Importance of advanced analytical techniques and methods for food quality control and pollution analysis for more sustainable future in the least developed countries

Chemistry education plays a central role in solving the world's pressing societal issues by equipping and inspiring chemists. However, chemistry education and research in the least developed countries (LDCs) is challenging due to a lack of physical infrastructure and human resources. Among a range of issues and challenges, food and herbal adulteration, microplastics pollution, and chemical hazards are more common in the LDCs, and these issues would impede the achievement of the United Nations sustainable development goals (SDGs). These societal issues are frequently highlighted in the academia and social media of the LDCs, and we believe that at least some aspects of these issues could be solved by advancement in chemistry education and research. However, the current chemistry education and research efforts are inadequate to address these societal issues in these countries. In this article, we have summarized the present scenario and offered examples where developments in chemistry education, particularly advancement in analytical techniques and methods, will provide technological solutions to these issues. Our analysis revealed that sophisticated analytical laboratories are unavailable or unaffordable in the LDCs. The governments of these countries are facing extreme challenges in their pursuit of sustainable development primarily due to a lack of financial resources and shortage of skilled personnel. Revised chemistry curricula and sophisticated analytical testing facilities are urgently required in higher education institutions (HEIs) in the LDCs in helping society achieve the SDGs. A concerted effort between policymakers, chemical societies, funding agencies, chemists, and industries is required to advance chemistry education.     

Characterization and Monitoring of a Novel Light-heavy-light Chain Mispair in a Therapeutic Bispecific Antibody

Site-specific cysteine engineering, along with other genetic mutations, is broadly implemented in bispecific antibodies (bsAb). Thus far, homodimer, half hole antibody, one-light chain mispaired and light chain swapped variants have been reported as chain-pairing variants for the asymmetric IgG-like bispecific antibodies. Here we report a novel mispair in which the C_H3 engineered cysteine on the hole heavy chain (HC) of a knob-into-hole (KiH) bsAb is linked to the engineered cysteine in C_L through a disulfide bond, forming a LHL species in a bsAb construct. Due to its impact on bioactivity, it is critical to implement an analytical strategy to monitor this CQA and mitigate risk for the future products. A set of orthogonal physicochemical assays that include hydrophobic interaction chromatography (HIC), capillary electrophoresis sodium dodecyl sulfate (CE-SDS), reverse phase liquid chromatography ultra-performance chromatography mass spectrometry (RP-UPLC MS) and disulfide bond mapping have been utilized to monitor and characterize this chain-pairing impurity for manufacturing process control and product release. Our data shows the LHL mispair in condition medium (CM) is approximately 1.3 - 1.9%. LambdaFabSelect affinity chromatography removes two major chain-pairing variants in CM – i.e. the hole-hole homodimer and hole half-antibody, while retaining the LHL species. Process improvement in Capto Q (anion exchange) and HS50 (cation exchange) chromatography steps removes LHL to as low as 0.2% in the final product. We have demonstrated an orthogonal analytical methodology that is capable of characterizing and monitoring bsAb mispairing, suitable for use in manufacturing process control and product release, and can be potentially implemented for similar bsAb constructs with engineered disulfide bonds.     

Quality of dorzolamide hydrochloride and timolol maleate containing eye drops distributed online

Patient safety risks associated with the online purchase of medications, especially in case of ophthalmic preparations, are significant. Our study aimed to carry out quality assessment of dorzolamide hydrochloride (DZA) and timolol maleate (TIM) eye drops preserved with benzalkonium chloride (BAC) via online test purchases.Three samples were purchased online, while control preparations were acquired through authorized national drug supply chain. Our method was based on the International Pharmaceutical Federation (FIP) Inspection Checklist and integrated the evaluation of packaging and labelling. Sterility was established according to the European Pharmacopoeia (Ph. Eur.), while qualitative and quantitative quality was assessed with high-performance liquid chromatographic (HPLC) analysis.Several signs of falsification were recognized upon visual inspection of the online samples. All the products were clear, colourless, slightly viscous solutions. They were free from visible contaminants. The samples were sterile as no evidence of microbial growth was found. A quick and inexpensive HPLC analysis, optimized by the authors showed that active ingredients and the preservative deviated significantly (p < 0,05) with more than 10% from the values stated on the labels for at least one component (DZA: 99.3–113.1%, TIM: 112.8–139.2%, BAC: 82.4–97.7%).Development of comprehensive and reliable quality assessment methods are vital to increase public safety of pharmaceutical products sold online. A complex approach, integrating visual inspection, labelling assessment, microbiological analysis coupled with qualitative and quantitative methods provide a most reliable method. Due to its limited feasibility and cost-effectiveness, raising public awareness and limiting illegal online sellers should be the primary approaches to protect patients from substandard and falsified medicinal products sold via the internet. Particularly important for health professionals to understand this market and its public health concern, and to raise patient awareness of the risks associated with uncontrolled online purchase of medication.     

Pure drug nano-assemblies: A facile carrier-free nanoplatform for efficient cancer therapy

Nanoparticulate drug delivery systems (Nano-DDSs) have emerged as possible solution to the obstacles of anticancer drug delivery. However, the clinical outcomes and translation are restricted by several drawbacks, such as low drug loading, premature drug leakage and carrier-related toxicity. Recently, pure drug nano-assemblies (PDNAs), fabricated by the self-assembly or co-assembly of pure drug molecules, have attracted considerable attention. Their facile and reproducible preparation technique helps to remove the bottleneck of nanomedicines including quality control, scale-up production and clinical translation. Acting as both carriers and cargos, the carrier-free PDNAs have an ultra-high or even 100% drug loading. In addition, combination therapies based on PDNAs could possibly address the most intractable problems in cancer treatment, such as tumor metastasis and drug resistance. In the present review, the latest development of PDNAs for cancer treatment is overviewed. First, PDNAs are classified according to the composition of drug molecules, and the assembly mechanisms are discussed. Furthermore, the co-delivery of PDNAs for combination therapies is summarized, with special focus on the improvement of therapeutic outcomes. Finally, future prospects and challenges of PDNAs for efficient cancer therapy are spotlighted.     

GC/MS-screening analyses of valuable products in the aqueous phase from microwave-assisted hydrothermal processing of Lemna minor

Microwave-assisted hydrothermal treatment of Lemna minor plant material was studied under subcritical conditions. The water phases of this process were extracted with polar and nonpolar solvents and the extracts were investigated by GC/MS screening analyses for value added products. The polar extracts were dominated by compounds known as flavouring agents and partly as platform chemicals, e.g. hydroxyacetone, 1-hydroxybutan-2-one, furfural, cyclotene and pyrrolidone. Pyridinol, an important educt for heterocyclic chemistry, was another interesting product. Benzaldehyde and oleamide were detected as main products in nonpolar n-hexane extracts. An essential aspect is that a large proportion of compounds like aldehyds and ketones were obtained when mild hydrothermal processing was applied.     

Variance Between Different Light Obscuration and Flow Imaging Microscopy Instruments and the Impact of Instrument Calibration

Subvisible particles (SVPs) are an obligatory critical quality attribute of the product, and yet, they are found in all biopharmaceutical products intended for infusion or injection. Light obscuration (LO) is the primary pharmacopeial method used to quantify SVPs. However, the method may not be equally sensitive toward all particles that can possibly occur. Calibration of LO instruments is usually performed using polystyrene beads suspended in water. In this study, the dependence of the sizing accuracy of LO analysis was evaluated by using a calibration suspension of lower refractive index beads made of silica suspended in sucrose solution. It was demonstrated that the sizing accuracy was strongly dependent on the reference material’s properties used for calibration. It was also demonstrated that flow imaging microscopy suffered from the same artifact, albeit to a smaller extent. We further tested different LO sensors and instruments. Interestingly, our results show that the sizing accuracy varied from instrument to instrument, strongly depending on the properties of the sensor.To summarize, sizing and counting accuracies were dependent on the material used for calibration and its optical properties as well as the calibration curve, the sensor, and the instrument supplier. Closer match of optical properties between calibration system and test system seems to improve the sensitivity of the measurement. The results of this study raise the following major practical implications: (1) LO and flow imaging microscopy are not truly orthogonal analytical methods, (2) while matching optimal properties of material used for calibration and test items increased sensitivity, this is of poor practical applicability given that analytes contain multiple particles, and (3) setting product-specific limits for SVPs require special considerations with regard to the data sets used.     

Probing the Tryptophan Environment in Therapeutic Proteins: Implications for Higher Order Structure on Tryptophan Oxidation

Tryptophan (Trp) oxidation in proteins leads to a number of events, including changes in color, higher order structure (HOS), and biological activity. We describe here a number of new findings through a comprehensive characterization of 6 monoclonal antibodies (mAbs) following selective oxidation of Trp residues by 2,2'-azobis(2-amidinopropane) dihydrochloride. Fluorescence spectroscopy, in combination with second derivative analysis, demonstrates that the loss of Trp fluorescence intensity is a sensitive indicator of Trp oxidation in mAbs. Size-exclusion chromatography with UV and intrinsic Trp fluorescence detection was demonstrated to be a useful method to monitor Trp oxidation levels in mAbs. Furthermore, the Trp oxidation levels measured by size-exclusion chromatography with UV and intrinsic Trp fluorescence detection were found to be in agreement with the values obtained from tryptic peptide mapping by liquid chromatography with mass spectrometric detection and correlate with the total solvent accessible surface area of the exposed Trp residues from in silico modeling. Finally, near-UV circular dichroism and Raman spectroscopy were used to evaluate the impact of Trp oxidation on HOS and identify specific oxidation products, respectively. This work demonstrates that protein HOS is altered on Trp oxidation in mAbs and multiple spectroscopic markers can be used to monitor the molecule-dependent Trp oxidation behavior.     

Modeling of the Biological Activity of Monoclonal Antibodies Based on the Glycosylation Profile

The influence of the glycosylation profile of IgG on biological activity is known, but it is not clear which glycoforms have the highest impact on the main mechanism of action. The aim of this study was to design a mathematical model for predicting the antibody-dependent cellular cytotoxicity (ADCC) activity and the Fc gamma IIIa receptors' (Fc?RIIIa) relative binding of rituximab drug products based on their glycosylation profile. An additional goal was to identify the glycoforms that have the greatest impact on these mechanisms of action. For these purposes, the glycosylation profile was examined by hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC), ADCC was assessed using a Promega kit, and Fc?RIIIa's binding affinity was assessed by surface plasmon resonance (SPR) analysis of a group of >50 rituximab drug products. Based on the results, mathematical models for the ADCC and Fc?RIIIa binding affinity prediction were designed using JMP 13.2.0. The quality of the model and the influence of sample size and heterogeneity on the reliability were verified. The results allow for the evaluation of rituximab drug products' activity based on their glycosylation profile and show that with a sufficiently large and differentiated dataset, it is possible to generate models for different monoclonal antibodies.     

An Industry Perspective on Compatibility Assessment of Closed System Drug-Transfer Devices for Biologics

The Formulation Workstream of the BioPhorum Development Group (BPDG), an industry-wide consortium, has identified the increased use of closed system drug-transfer devices (CSTDs) with biologics, without an associated compatibility assessment, to be of significant concern. The use of CSTDs has increased significantly in recent years due to the recommendations by NIOSH and USP that they be used during preparation and administration of hazardous drugs. While CSTDs are valuable in the healthcare setting to reduce occupational exposure to hazardous compounds, these devices may present particular risks that must be adequately assessed prior to use to ensure their compatibility with specific types of drug products, such as biologic drugs, which may be sensitive. The responsibility of ensuring quality of biologic products through preparation and administration to the patient lies with the drug product sponsor. Due to the significant number of marketed CSTD systems, and the large variety of components offered for each system, a strategic, risk-based approach to assessing compatibility is recommended herein. In addition to traditional material compatibility, assessment of CSTD compatibility with biologics should consider additional parameters to address specific CSTD-related risks. The BPDG Formulation Workstream has proposed a systematic risk-based evaluation approach as well as a mitigation strategy for establishing suitability of CSTDs for use.     

Recent advances in column switching sample preparation in bioanalysis

Column switching techniques, using two or more stationary phase columns, are useful for trace enrichment and online automated sample preparation. Target fractions from the first column are transferred online to a second column with different properties for further separation. Column switching techniques can be used to determine the analytes in a complex matrix by direct sample injection or by simple sample treatment. Online column switching sample preparation is usually performed in combination with HPLC or capillary electrophoresis. SPE or turbulent flow chromatography using a cartridge column and in-tube solid-phase microextraction using a capillary column have been developed for convenient column switching sample preparation. Furthermore, various micro-/nano-sample preparation devices using new polymer-coating materials have been developed to improve extraction efficiency. This review describes current developments and future trends in novel column switching sample preparation in bioanalysis, focusing on innovative column switching techniques using new extraction devices and materials.     

Construction of [C4MIM]BF4 based in situ alkaline aqueous biphasic systems by NaOH for efficient extraction saponins from Xanthoceras sorbifolium Bunge leaves

In this study, a novel method for extraction saponins from Xanthoceras sorbifolium Bunge leaves (XLs) was developed using ultrasonic-assisted ionic liquid combined with in situ alkaline aqueous biphasic systems (UAE-IL-AABS). UAE-IL was firstly used to extract saponins from XLs, followed by removal of the phenolic impurities using in situ IL-AABS. A Box-Behnken Design (BBD) was used to analyze the [C₄MIM]BF₄ concentration, time, liquid/solid ratio, and power on the saponins yield. Under the optimal conditions with [C₄MIM]BF₄ concentration 2.3 M, time 19.9 min, liquid/solid ratio 24.9:1 (mL/g), and power 165 W, the saponins yield was 8.17%, which was 11.01% higher than ultrasonic assisted ethanol extraction method. The major compounds in UAE-IL were also identified using UPLC-MS/MS. Moreover, the optimized conditions of IL-AABS with recovery rate of saponins was 96.70%. Finally, a low extraction time, and favorable impurities removal efficiency and recyclability suggested UAE-IL-AABS is greener for production in the food, medicine, and chemical industry.     

Liquid Oil Marbles: Increasing the Bioavailability of Poorly Water-Soluble Drugs

Many new therapeutic candidates and active pharmaceutical ingredients (APIs) are poorly soluble in an aqueous environment, resulting in their reduced bioavailability. A promising way of enhancing the release of an API and, thus, its bioavailability seems to be the use of liquid oil marbles (LOMs). An LOM system behaves as a solid form but consists of an oil droplet in which an already dissolved API is encapsulated by a powder. This study aims to optimize the oil/powder combination for the development of such systems. LOMs were successfully prepared for 15 oil/powder combinations, and the following properties were investigated: particle mass fraction, dissolution time, and mechanical stability. Furthermore, the release of API from both LOMs and LOMs encapsulated into gelatine capsules was studied.     

Recent advances in screening active components from natural products based on bioaffinity techniques

Natural products have provided numerous lead compounds for drug discovery. However, the traditional analytical methods cannot detect most of these active components, especially at their usual low concentrations, from complex natural products. Herein, we reviewed the recent technological advances (2015–2019) related to the separation and screening bioactive components from natural resources, especially the emerging screening methods based on the bioaffinity techniques, including biological chromatography, affinity electrophoresis, affinity mass spectroscopy, and the latest magnetic and optical methods. These screening methods are uniquely advanced compared to other traditional methods, and they can fish out the active components from complex natural products because of the affinity between target and components, without tedious separation works. Therefore, these new tools can reduce the time and cost of the drug discovery process and accelerate the development of more effective and better-targeted therapeutic agents.     

Application of a High Throughput and Automated Workflow to Therapeutic Protein Formulation Development

Rapid and efficient formulation development is critical to successfully bringing therapeutic protein drug products into a competitive market under increasingly aggressive timelines. Conventional application of high throughput techniques for formulation development have been limited to lower protein concentrations, which are not applicable to late stage development of high concentration therapeutics. In this work, we present a high throughput (HT) formulation workflow that enables screening at representative concentrations via integration of a micro-buffer exchange system with automated analytical instruments. The operational recommendations associated with the use of such HT systems as well as the efficiencies gained (reduction in hands-on time and run time by over 70% and 30%, respectively), which enable practical characterization of an expanded formulation design space, are discussed. To demonstrate that the workflow is fit for purpose, the formulation properties and stability profiles (SEC and CEX) from samples generated by the HT workflow were compared to those processed by ultrafiltration/diafiltration, and the results were shown to be in good agreement. This approach was further applied to two case studies, one focused on a formulation screen that studied the effects of pH and excipient on viscosity and stability, and the other focused on selection of an appropriate viscosity mimic solution for a protein product.     

Recent progress of hypoxia-modulated multifunctional nanomedicines to enhance photodynamic therapy: opportunities,challenges, and future development

Hypoxia, a salient feature of most solid tumors, confers invasiveness and resistance to the tumor cells. Oxygen-consumption photodynamic therapy (PDT) suffers from the undesirable impediment of local hypoxia in tumors. Moreover, PDT could further worsen hypoxia. Therefore, developing effective strategies for manipulating hypoxia and improving the effectiveness of PDT has been a focus on antitumor treatment. In this review, the mechanism and relationship of tumor hypoxia and PDT are discussed. Moreover, we highlight recent trends in the field of nanomedicines to modulate hypoxia for enhancing PDT, such as oxygen supply systems, down-regulation of oxygen consumption and hypoxia utilization. Finally, the opportunities and challenges are put forward to facilitate the development and clinical transformation of PDT.     

Neutralising effects of small molecule toxin inhibitors on nanofractionated coagulopathic Crotalinae snake venoms

Repurposing small molecule drugs and drug candidates is considered as a promising approach to revolutionise the treatment of snakebite envenoming. In this study, we investigated the inhibiting effects of the small molecules varespladib (nonspecific phospholipase A₂ inhibitor), marimastat (broad spectrum matrix metalloprotease inhibitor) and dimercaprol (metal ion chelator) against coagulopathic toxins found in Crotalinae (pit vipers) snake venoms. Venoms from Bothrops asper, Bothrops jararaca, Calloselasma rhodostoma and Deinagkistrodon acutus were separated by liquid chromatography, followed by nanofractionation and mass spectrometry identification undertaken in parallel. Nanofractions of the venom toxins were then subjected to a high-throughput coagulation assay in the presence of different concentrations of the small molecules under study. Anticoagulant venom toxins were mostly identified as phospholipases A₂, while procoagulant venom activities were mainly associated with snake venom metalloproteinases and snake venom serine proteases. Varespladib was found to effectively inhibit most anticoagulant venom effects, and also showed some inhibition against procoagulant toxins. Contrastingly, marimastat and dimercaprol were both effective inhibitors of procoagulant venom activities but showed little inhibitory capability against anticoagulant toxins. The information obtained from this study aids our understanding of the mechanisms of action of toxin inhibitor drug candidates, and highlights their potential as future snakebite treatments.     

Cyrene™ as an Alternative Sustainable Solvent for the Preparation of Poly(lactic-co-glycolic acid) Nanoparticles

Toxic and environmental harmful organic solvents are widely applied to prepare poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NP) in standard preparation methods. Alternative non-toxic solvents suffer from disadvantages like high viscosity and plasticizing effects. To overcome these hurdles, Cyrene? as a new sustainable, non-toxic and low viscous solvent was used to formulate PLGA NPs. A new preparation method was developed and optimized. Small sized blank NPs around 220 nm with a narrow size distribution and highly negative charge (<?23 mV) were obtained. To test the application for drug delivery, the lipophilic model drug atorvastatin was encapsulated in high drug loads with comparable physicochemical characteristics as the blank NPs, and a total drug release within 24 h. No changes of the crystallinity or plasticizing effects could be observed. Highly purified NPs were obtained with a residual Cyrene? content <2.5%. Finally, the biocompatibility of Cyrene? itself and of the NPs formed in the presence of Cyrene? was demonstrated in a hen's egg test. Conclusively, the use of Cyrene? as solvent offers a simple, fast and non-toxic procedure for preparation of PLGA NPs as drug delivery systems circumventing the downsides of standard methods.     

MCDB: A comprehensive curated mitotic catastrophe database for retrieval,protein sequence alignment,and target prediction

Mitotic catastrophe (MC) is a form of programmed cell death induced by mitotic process disorders, which is very important in tumor prevention, development, and drug resistance. Because rapidly increased data for MC is vigorously promoting the tumor-related biomedical and clinical study, it is urgent for us to develop a professional and comprehensive database to curate MC-related data. Mitotic Catastrophe Database (MCDB) consists of 1214 genes/proteins and 5014 compounds collected and organized from more than 8000 research articles. Also, MCDB defines the confidence level, classification criteria, and uniform naming rules for MC-related data, which greatly improves data reliability and retrieval convenience. Moreover, MCDB develops protein sequence alignment and target prediction functions. The former can be used to predict new potential MC-related genes and proteins, and the latter can facilitate the identification of potential target proteins of unknown MC-related compounds. In short, MCDB is such a proprietary, standard, and comprehensive database for MC-relate data that will facilitate the exploration of MC from chemists to biologists in the fields of medicinal chemistry, molecular biology, bioinformatics, oncology and so on. The MCDB is distributed on http://www.combio-lezhang.online/MCDB/index_html/.