug delivery and also highlight the recent progress in chitosan based nanocarrier platforms in terms of their limitations and potentials.

Abstract: Luliconazole is a broad-spectrum antifungal agent with impactful fungicidal and fungistatic activity. It has shown exceptional potency against miscellaneous fungal strains like Candida, Aspergillus, Malassezia, Fusarium species and various dermatophytes. Luliconazole belongs to class II of the Biopharmaceutical Classification System with low aqueous solubility. Although it is available conventionally as 1% w/v topical cream, it has limitations of lower skin permeation and shorter skin retention. Therefore, nanoformulations based on various polymers and nanostructure carriers can be employed to overcome the impediments regarding topical delivery and efficacy of luliconazole. In this review, we have tried to provide insight into the literature gathered from authentic web resources and research articles regarding recent research conducted on the subject of formulation development, patents, and future research requisites of luliconazole. Nanoformulations can play a fundamental role in improving topical delivery by escalating dermal localization and skin penetration. Fabricating luliconazole into nanoformulations can overcome the drawbacks and can efficiently enhance its antimycotic activity. It has been concluded that luliconazole has exceptional potential in the treatment of various fungal infections, and therefore, it should be exploited to its maximum for its innovative application in the field of mycology.

Capillary electrophoresis (CE), after being introduced several decades ago, has carved out a niche for itself in the field of analytical characterization of biopharmaceutical products. It does not only offer fast separation, high resolution in miniaturized format, but equally importantly represents an orthogonal separation mechanism to high-performance liquid chromatography. Therefore, it is not surprising that CE-based methods can be found in all major pharmacopoeias and are recommended for the analysis of biopharmaceutical products during process development, characterization, quality control, and release testing. Different separation formats of CE, such as capillary gel electrophoresis, capillary isoelectric focusing, and capillary zone electrophoresis are widely used for size and charge heterogeneity characterization as well as purity and stability testing of therapeutic proteins. Hyphenation of CE with MS is emerging as a promising bioanalytical tool to assess the primary structure of therapeutic proteins along with any impurities. In this review, we confer the latest developments in capillary electrophoresis, used for the characterization of critical quality attributes of biopharmaceutical products covering the past 6 years (2015-2021). Monoclonal antibodies, due to their significant share in the market, have been given prioritized coverage.

Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non-enzymatic, post-translational modification of N-terminal HisTags. We report high-yield, site-selective in vitro α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pH 7.5 in 1 h. Conjugates slowly hydrolyse, but diol-masking with borate esters inhibits reversibility. In an example, we multimerise azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry, to give a COVID-19 vaccine. Compared to yeast antigen, HEK-derived RBD was immunologically superior, likely due to observed differences in glycosylation. We show the benefits of ordered over randomly oriented multimeric antigen display, by demonstrating single-shot seroconversion and best virus-neutralizing antibodies. Azidogluconoylation is simple, fast and robust chemistry, and should accelerate research and development.

Made available in DSpace on 2021-06-25T11:15:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-10-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Engineering and Physical Sciences Research Council Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Pharmaceutical co-crystals have been explored by many researchers as a strategy to optimize physicochemical properties of solid-state drugs. Their improvements of solubility, bioavailability, and the reduced tendency for phase transformation occurrence, are factors that highlight benefits of pharmaceutical co-crystals among other solid forms. According to the Biopharmaceutical Classification System (BCS), non-steroidal anti-inflammatory drugs (NSAIDs) are class II drugs, which have low aqueous solubility and therefore co-crystallization has the potential to optimize NSAID product properties. In this review, we highlight the recent progress made on NSAIDs co-crystals, their co-formers, synthesis, methods and use, while we underline some promising results on in vitro and in vivo co-crystal properties. A celecoxib-tramadol co-crystal reaches phase III clinical trials, showing greater analgesic activity than both individual APIs. The aqueous solubility of the co-crystal formed between L-proline and diclofenac is very high in comparison with the pure drug. Naproxen co-crystals with urea and thiourea have an increase of drug release of almost 60%. Co-crystal design brings a new perspective in drug development since the co-former used can also be a biologically active component allowing to combine different anti-inflammatory drugs, which have an incredible spectrum of application due to the analgesic, anti-pyretic and anti-inflammatory properties. São Paulo State University (UNESP) School of Pharmaceutical Sciences São Paulo State University (UNESP) Institute of Chemistry EPSRC Centre for Innovative Manufacturing in Continuous Manufacturing and Crystallisation University of Strathclyde São Paulo State University (UNESP) School of Sciences São Paulo State University (UNESP) School of Pharmaceutical Sciences São Paulo State University (UNESP) Institute of Chemistry São Paulo State University (UNESP) School of Sciences

Biopharmaceuticals (recombinant technology-based products, vaccines, whole blood and blood components, gene therapy, cells, tissues, etc.,) are described as biological medical products produced from various living sources such as human, microbial, animal, and so on by manufacturing, extraction, or semi-synthesis. They are complex molecules having high molecular weights. For their safety and efficacy, their structural, clinical, physicochemical, and chemical features must be carefully controlled, and they must be well characterized by analytical techniques before the approval of the final product. Capillary electrophoresis (CE) having versatile modes can provide valuable safety and efficacy information, such as amino acid sequence, size variants (low and high molecular weight variants), charged variants (acidic and basic impurities), aggregates, N-linked glycosylation, and O-linked glycosylation. There are numerous applications of CE in the literature. In this review, the most significant and recent studies on the analysis of recombinant DNA technology-based products using different CE modes in the last ten years have been overviewed. It was seen that the researches mostly focus on the analysis of mAbs and IgG. In addition, in recent years, researchers have started to prefer CE combined mass spectrometry (MS) techniques to provide a more detailed characterization for protein and peptide fragments.

Vesicular drug delivery systems including niososmes, liposomes, pharmacosomes, transferosomes, electrosomes, ethosomes, etc have been widely accepted for controlled delivery of the drug. Amongst, all these drug delivery systems pharmacosomes are gaining more attention of the researchers due to several benefits such as high entrapment efficiency, improved biopharmaceutical properties, and pharmacokinetic performance, no leakage or loss of drug, stability, etc. Pharmacosomes are amphiphilic phospholipid complexes of drugs having active hydrogen that bind to phospholipids and self-assembled into vesicles in an aqueous medium. Both hydrophilic and lipophilic drugs have been formulated into pharmacosomes that caused improved solubility and permeability of drugs. Pharmacosomes are prepared by using various techniques such as hand shaking method, ether injection, solvent evaporation method, supercritical fluid approach, etc and are characterized for prodrug confirmation, surface morphology, crystal state measurement, in vitro drug release, and stability, etc. Despite wide research and highly encouraging results in the preclinical studies, translation of these nanomedicines from laboratory to market has been very limited. The main aim of this review is to describe comprehensively the potential of pharmacosomes as a vesicular drug delivery system focusing mainly on their conventional and advanced methods of preparation, different characterization techniques, and their applications in the delivery of different types of drugs with improved biopharmaceutical properties and pharmacokinetic performance.

The development of efficient, high-quality, harmless and affordable pharmaceutical forms for drug treatment in pediatrics is a current task of contemporary pharmacy. Currently, psychiatrists and neurologists from various curative-prophylactic institutions prescribe for children and adolescents nootropic preparations in the form of tablets, powders and syrups, in various neurological diseases, the share of which, according to national and international statistics, is constantly increasing. Currently, the prescription of hopanthenic acid is gaining popularity, thanks to its neuroprotective, neurotrophic, neurometabolic, antidepressant, anxiolytic and analgesic effects. The presented paper has as starting point the analysis of the structural-molecular properties of hopanthenic acid, exposed by biopharmaceutical study, pharmacokinetic parameters and strategies for optimizing pharmaceutical formulations with gastric release.

Salification is one of the powerful and widely employed approaches to improve the biopharmaceutical properties of drugs. The FDA's eighty-year trajectory of new drug approvals depicts around one-third of the drugs clinically used as their pharmaceutical salts. Among various cationic and anionic counterions used in FDA-approved pharmaceutical salts, the carboxylic acids have significantly contributed. A total of 94 pharmaceutical salts discovered during 1943-2020 comprises carboxylic acids as counterions with a major contribution of acetate, maleate, tartrate, fumarate, and succinate. Hydrocodone tartrate is the first FDA-approved carboxylate salt approved in 1943. Overall, the analysis shows that fifteen carboxylic acid counterions are present in FDA-approved pharmaceutical salts with a major share of acetate (18 drugs). This review provides an account of FDA-approved carboxylate salts from 1939 to 2020. The decade-wise analysis indicates that 1991-2000 contributed a maximum number of carboxylate salts (24) and least (3) in 1939-1950. The technical advantage of carboxylate salts over free-base or other counterions is also discussed. Graphical Abstract.

Nanocochleates (NCs) are amongst the nanoformulations gaining wide popularity owing to their ability to improve biopharmaceutical performance of drug molecules remarkably. However, biggest drawback...

Therapeutic antibodies have many biopharmaceutical applications; however, characterization of their higher-order structures is a major concern in quality control. We have developed AF.2A1, an artificial protein, that specifically recognizes non-native, structured IgGs. We performed binding assays using various types of IgGs and fragments to investigate the mechanisms by which AF.2A1 interacts with the non-native IgG. AF.2A1 recognized the acid-stressed IgGs from human, mouse, and rat, but not rabbit. Binding assays using the human IgG1 fragments revealed that an interface emerged by deleting five C-terminal residues. We conclude that AF.2A1 recognizes an exposed hydrophobic core centered on the Trp417. Our results concur with those of the previous studies showing that C-terminal structural changes occur early during antibody denaturation and aggregation. Our findings explain the molecular rationale for using AF.2A1 in quality control of biopharmaceutical IgGs.

Drug delivery in conventional dosage forms often suffers from the drawbacks of repeated drug administration and large fluctuations in blood drug levels. Controlled drug delivery systems are a convenient way of controlling the dosing frequency responsible for rapid absorption and distribution of drug in conventional dosage forms, and are dependent upon two intrinsic properties of the drug, namely, elimination half-life (t1/2) and therapeutic index (TI). The goal is to give a drug at a sufficient rate, frequency and dose so that the ratio Cmax/Cmin in plasma at steady state is always maintained at effective concentrations during the course of therapy, reducing side effects or improving physicochemical and biopharmaceutical properties. The use of polymers provides the potential to control drug delivery both temporally and spatially. The objective of the present investigation was to develop bilayered tablets of orlistat to achieve controlled release and immediate release. The process is predetermined in such a way to release the drug at an IR and CR by using different polymers. Half life of orlistat is 1-2 hrs, as it has been released immediately. This paper mainly focuses on designing the process to release the drug in a controlled manner by using different polymers like sodium alginate, ethyl cellulose, HPMC.

Photooxidation of methionine (Met) and tryptophan (Trp) residues is common and includes major degradation pathways that often pose a serious threat to the success of therapeutic proteins. Oxidation impacts all steps of protein production, manufacturing, and shelf life. Prediction of oxidation liability as early as possible in development is important because many more candidate drugs are discovered than can be tested experimentally. Undetected oxidation liabilities necessitate expensive and time-consuming remediation strategies in development and may lead to good drugs reaching patients slowly. Conversely, sites mischaracterized as oxidation liabilities could result in overengineering and lead to good drugs never reaching patients. To our knowledge, no predictive model for photooxidation of Met or Trp is currently available. We applied the random forest machine learning algorithm to in-house liquid chromatography-tandem mass spectrometry (LC-MS/MS) datasets (Met, n = 421; Trp, n = 342) of tryptic therapeutic protein peptides to create computational models for Met and Trp photooxidation. We show that our machine learning models predict Met and Trp photooxidation likelihood with 0.926 and 0.860 area under the curve (AUC), respectively, and Met photooxidation rate with a correlation coefficient (Q2) of 0.511 and root-mean-square error (RMSE) of 10.9%. We further identify important physical, chemical, and formulation parameters that influence photooxidation. Improvement of biopharmaceutical liability predictions will result in better, more stable drugs, increasing development throughput, product quality, and likelihood of clinical success., Graphical abstract, We present machine learning strategies to accurately predict methionine and tryptophan photooxidation. We identify important physicochemical and formulation parameters that influence photooxidation. There is limited AI-based support in biopharmaceutical development and no available photooxidation prediction for proteins. Better predictive tools result in increased development throughput, product quality, and clinical success.

Introduction. The presence of biologically active substances in the extract from the roots of turmeric extract (Curcuma longa L.) makes it reasonable and makes it possible to predict their effectiveness in pathological conditions caused by oxidative stress and immune disorders arising against its background. Based on data obtained, a pattern was revealed in improving the solubility of the combination of methionine and turmeric extract, which includes auxiliary substances. Dosage form – granules with methionine and liquid turmeric extract, with enteric coat can be used to eliminate the consequences of oxidative and immune disorders, treatment and prevention of systemic diseases caused by oxidative stress.Aim. Biopharmaceutical and in-process tests of the pharmaceutical composition of granules with methionine and liquid turmeric extract.Materials and methods. The object of the trials was the multicomponent drug containing methionine and a liquid turmeric extract of the roots. The proposed composition of plant compositions is original, had no analogues and protected by a patent for an invention (RU 268 4111). To assess the pharmaceutical properties, biopharmaceutical trials carried out by the UV-spectrophotometric method using biorelevant medium. The research results statistically processed using Statistica for Windows, version 10.Results and discussion. Based on the data obtained, a pattern revealed in improving the solubility of the combination of methionine and turmeric extract, in contrast to the multicomponent composition containing only turmeric extract.Conclusion. The developed multicomponent pharmaceutical composition will expand the register of herbal medicines of domestic production for the prevention and treatment of diseases caused by oxidative stress.

The pharmaceutical industry and most particularly the biopharmaceutical industry are demanding advancements in analytical techniques. The field of proteomics and peptide mapping needs sensitive analysis and sophisticated instrumentation, which needs hyphenated analytical techniques to be explored. In addition to effective detection and characterization, Due to efficient and selective separation, predominant focus has been drawn to capillary electrophoresis/mass spectrometry (CE/MS) compared to widely used techniques such as slab-gel electrophoresis (SGE), liquid chromatography (LC) and capillary electrophoresis (CE) Several significant advances in CE/MS instrumentation and software have made CE/MS a competitive tool in the last decade to solve a wide variety of analytical and biopharmaceutical problems. This analysis discusses the technological aspects of CE / MS interfaces, analytical separation modes, MS analyzer, recent CE / MS approaches and applications specific to biopharmaceutical. Applications using some of the more common electrophoretic separation modes, such as conventional electrophoresis of the capillary zone (CZE) and capillary isoelectric focusing (CIEF), have been highlighted. The peptide analysis also includes recent studies on capillary electro-chromatography (CEC) / MS and CE-MS applications. Keywords: Capillary electrophoresis, Mass spectrometry, Pharmaceutical applications, Peptide analysis, Proteomics, Intact protein.

Mass spectrometry (MS) plays a key role throughout all stages of drug development and is now as ubiquitous as other analytical techniques such as surface plasmon resonance, nuclear magnetic resonance, and supercritical fluid chromatography, among others. Herein, we aim to discuss the history of MS, both electrospray and matrix-assisted laser desorption ionization, specifically for the analysis of antibodies, evolving through to denaturing and native-MS analysis of newer biologic moieties such as antibody-drug conjugates, multispecific antibodies, and interfering nucleic acid-based therapies. We discuss challenging therapeutic target characterization such as membrane protein receptors. Importantly, we compare and contrast the MS and hyphenated analytical chromatographic methods used to characterize these therapeutic modalities and targets within biopharmaceutical research and highlight the importance of appropriate MS deconvolution software and its essential contribution to project progression. Finally, we describe emerging applications and MS technologies that are still predominantly within either a development or academic stage of use but are poised to have significant impact on future drug development within the biopharmaceutic industry once matured. The views reflected herein are personal and are not meant to be an exhaustive list of all relevant MS performed within biopharmaceutical research but are what we feel have been historically, are currently, and will be in the future the most impactful for the drug development process.

Size exclusion chromatography (SEC) is a powerful tool for analyzing size variants of proteins in academic and biopharmaceutical laboratories. Recent advancements in SEC methodology have improved s...

From traditional monoclonal antibodies to more and more complex mAb-based formulations, biopharmaceutical faces one challenge after another. To avoid these issues, identification of therapeutic proteins in the initial discovery process that has high stability and low self-interaction would simplify the development of safe and effective antibody therapeutics. Affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) is a new prediction method capable of identifying mAbs with different self-association propensity. In this study, 10 formulated monoclonal antibody (mAb) therapeutics include different mAb isotypes and co-formulated antibodies were measured by AC-SINS and some biophysical methods to predict protein stability. The prediction results of all 10 mAbs were compared to their stability data (Δ%monomer and Δ%HMWs) at accelerated (25°C and 40°C) and long-term storage conditions (4°C) as measured by size exclusion chromatography. AC-SINS method has a good predictive correlation with each mAbs and co-formulated antibodies. There were no physicochemical, intermolecular, or biological interactions that occurred between the two components of co-formulated antibodies which confirmed by Analytical ultracentrifugation (AUC). Here we discuss the correlation between each method and protein stability, and also use AC-SINS assay to predict the stability of co-formulated antibodies for the first time. This may be an effective way to predict the stability of these complex mAb-based formulations such as co-formulated mAbs.

Objectives. In this biosimilar research, we compare the monoclonal antibody eculizumab obtained from different drugs [original Soliris® (Alexion Pharmaceuticals) and candidate Elizaria® (Generium)] by intact mass measurement and middle-up mass spectrometry analysis to enhance the role of mass spectrometry methods in biopharmaceutical development processes.Methods. The intact mass measurement is performed using a high-resolution ESI-MS. The middle-up analysis is performed by reversed-phase high-performance liquid chromatography with ESI-MS detection, subsequent IdeS treatment of antibodies, and disulfide bond reduction.Results. We have shown some small differences between the original and candidate drugs in the minor glycans level. Man5 glycan is only found in the original Soliris, and G0 is only found in the Elizaria. Glycation sites are also found in the light chain and Fd subunits of the original Soliris. The glycation level does not exceed 4.4%. The non-clipped C-end lysine level and G0F glycan levels are slightly lower in the original Soliris. All registered differences are not crucial for eculizumab’s quality and do not affect its effectiveness and preclinical safety. Generally, the results show a high level of similarity between the original and candidate drugs.Conclusions. The comparative mass spectrometry analysis of eculizumab in the original Soliris and Elizaria allows us to estimate their high degree of similarity by molecular masses and major modification profiles.

Most functional biopharmaceuticals such as antibodies are glycoproteins carrying N-linked oligosaccharides (N-glycans). In animal cells, these glycans are generally expressed as heterogeneous glycoforms that are difficult to separate into a pure form. The structure of these glycans directly affects several biological aspects of the glycoproteins, especially binding affinity. Therefore, the preparation of glycoproteins with well-defined and homogeneous glycoforms is necessary for functional studies and improved efficacy, particularly for biopharmaceuticals. This review describes the recent remarkable progress in the development and production of biopharmaceutical glycan-modified antibodies, through the use of glycan remodeling using microbial endoglycosidases and sophisticated glycoengineering techniques utilizing microbial enzymatic reaction mechanisms.

The Nano ionization technology is particularly promising for improving the oral absorption and bioavailability of BCS class II drugs. One such approach is Nano ionization thereby increasing surface area and hence dissolution and ultimately the bioavailability. Ritonavir is a BCS class II and IV drug. Valsartan is a commonly used antihypertensive agent belonging to the family of angiotensin II receptor antagonists. Valsartan is a BCS class II drug being slightly soluble in water. It has a very low oral bioavailability of 25-35% due to its limited solubility in biological fluids. Poor water solubility of drugs shows insufficient bioavailability and fluctuating plasmalevels. Based on their physicochemical and biopharmaceutical properties, Ritonavir and Valsartan were selected as a drug candidates for developing Nanosuspension based formulations for improving the solubility and bioavailability by enhancing the rate and extent ofdissolution.The scope of the present work is to enhance the solubility, dissolution rate and oral bio availability of poorly soluble drugs Ritonavir and Valsartan by formulating them into Nanosuspension with Poloxamer as a stabilizing agent and subsequently converting the optimized Nanosuspension of Ritonavir/Valsartan drug molecule into tablets and capsuleswith the help of various extragranular agents.

Hydrolytic degradation of polysorbate during 2–8°C storage of monoclonal antibody drug products has been attributed to residual enzymes (e.g., esterases) from bioprocessing steps. Robust detection of esterase activity using sensitive, non-polysorbate surrogate substrates can provide an alternate method to assess polysorbate degradation risk, if the correlation between the esterase activity and polysorbate degradation is established. A general esterase activity assay was developed as a monitoring and characterization tool during bioprocess development of monoclonal antibodies. We report a fluorescence plate-based assay for quantifying esterase activity, utilizing 4-methylumbelliferyl caprylate (MU-C8) as the esterase substrate. The assay was first assessed for substrate, inhibitor and pH specificity using both model enzymes and purified protein samples. The assay was then extensively tested to understand sample matrix effects on activity rates. The use of this high-throughput method will allow for rapid characterization of protein samples in under three hours. The esterase activity correlated directly with polysorbate degradation and can provide valuable information on polysorbate degradation risk throughout drug development.

Generic medicines occupy dominant positions both in the pharmaceutical market of Ukraine and in industrial production by domestic pharmaceutical enterprises. The use of generic drugs in medical practice is of significant medical and social importance for expanding the accessibility of the general population to essential drugs. In Ukraine, more than twenty generic medicines based on clopidogrel, both foreign and domestic, are registered. All generic drugs containing clopidogrel bisulfate must comply with pharmaceutical bisulfate must comply with pharmaceutical equivalence, the kinetics of release of the active pharmaceutical ingredient using the Dissolution test in vitro, and pharmacokinetic parameters in vivo. The aim of the work was to carry out comparative studies of the dissolution kinetics of four samples of generic drugs based on clopidogrel with the dissolution kinetics of the original drug Plavix®, to evaluate the similarity factor of dissolution profiles and to determine the effect of biopharmaceutical factors on the equivalence of generics. Comparative studies of the kinetics of dissolution were carried out by the in vitro method according to the «Dissolution» test using a device with a blade with a rotation speed of 50 rpm, a dissolution medium with a pH value of 2.0 in a volume of 900 ml at a temperature of 37 ± 1 °C. The determination of the quantitative content of clopidogrel, which passed into the dissolution medium, was carried out by the method of adsorption spectrophotometry in the ultraviolet region at a wavelength of about 240 ± 2 nm. Based on the data obtained, the dissolution profiles of the original drug Plavix® and the studied samples of generic drugs were constructed, the similarity of which was assessed by the value of the similarity factor. According to the research results, it was found that one sample of the generic drug proved its equivalence by the in vitro method to Plavix®, and three other samples of generics had differences in dissolution kinetics in comparison with the original drug. Biopharmaceutical factors were analyzed that could affect the dissolution kinetics of the studied generic drugs, from which the physicochemical characteristics of clopidogrel bisulfate, the qualitative and quantitative composition of excipients and the features of the technological process were determined. Thus, on the basis of the comparative studies of the dissolution kinetics of drugs based on clopidogrel, generics were found that did not correspond to the in vitro equivalence according to the Dissolution test to the original drug, which could be due to the influence of a combination of biopharmaceutical factors.

Biopharmaceuticals, such as proteins, peptides, nucleic acids and vaccines, bring about great hopes for the prevention and treatment of various diseases, but the industrialization of these products still faces challenges such as structural instability, inefficient bioactivity and low bioavailability. Ionic liquids (ILs), the marvelous solvent media with inimitable and tunable properties, may provide alternative solutions to overcome the above problems of biopharmaceutical industry. Progress has gradually been made through studies by combination of ILs with biomacromolecules. The applications involved the stabilization, protection, and delivery of biopharmaceuticals. Recent trends are being forwarded to using ILs in vaccines and nucleic acid drugs. However, challenges remain on the toxicity and safety issues. Besides, the cost of adding ILs to the benefits of biopharmaceuticals need to be considered.

The rapid rise in research interest in carbohydrate-based polymers is undoubtedly due to the nontoxic nature of such materials in an in vivo environment and the versatile roles that the polymers can play in cellular functions. Such polymers have served as therapeutic tools for drug delivery, including antigens, proteins, and genes, as well as diagnostic devices. Our focus in the first half of this Review is on synthetic methods based on ring-opening polymerization and enzyme-catalyzed polymerization, along with controlled radical polymerization. In the second half of this Review, sugar-based polymers are discussed on the basis of their remarkable success in competitive receptor binding, as multifunctional nanocarriers of targeting inhibitors for cancer treatment, in genome-editing delivery, in immunotherapy based on endogenous antibody recruitment, and in treatment of respiratory diseases, including influenza A. Particular emphasis is put on the synthesis and biopharmaceutical applications of sugar-based polymers published in the most recent 5 years. A noticeable attribute of carbohydrate-based polymers is that the sugar-receptor interactions can be facilitated by the cooperative effect of multiple sugar units. Their diversified topology and structures will drive the development of new synthetic strategies and bring about important applications, including coronavirus-related drug therapy.

Ellagic acid (EA) is a polyphenolic active compound with antimalarial and other promising therapeutic activities. However, its solubility and its permeability are both low (BCS IV). These properties greatly compromise its oral bioavailability and clinical utilizations. To overcome these limitations of the physicochemical parameters, several formulation approaches, including particle size reduction, amorphization and lipid-based formulations, have been used. Although these strategies have not yet led to a clinical application, some of them have resulted in significant improvements in the solubility and bioavailability of EA. This critical review reports and analyses the different formulation approaches used by scientists to improve both the biopharmaceutical properties and the clinical use of EA.

Protein instability caused by exposure to external additives or severe stress may result in different diseases. Of these diseases, many are triggered by protein misfolding and denaturation, leading to neurodegenerative disorders. Additionally, many other diseases are treated by biopharmaceutical approaches using proteins as drugs, which is again prone to denaturation. Numerous reagents and methods have been developed to understand protein instability and protection. However, the development of polymer-based protein protection agents as well as small molecules and peptides for combatting protein instability by inhibiting aggregation and facilitating protein refolding only gained attention in the last decade. In this review, we discuss protein aggregation inhibition and the role of polymers in protein protection. Further, we outline the significance of chemical additives in facilitating protein refolding and their importance in the use of recombinant proteins for treating neurodegenerative disorders.

Core-shell structured nanoparticles (NPs) render the simultaneous coloading capacity of both hydrophobic and hydrophilic drugs and may eventually enhance therapeutic efficacy. In this study, we employed a facile squalenoylation technology to synthesize a new amphiphilic starch derivative from partially oxidized starch, which self-assembled into core-shell starch NPs (StNPs) only at a squalenyl degree of substitution (DoS) of ∼1%. The StNPs characteristics could be tuned as the functions of the polymer molecular weight, DoS, and NPs concentration. The biopharmaceutical features of the StNPs, including colloidal stability, carrier properties, and biocompatibility, were carefully investigated. The interaction study between StNPs and mucin glycoproteins, the main organic component of mucus, revealed a moderate mucin interacting profile. Furthermore, the StNPs also showed good penetration through Pseudomonas aeruginosa biofilms. These results nominate StNPs as a versatile drug delivery platform with potential applications for mucosal drug delivery and the treatment of persistent infections.

Several types of cutaneous fungal infections can affect the population worldwide, such as dermatophytosis, cutaneous candidiasis, onychomycosis, and sporotrichosis. However, oral treatments have pronounced adverse effects, making the topical route an alternative to avoid this disadvantage. On the other hand, currently available pharmaceutical forms designed for topical application, such as gels and creams, do not demonstrate effective retention of biomolecules in the upper layers of the skin. An interesting approach to optimise biomolecules' activity in the skin is the use of nanosystems for drug delivery, especially solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), which in the past decade has shown advantages like increased adhesiveness, great occlusive properties and higher biomolecule deposition in stratum corneum when designed for topical application. Considering the demand for more effective therapeutic alternatives and the promising characteristics of SLN and NLC for topical application, the present study sought to gather studies that investigated the potential of using SLN and NLC for the treatment of cutaneous fungal infections. Studies demonstrated that these nanosystems showed optimisation, mostly, of the effectiveness of biomolecules besides other biopharmaceutical properties, in addition to offering potential occlusion and hydration of the applied region.

The present work was aimed at formulating solidified reverse micellar microparticles (SRMMs) using templated lipids (Softisan 154, stearic acid, and Phospholipon ®90H) for improved biopharmaceutical performance, dissolution of miconazole nitrate. SRMMs containing miconazole nitrate (MN) (1, 2, and 3% w/w) were formulated using Softisan® 154 (SOFT) and stearic acid (ST) and their combinations, containing Phospholipon® 90H (P90H) by melt homogenization employing Polysorbate 80 as the surfactant. The microparticles were freeze-dried, characterized, and optimized batches evaluated for in vitro dissolution in methanolic HCl and antifungal activity. The average SRMM yield was 70%. Entrapment efficiency and loading capacity determination showed that the lipid matrix composed of Softisan® 154 and Phospholipon® 90H (7:3) possessed greater entrapment than the other matrices with the 5% SOFT + P90H matrix containing 3% MN entrapping 98.48% of the drug. Preliminary anticandidal evaluation of the formulations showed significant activity of all the formulations containing drug. DSC analysis of the lipid excipients and matrices showed slight modifications in the physical nature of the excipients. Further analysis using X-ray diffraction showed retention of drug form in the formulations though with slight alterations in d-spacings of the X-ray diffractograms. In vitro dissolution studies of the optimized batches revealed a significant (p

We have adopted the concept of bispecific antibodies, which can simultaneously block or cross-link two different biomolecular targets, to create bispecific enzymes by exploiting the homodimeric quaternary structure of bacterial phosphotriesterases (PTEs). The PTEs from Brevundimonas diminuta and Agrobacterium radiobacter, whose engineered variants can efficiently hydrolyze organophosphorus (OP) nerve agents and pesticides, respectively, have attracted considerable interest for the treatment of the corresponding intoxications. OP nerve agents and pesticides still pose a severe toxicological threat in military conflicts, including acts of terrorism, as well as in agriculture, leading to >100000 deaths per year. In principle, engineered conventional homodimeric PTEs may provoke hydrolytic inactivation of individual OPs in vivo, and their application as catalytic bioscavengers via administration into the bloodstream has been proposed. However, their narrow substrate specificity would necessitate therapeutic application of a set or mixture of different enzymes, which complicates biopharmaceutical development. We succeeded in combining subunits from both enzymes and to stabilize their heterodimerization by rationally designing electrostatic steering mutations, thus breaking the natural C2 symmetry. The resulting bispecific enzyme from two PTEs with different bacterial origin exhibits an ultrabroad OP substrate profile and allows the efficient detoxification of both nerve agents and pesticides. Our approach of combining two active sites with distinct substrate specificities within one artificial dimeric biocatalyst-retaining the size and general properties of the original enzyme without utilizing protein mixtures or much larger fusion proteins-not only should facilitate biological drug development but also may be applicable to oligomeric enzymes with other catalytic activities.

Metabolic engineering of mammalian cells has to-date focused primarily on biopharmaceutical protein production or the manipulation of native metabolic processes towards therapeutic aims. However, significant potential exists for expanding these techniques to diverse applications by looking across the taxonomic tree to bioactive metabolites not synthesized in animals. Namely, cross-taxa metabolic engineering of mammalian cells could offer value in applications ranging food and nutrition to regenerative medicine or gene therapy. Towards the former, recent advances in meat production through cell culture suggest the potential to produce meat with fine cellular control, where tuning composition through cross-taxa metabolic engineering could enhance nutrition and food-functionality. Here we demonstrate this possibility by engineering primary bovine and immortalized murine muscle cells with prokaryotic enzymes to endogenously produce the antioxidant carotenoids phytoene, lycopene and β-carotene. These phytonutrients offer general nutritive value and protective effects against diseases associated with red and processed meat consumption, and so offer a promising proof-of-concept for nutritional engineering in cultured meat. We demonstrate the phenotypic integrity of engineered cells, the ability to tune carotenoid yields, and antioxidant functionality of these compounds in vitro towards both nutrition and food-quality objectives. Our results demonstrate the potential for tailoring the nutritional profile of cultured meats. They further lay a foundation for heterologous metabolic engineering of mammalian cells for applications outside of the clinical realm., This document is the Authors Accepted Manuscript (or "postprint") and may have minor differences from the Version of Record due to final copyedits. The final published version can be accessed at https://doi.org/10.1016/j.ymben.2020.07.011.

Controlling the viscosity of concentrated protein solutions – usually reducing – is an open challenge, with major recent relevance in protein formulations for biopharmaceutical, medical, food, and other applications. The addition of viscosity-reducing additives generally not only changes the viscosity of the protein solutions but also the actual secondary/tertiary structure of the proteins, which is usually highly undesirable, and can be even toxic in systems, such as for biopharmaceutical applications. Therefore, it is of major importance to be able to establish control over the combination of viscosity-affecting additives and adequate protein stability, usually at high protein concentrations. Here, we demonstrate the control and manipulation of the viscosity profile of a selected protein solution (monoclonal antibody of immunoglobulin gamma type IgG) of direct biopharmaceutical relevance, by identifying elementary viscosity contributions via selected additives that target different protein–protein interactions. Specifically, a combined study of viscosity control and protein aggregation is performed with viscosity characterized by microfluidic measurements and protein aggregation by size-exclusion chromatography, where aggregation data is further supplemented with conformational stability measurements via thermal and chemical protein denaturation. A dissection of contributions to total viscosity – steric, electrostatic, hydrophobic, van der Waals – is performed. A novel mechanism of the impact of electrostatic interactions on the viscosity of IgG solutions is proposed based on interacting charged protein patches subjected to orientational alignment under flow birefringence. More generally, we show a control over the interplay of viscosity, potency and stability in a distinct protein system, as a general contribution to understanding the viscosity in different colloidal, biological, and soft materials.

Glycosylated biopharmaceuticals are important in the global pharmaceutical market. Despite the importance of their glycan structures, our limited knowledge of the glycosylation machinery still hinders controllability of this critical quality attribute. To facilitate discovery of glycosyltransferase specificity and predict glycoengineering efforts, here we extend the approach to model N-linked protein glycosylation as a Markov process. Our model leverages putative glycosyltransferase (GT) specificity to define the biosynthetic pathways for all measured glycans, and the Markov chain modelling is used to learn glycosyltransferase isoform activities and predict glycosylation following glycosyltransferase knock-in/knockout. We apply our methodology to four different glycoengineered therapeutics (i.e., Rituximab, erythropoietin, Enbrel, and alpha-1 antitrypsin) produced in CHO cells. Our model accurately predicted N-linked glycosylation following glycoengineering and further quantified the impact of glycosyltransferase mutations on reactions catalyzed by other glycosyltransferases. By applying these learned GT-GT interaction rules identified from single glycosyltransferase mutants, our model further predicts the outcome of multi-gene glycosyltransferase mutations on the diverse biotherapeutics. Thus, this modeling approach enables rational glycoengineering and the elucidation of relationships between glycosyltransferases, thereby facilitating biopharmaceutical research and aiding the broader study of glycosylation to elucidate the genetic basis of complex changes in glycosylation.

Recently, biologics including peptides, proteins, antibodies, and nucleic acids have attracted interest as drug candidates for new modalities, since these compounds can act on target molecules that are not be affected by conventional drugs with a small molecular weight to promote greater selectivity, potency, and safety. Generally, to administer biologics, parenteral routes like intravenous and intramuscular injections have been mainly selected due to their poor oral absorbability and stability in the gastrointestinal tract, which can adversely affect patient compliance. Depending on the target diseases, inhalable formulations can be used to achieve both topical effects in the respiratory tracts and systemic actions due to the characteristics of the pulmonary site, including a large surface area, abundant capillary network, thin membrane with adequate permeability for macromolecules, reduced enzymatic degradation, and a lack of first-pass metabolism. In this study, to achieve desirable delivery of peptide drugs with an inhalable formulation to target sites in the respiratory tract and/or absorption sites in the lung, peptide-loaded inhalable formulations were designed by the application of flash nanoprecipitation, one of the precipitation methods to prepare functional nanoparticles, and the fine droplet drying process, a powderization technique using printing technology, to control the pharmacokinetic behavior. From the findings of the study, the strategic applications of these techniques could contribute to provide peptide-loaded inhalable formulations to enhance their biopharmaceutical potentials.

In recent decades, in addition to existing small-molecule drug therapies, biomedical technology has also rapidly progressed, leading to the development of various therapies based on biopharmaceuticals and therapeutic cells. However, these materials require effective separation methods for their analysis and production. A representative separation method, which has been extensively studied, is the temperature-responsive chromatography system using poly(N-isopropylacrylamide) and its copolymers. Over the last 20 years, various temperature-responsive chromatographic techniques have been developed for the separation of different types of analytes by changing the copolymer composition, the polymer graft configuration, and the base materials of the stationary phase. The developed methods have been successfully applied for the separation of small-molecule drugs, peptides, and proteins, without affecting their biological activity, simply by changing the column temperature. Furthermore, temperature-modulated cell separation columns have been investigated for the separation of cells without changing their properties. Therefore, the developed methods can serve as effective tools for the current and future bioseparation of various biological compounds, biopharmaceutical proteins, and therapeutic cells that are currently used in therapies.

Diacerein (DIA) is an approved treatment for osteoarthritis. However, its clinical effectiveness is limited because of its poor aqueous solubility, which causes bioavailability issues. The current ...

Background: Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) are useful drug delivery systems for dermal application. Thanks to their biocompatible and biodegradable profile, these carriers offer many advantages such as enhanced bioavailability, low toxicity, viable drug targeting and controlled release. SLN and NLC are composed of well-tolerated lipids, including natural fats and oils that are successfully used in the pharmaceutical and cosmetic dermal formulation. Objective: This article presents an overview of the benefits of selecting natural fats and oils as structural components of SLN and NLC for topical application. Methods: This review is based on data published over the past 20 years about the development of stable and nontoxic lipid nanoparticles with natural lipids. We shed light on the role of natural fats in skin restoration, as well as on the contributed penetration and occlusive properties of SLN and NLC. Results: The deliberate selection of excipients (type and lipid ratio) influences the quality of the final dermal formulation. Natural lipids show good compatibility with different active molecules and are able to create stable lipid matrices that facilitate the biopharmaceutical properties of lipid nanoparticles. Patents involving natural fats and oils in SLN and NLC composition are listed, yet it is important to note that the approved marketed formulations are mainly cosmetic, not pharmaceutical, products. Conclusion: Natural lipids can enhance topical drug delivery by adding their ability of improving skin penetration and hydration to the permeation and occlusion properties of SLN and NLC.

Catalase is an antioxidant enzyme with potential application in photoprotection, skin aging, and skin disorders as vitiligo. However, as a large and hydrophilic molecule, catalase topical applicati...

Nucleic acid therapeutics for RNA interference (RNAi) are gaining attention in the treatment and management of several kinds of the so-called "undruggable" tumors via targeting specific molecular pathways or oncogenes. Synthetic ribonucleic acid (RNAs) oligonucleotides like siRNA, miRNA, shRNA, and lncRNA have shown potential as novel therapeutics. However, the delivery of such oligonucleotides is significantly hampered by their physiochemical (such as hydrophilicity, negative charge, and instability) and biopharmaceutical features (in vivo serum stability, fast renal clearance, interaction with extracellular proteins, and hindrance in cellular internalization) that markedly reduce their biological activity. Recently, several nanocarriers have evolved as suitable non-viral vectors for oligonucleotide delivery, which are known to either complex or conjugate with these oligonucleotides efficiently and also overcome the extracellular and intracellular barriers, thereby allowing access to the tumoral micro-environment for the better and desired outcome in glioblastoma multiforme (GBM). This Review focuses on the up-to-date advancements in the field of RNAi nanotherapeutics utilized for GBM treatment.

N-linked glycosylation is a post-translational modification that occurs on many proteins during biosynthesis. The profile of different glycans on the protein is a critical quality attribute of some recombinant biopharmaceutical proteins including monoclonal antibodies (mAbs). Methods for profiling glycan should be robust, fast, and sensitive. Isolating glycans from proteins and tagging a label on glycans is the most commonly used technique for glycan profiling. Currently, existing protocols for sample preparation can be complicated, time-consuming, and expensive, which can limit the wide adaptation of glycan profiling methods. As a further barrier to use, an expensive ultra-high-pressure liquid chromatography (UHPLC) system is frequently required for the profile. In this article, a low cost and easily-used workflow of sample preparation is coupled with a standard high-performance liquid chromatography (HPLC) system to achieve comparable results to UHPLC. The number of steps required in the protocol and the time, as well as the cost associated with the sample preparation, is significantly reduced, while maintaining robust analytical performance. We describe the creation and validation of a human serum IgG glycan library to be used as the calibration standard, and successful profiling of glycoforms from a variety of mAbs.

Solubility controlling peptide tags (SCP‐tags) constructed with 5‐proline (C5P) and 5‐arginine (C5R) residues did not alter the secondary structure content nor the tertiary structure nor the monomeric state of BPTI‐19A, but significantly increased its in vivo immunogenicity, by up to 240‐ and 73‐fold, respectively, suggesting the potential of non‐oligomerizing SCP‐tags for increasing the immunogenicity of a non‐immunogenic protein., Poor immunogenicity of small proteins is a major hurdle in developing vaccines or producing antibodies for biopharmaceutical usage. Here, we systematically analyzed the effects of 10 solubility controlling peptide tags (SCP‐tags) on the immunogenicity of a non‐immunogenic model protein, bovine pancreatic trypsin inhibitor (BPTI‐19A; 6 kDa). CD, fluorescence, DLS, SLS, and AUC measurements indicated that the SCP‐tags did not change the secondary structure content nor the tertiary structures of the protein nor its monomeric state. ELISA results indicated that the 5‐proline (C5P) and 5‐arginine (C5R) tags unexpectedly increased the IgG level of BPTI‐19A by 240‐ and 73‐fold, respectively, suggesting that non‐oligomerizing SCP‐tags may provide a novel method for increasing the immunogenicity of a protein in a highly specific manner.

Poor aqueous solubility is the principle obstacle in accomplishing suitable oral bioavailability for huge proportion of drug composites in drug development todays. Nano-suspension is an emerging field of research in the scientific community to provide a new solution for poorly water-soluble active constituents. The objective of the nano-suspension is to reduce the drug particle size (100-200 nm) range, which enhance solubility and bioavailability of biopharmaceutical active compounds. Crude extract of Piper nigrum shows various biological activities but poor aqueous solubility so, there is a need to isolate the piperine from Piper nigrum and prepare its nano-suspension to reduce particle size and enhance its bioavailability. This review defines the principles behind nanosizing, the synthesis and characterization of piperine nano-suspension as well as the recent practice withapplication of such formulations in-vivo can be used as a better alternative to treat cardiovascular diseases with improved therapeutic efficacy compared to extract.

The discovery and development of drugs that can compete in effectiveness with direct anticoagulants is a critical problem. Fucoidan is a complex branched heteropolysaccharide with anticoagulant and antithrombic activity. Fucoidan drug substance is hygroscopic and possesses poor technological properties. This study was aimed at development of the optimum composition and technology of fucoidan tablets and their biopharmaceutical evaluation. The generalized Harrington desirability function and dispersion and regression analyses were used for optimization of the fucoidan tablet formulation. The contents of crospovidone and lactose were the most significant factors affecting the disintegration and compressibility of model tablets. The lactose content had the greatest impact on the Carr index and Hausner ratio. The in vitro dissolution curves of the tablets were compared (at pH 1.2, 5.7, and 6.8). The in vitro release of fucoidan from the developed tablets obeyed a first-order kinetic equation.

Proteins have attracted increasing attention as biopharmaceutics and diagnostics due to their high specificity, biocompatibility, and biodegradability. The biopharmaceutical sector in particular is experiencing rapid growth, which has led to an increase in the production and sale of protein drugs and diagnostics over the last two decades. Since the first‐generation biopharmaceutics dominated by native proteins, both recombinant and chemical technologies have evolved and transformed the outlook of this rapidly developing field. This review article presents updates on the fabrication of covalent and supramolecular fusion hybrids, as well as protein‐polymer hybrids using solid‐phase approaches that hold great promise for preparing protein hybrids with precise control at the macromolecular level to incorporate additional features. In addition, the applications of the resultant protein hybrids in medicine and diagnostics are highlighted where possible., Molecular precision in macromolecular engineering: This review summarizes solid‐phase strategies that have emerged for selective protein modification and nanofabrication of protein–protein conjugates. The applications of the resultant protein hybrids in medicine and diagnostics are also highlighted.

Objectives. We hereby describe an improvement of a previously developed quantification technique for polysorbate 80 in biopharmaceutical formulations (darbepoetin alfa and eculizumab) and report the validation of the new approach.Methods. Polysorbate was isolated from analyte samples by protein precipitation using an organic solvent, followed by supernatant evaporation in vacuum. Polysorbate was derivatized using a ferric thiocyanate reagent and extracted into an organic phase; the relevant optical density measurements were performed.Results. We established the optimal conditions for each step of the analysis procedure. The accuracy was 97–102% in the tested analytical range, the relative standard deviation did not exceed 5%, and the limit of quantification was 0.01 mg/mL.Conclusions. The reported approach is highly sensitive; polysorbate isolation and quantification do not depend on the matrix or, most importantly, the protein.