Bevin Gangadharan

For many decades, there have been numerous reported cases of falsified liquid medical products, including vaccine and insulin preparations worldwide, but to date, there has been a lack of affordable and accessible analytical methods for biological medicines and vaccine authenticity testing. A conventional clinical chemistry analyser (Abbott Architect c16000) was used to determine the concentrations of analytes in genuine liquid biological products (vaccines and insulin) and falsified vaccine surrogates. Eight analytes were measured for each sample: sodium, potassium, chloride, calcium, magnesium, phosphate, glucose and protein. Each genuine liquid product had unique concentrations of analytes when tested using the eight methods applied, allowing clear differentiation from the falsified surrogates. In a blinded study, reproducibility was significantly high when the samples were run intra- and inter-batch up to 9 times over 9 different days, and it was possible to identify most of the samples by analyte presence alone. Imprecision was < 1.0 CV% for ion-selective electrode methods and typically < 5 CV% for spectrophotometric methods. A decision tree was created which was able to identify all samples. We demonstrate for the first time that a conventional clinical chemistry analyser provides a low-cost method to accurately differentiate genuine products from falsified surrogate liquid medicines and vaccines. This novel method has the potential to be used globally due to widespread use of clinical chemistry analysers in hospitals across the world, including in low- and middle-income countries where many cases of falsified medicines have been identified.

There have been hundreds of child deaths due to contamination of medicinal syrups with diethylene glycol (DEG) and ethylene glycol (EG). Detection of DEG and EG is usually performed by gas chromatography, a method that is costly, laborious, time-consuming, and not readily available in many low- and middle-income countries (LMICs). Thin-layer chromatography is relatively lower cost and portable; however, as with gas chromatography, it requires time and trained personnel. Alternative rapid, low-cost and simple methods to determine DEG/EG contamination are needed. We tested the suitability of enzymatic, chemical and antibody-based assays to determine DEG/EG. Assays using alcohol dehydrogenase and aldehyde dehydrogenase alone as well as in combination with glycolate oxidase could determine EG in raw materials and at less than 0.1% m/m in some finished products. Saliva and breast milk alcohol test strips, containing alcohol oxidase and costing $1, could determine EG with a detection limit of 0.5 to 2% m/m in under 2 minutes. Disposable breathalysers, costing only $1, could determine both DEG and EG from other alcohols in only 10 seconds. The methods described provide simple, rapid and low-cost alternatives to help determine DEG and EG contamination in pharmaceutical supply chains to help prevent deaths where equipment and trained human resources are limited.

Maintaining cold-chain integrity is vital for vaccines to ensure they remained within the recommended temperature limits to ensure stability and avoid degradation as storage temperature is one of the key factors contributing to rendering products substandard or ‘out of specification’. Heat-exposed vaccines closely resemble the chemical composition of the stable product making them very difficult to detect and testing as such is not routinely carried out at various points in the supply chain due to the lack of tools to identify effects of heat exposure in the field, a particular issue in countries with high-temperature climates. Here, we propose rapid and low-cost tests based on simple glucose assays to detect heat-exposed degraded sucrose-containing vaccines through its inherent gradual conversion to glucose at elevated temperatures. Bioluminescent and colorimetric assays and a clinical biochemical analyser for urine samples could successfully determine effects of heat exposure by detecting a significant increase in glucose levels. We show that this increase in glucose also correlates with the loss of vaccine potency. When vaccines were incubated at 37 and 45°C, the bioluminescent assay was able to detect an increase in glucose levels from 12 hours of heat exposure. The biochemical analyser could successfully detect if a COVID-19 vaccine had been exposed to 37 and 45°C. Most importantly, the colorimetric assay has the advantage of noticing a colour change by eye upon simply mixing the vaccine with a reagent without the need for a plate reader or any other sophisticated devices. To our knowledge, this is the first device-free test of its kind to determine the heat-exposed vaccines, making it an ideal test for deploying at various points in the supply chain in low- and middle-income countries to ensure the integrity of vaccine cold-chain. Although this test does not replace the more definitive potency assays, it could initially be used as a rapid and low-cost test to identify substandard sucrose-containing vaccines within supply chains, in support of WHO’s Prevent, Detect, and Respond strategy.

Infections caused by dengue virus (DENV) result in significant morbidity and mortality. A proportion of infected individuals develop dengue haemorrhagic fever (DHF) characterized by circulatory collapse and multiorgan failure. Early detection of individuals likely to develop DHF could lead to improved outcomes for patients and help us use healthcare resources more efficiently. We identified proteins that are differentially regulated during early disease in peripheral blood mononuclear cells (PBMCs) of patients who subsequently developed DHF. Four dengue fever (DF), four DHF and two healthy control PBMCs were subjected to tandem mass tag mass spectrometry. Differentially regulated proteins were used to identify up- or down-regulated Gene Ontology pathways. One hundred and sixty proteins were differentially expressed in DENV-infected samples compared to healthy controls. PBMCs from DHF patients differentially expressed 90 proteins compared to DF; these were involved in down-regulation of platelet activation and aggregation, cell adhesion, and cytoskeleton arrangement pathways. Proteins involved in oxidative stress and p38 MAPK signalling were upregulated in DHF samples during early infection compared to DF. This study has identified 90 proteins differentially regulated in PBMCs that could potentially serve as biomarkers to identify patients at risk of developing DHF at an early disease stage.

The rapid development and worldwide distribution of COVID-19 vaccines is a remarkable achievement of biomedical research and logistical implementation. However, these developments are associated with the risk of a surge of substandard and falsified (SF) vaccines, as illustrated by the 184 incidents with SF and diverted COVID-19 vaccines which have been reported during the pandemic in 48 countries, with a paucity of methods for their detection in supply chains. In this context, matrix-assisted laser desorption ionisation-time of flight (MALDI-ToF) mass spectrometry (MS) is globally available for fast and accurate analysis of bacteria in patient samples, offering a potentially accessible solution to identify SF vaccines. We analysed the COVISHIELD™ COVID-19 vaccine; falsified versions of which were found in India, Myanmar and Uganda. We demonstrate for the first time that analysis of spectra from the vaccine vial label and its adhesive could be used as a novel approach to detect falsified vaccines. Vials tested by this approach could be retained in the supply chain since it is non-invasive. We also assessed whether MALDI-ToF MS could be used to distinguish the COVISHIELD™ vaccine from surrogates of falsified vaccines and the effect of temperature on vaccine stability. Both polysorbate 80 and L-histidine excipients of the genuine vaccine could be detected by the presence of a unique combination of MALDI-ToF MS peaks which allowed us to distinguish between the genuine vaccines and falsified vaccine surrogates. Furthermore, even if a falsified product contained polysorbate 80 at the same concentration as used in the genuine vaccine, the characteristic spectral profile of polysorbate 80 used in genuine products is a reliable internal marker for vaccine authenticity. Our findings demonstrate that MALDI-ToF MS analysis of extracts from vial labels and the vaccine excipients themselves can be used independently to detect falsified vaccines. This approach has the potential to be integrated into the national regulatory standards and WHO’s Prevent, Detect, and Respond strategy as a novel effective tool for detecting falsified vaccines.

The global population is increasingly reliant on vaccines to maintain population health with billions of doses used annually in immunisation programmes. Substandard and falsified vaccines are becoming more prevalent, caused by both the degradation of authentic vaccines but also deliberately falsified vaccine products. These threaten public health, and the increase in vaccine falsification is now a major concern. There is currently no coordinated global infrastructure or screening methods to monitor vaccine supply chains. In this study, we developed and validated a matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) workflow that used open-source machine learning and statistical analysis to distinguish authentic and falsified vaccines. We validated the method on two different MALDI-MS instruments used worldwide for clinical applications. Our results show that multivariate data modelling and diagnostic mass spectra can be used to distinguish authentic and falsified vaccines providing proof-of-concept that MALDI-MS can be used as a screening tool to monitor vaccine supply chains.