Ensuring vaccine cold chain integrity: A rapid and low-cost test for identifying heat-exposed sucrose-containing vaccines

Ensuring vaccine cold chain integrity: A rapid and low-cost test for identifying heat-exposed sucrose-containing vaccines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ensuring vaccine cold chain integrity: A rapid and low-cost test for identifying heat-exposed sucrose-containing vaccines Benediktus Yohan Arman, Andrea Magri, Matteo Barbaglia, Lawrence Petherbridge, and 22 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6839799/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. Biological sciences/Biochemistry Biological sciences/Immunology/Vaccines Biological sciences/Microbiology/Vaccines Health sciences/Health care/Public health Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The surge of substandard and falsified (SF) medicines and vaccines coupled with the lack of accessible and affordable methods for their screening and detection in supply chains is a significant problem. 1 One in 10 medical products are reported to be either substandard or falsified. 2 Substandard products are genuine products that fail to meet either quality standards or specifications, or both. In contrast, falsified products are where the products’ identity, composition or source is deliberately and fraudulently misrepresented by criminals. 2 SF medical products have become significant health threats and potentially lead to higher rates of illness and death, as well as eroding public trust in the form of hesitancy as they are unsafe and ineffective. 3 Our Vaccine Identity Evaluation (VIE) consortium has been evaluating novel techniques for detecting SF vaccines in supply chains. 4 We have successfully used spatially offset Raman spectroscopy (SORS) 5 , rapid diagnostic tests 6 , and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) 7 , 8 to differentiate genuine vaccines from falsified vaccine surrogates. However, complex devices or tests are required to determine substandard vaccines due to heat exposure. The World Health Organisation (WHO) estimated that over half of vaccines are wasted globally each year due to temperature control failures, logistics challenges, and shipment-related issues. 9 Environmental factors, such as temperature and light, should be examined in vaccine stability studies 10 with heat exposure being the greatest problem since light exposure can be more easily avoided by the packaging of vaccine vials and syringes. Cold chain is a crucial aspect of protecting vaccines from deterioration, as improper temperature storage could affect vaccine stability and cause harmful effects of reduced efficacy and changes in the safety profile. 11 – 13 Maintaining the cold chain is deemed expensive and contributes substantially to costs with the pharmaceutical cold chain logistics market size of US $ 18.61 billion in 2024 and expected to reach $ 27.11 billion by 2033. 14 , 15 Successful strategies are needed to generate stable and efficacious dosage forms; these include efforts to stabilise the antigen, selection of adjuvants, and advanced analytical methods to monitor vaccine stability. 16 Potency assays for vaccines and medical products rely on biological assays in the form of in vivo (animal immunogenicity tests) or in vitro (cell-based or antibody-binding-based) assays. 17 Vaccine potency is an indicator of vaccine stability and is a regulatory requirement for release into the market. 18 However, these assays are complex, expensive, time-consuming and can be variable with the need for specialist laboratory instruments and trained personnel. 11 , 18 – 21 . Vaccine vial monitors (VVM) are thermochromic labels on vaccine vials which change in colour when exposed to elevated temperatures. 22 However, many vaccines do not have VVM attached and there are no simple, rapid and low-cost methods to screen for substandard heat-exposed vaccines in supply chains and evaluate the accuracy of VVM. Furthermore, VVM do not show whether a vaccine has deteriorated and only indicates if a vaccine has been cumulatively exposed to elevated temperature. Sugars, such as sucrose and lactose, are common ingredients in approved vaccines where they are used as stabilisers from natural sources 23 , 24 due to their antigen-stabilising and immunogenicity-maintaining properties. 25 – 27 When an aqueous solution of the disaccharide sucrose is heated, it breaks down into its monosaccharides glucose and fructose. 28 Here we demonstrate three novel, simple, and sensitive approaches to detect heat-exposed sucrose-containing vaccines based on the detection of the glucose formed as a product of degraded sucrose molecules: 1) a quantitative bioluminescent glucose assay, 2) a semi-quantitative colorimetric glucose assay which involves mixing the vaccine with a reagent and then observing a colour change by eye, and 3) a quantitative biochemical analyser which is available in most hospitals worldwide was used to determine eight analytes, including the levels of glucose in heat-exposed vaccines. These quantitative and semi-quantitative glucose assays are not intended to replace the standard vaccine potency assay and are only presented as surrogatecost approaches. These assays can be used as predictors of unstable heat-exposed vaccines within supply chains in accordance with the WHO’s Prevent, Detect, and Respond strategy. Of most importance is the colorimetric assay since it does not require a plate reader or other spectrophotometer, is of very low-cost and therefore could be easily deployed in low- and middle-income countries which may not have access to such instrumentation. Results Glucose levels in sucrose-containing vaccines after exposure to different temperature conditions Using the bioluminescent assay, glucose concentrations were measured as an indicator of thermal degradation of sucrose after the vaccines were exposed to different temperatures for seven days and compared to vaccines correctly stored at 2–8°C as recommended by the manufacturer. Among seven vaccines tested, three vaccines (Bexsero™, COMIRNATY™ and COVISHIELD™) showed a clear and statistically significant increase in glucose concentration after exposure to elevated temperatures of 37 and 45°C (Fig. 1 . A-C). A smaller increase was observed for Nimenrix™ but was significant at both 37 and 45°C (Fig. 1 D). The increase was recorded as 4.5-, 9.6-, 2.8-, and 1.1- fold in Bexsero™, COMIRNATY™, COVISHIELD™, and Nimenrix™ respectively, after seven days of incubation at 37°C compared to the vaccine samples correctly stored at 2–8°C. After incubation at 45°C, a higher increase in glucose was observed compared to incubation at 37°C, with a 9.2-, 27.6-, and 6.3-fold change in Bexsero™, COMIRNATY™, and COVISHIELD™, respectively (Supplementary Table S1) compared to the 2–8°C storage condition. However, no significant increase in glucose after heat exposure was observed for Rabipur™, Rotarix™, and Ticovac™ (Fig. 1 E-G). Exposure to three freeze-thaw cycles and room temperature conditions after seven days did not result in significant changes in glucose levels in any of the vaccines tested (Fig. 1 ). Analysis of thermal degradation of sucrose over time using the bioluminescent glucose assay After establishing which vaccines gave rise to measurable glucose levels upon heat treatment after a week, we proceeded to measure those vaccines (Bexsero™, COMIRNATY™, and COVISHIELD™) over a seven-day time-course to determine when sucrose degradation could be first detected by the bioluminescent glucose assay. Thermal degradation of sucrose in all three vaccines could be observed by increasing levels of glucose when exposed to 37 and 45°C. Significant increases in glucose levels were detected after 12 hours for Bexsero™ and COMIRNATY™, and 3 hours for COVISHIELD™ (Fig. 2 ). Colorimetric glucose assay to detect glucose levels in sucrose-containing vaccines A simpler colorimetric glucose assay was used to determine the glucose levels in COMIRNATY vaccines for both the 30 µg adult and 10 µg children’s vaccines (Fig. 3 , Table 1 ). The development of colour intensity along with the increasing level of glucose could be observed visually by eye (Fig. 3 a). The assay could significantly detect vaccine vials exposed to the elevated temperatures 37 and 45°C (Fig. 3 b). A similar increase in glucose was observed in both 30 µg adult and 10 µg children's vaccines which are manufactured with the same amount of sucrose in both vaccines. Table 1 Sucrose-containing vaccines used in the study. Data were retrieved from the Electronic Medicine Compendium (EMC), available at https://www.medicines.org.uk/emc and other sources in the public domain. Vaccine trade name Description and usage Manufacturer Pharmaceutical form Excipients including sucrose concentration in bold Rotarix™ Live attenuated rotavirus vaccine GlaxoSmithKline (GSK) Oral suspension in squeezable tube, clear and colourless viscous liquid Sucrose ( 715 mg/mL ), disodium Adipate, Dulbecco's Modified Eagle Medium/DMEM (containing phenylalanine, sodium, glucose, and other substances), sterile water Nimenrix™ Meningococcal groups A, C, W-135 and Y conjugate vaccine Pfizer Ltd Powder and solvent for solution for injection in pre-filled syringe Powder : Sucrose ( 56 mg/ml after reconstitution), trometamol Solvent : Sodium chloride, water for injection Rabipur™ Rabies vaccine (inactivated, strain Flury LEP) Bavarian Nordic A/S Powder and solvent for solution for injection in pre-filled syringe Powder : Trometamol, sodium chloride, disodium edetate, potassium-L-glutamate, polygeline, sucrose ( 60 mg/mL after reconstitution a ) Solvent : Water for injection Bexsero™ Meningococcal group-B vaccine (rDNA, component, adsorbed) GSK Suspension for injection in pre-filled syringe Sodium chloride, histidine, sucrose ( 20 mg/mL ), water for injections, aluminium hydroxide (hydrated) TicoVac™ Junior Tick-Borne Encephalitis Vaccine (whole virus, inactivated) Pfizer Ltd Suspension for injection in a pre-filled syringe Human albumin, sodium chloride, disodium phosphate-dihydrate, potassium dihydrogenphosphate, water for Injection, sucrose (expected to be ≤ 30 mg/mL as for TicoVac™ adult dose), aluminium hydroxide, hydrated. COVISHIELD™ COVID-19 vaccine; Recombinant, replication-deficient chimpanzee adenovirus vector encoding the SARS-CoV-2 Spike (S) glycoprotein Serum Institute of India Solution for injection, colourless to slightly brown, clear to slightly opaque and particle-free with a pH of 6.6 L-Histidine, L-Histidine hydrochloride monohydrate, magnesium chloride hexahydrate, polysorbate 80, ethanol, sodium chloride, disodium edetate dihydrate (EDTA), sucrose ( 75 mg/mL ) b COMIRNATY™ COVID-19 vaccine; mRNA vaccine (nucleoside modified): 10 micrograms per dose (children 5 to 11 years) and 30 micrograms per dose (ages 12 years and older) Pfizer Ltd Concentrate for dispersion for injection ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC); cholesterol, trometamol; trometamol hydrochloride; water for injections; sucrose ( 20 mg/mL 35 ) a information obtained from https://www.medsafe.govt.nz/profs/datasheet/r/rabipurinj.pdf b information obtained from https://www.seruminstitute.com/health_faq_covishield.php Correlation of potency with glucose for heat-exposed vaccines To assess whether glucose levels could correlate with vaccine potency, a human Adenovirus 5-based vaccine formulated in a buffer containing 7.5% sucrose was exposed to various temperature-altered conditions. Storage at 4°C, room temperature, or freeze/thaw (F/T) three times had no impact on viral potency compared to the reference sample stored at -80°C (Fig. 4 A). Conversely, the detected glucose levels showed no variation compared to the reference sample. Exposure of samples to higher temperatures (37°C and 45°C) resulted in reduced levels of potency, with a 2-log decrease for 37°C incubation and a 3-log reduction for the 45°C storage. Glucose levels from those samples were elevated, between 5- and 11-fold (Fig. 4 A). Furthermore, the correlation analysis confirmed that degradation-mediated glucose levels correlate significantly with vector infectivity, confirming that glucose is potentially a good predictor for vaccine potency (Fig. 4 B). Glucose assays using a biochemical analyser Using the biochemical analyser, glucose could be successfully detected in COVISHIELD™ exposed to 37°C (at the lower limit of quantitation 60 µM) and 45°C (140 µM). Both of these measured glucose concentrations were similar to the levels detected by bioluminescent glucose assay (Fig. 1 C). Glucose could also be detected at even higher levels in COVISHIELD™ samples exposed to 100°C (data not shown since vaccines would never be exposed to such a high temperature on the supply chain). However, glucose could not be detected and quantified in Bexsero™ and COMIRNATY™ samples. Discussion Almost all vaccines currently need to be either refrigerated or frozen during storage and shipment. This is an essential requirement to maintain their potencies which could be reduced or lost if vaccines are exposed to high ambient temperature which in many countries can exceed 45°C. 16 Potency assays are difficult to carry out rapidly in the supply chain since they require sophisticated laboratory instruments and sometimes reagents manufactured by the vaccine manufacturer which would further increase costs. VVM can help to determine heat exposure, but they are not always used on vaccine vials and syringes. All seven vaccines (Fig. 1 ) used in this study did not have VVM highlighting the importance of tests on the vaccine itself. Furthermore, VVMs only indicate if vaccine is likely to have been degraded with no correlation to potency. In contrast, we show that the glucose assay has good correlation to potency highlighting that our reported rapid test adds value to even products which already have VVM. There is a need for faster and easier methods which could be used without the need for devices. Such rapid tests would help to initially screen for substandard vaccines that have been exposed to elevated temperature, before being sent for reference potency assays, facilitating timely and more cost-effective detection. Sugars, such as sucrose and lactose, are often used as stabilisers in injectable vaccines but sucrose has also been used in oral vaccines to improve taste. We have demonstrated that the surrogate measurement of glucose, as a product of sucrose degradation, could be used as a predictor of vaccines exposed to elevated temperatures. This novel approach was found to successfully work for most of the vaccines tested. Importantly, we have shown that the increase in glucose correlates significantly with vector infectivity, confirming that this novel marker for heat-exposed sucrose-containing vaccines is a good predictor for vaccine potency (Fig. 4 ). We have recently shown that SORS and MALDI-ToF MS are unable to detect if a vaccine, which contained sucrose, was exposed to heat. 5 , 8 SORS, MALDI-ToF MS and rapid diagnostic tests can aid in determining falsified vaccines whereas the glucose assays can help in identifying substandard heat-exposed sucrose-containing vaccines, highlighting that these techniques in combination can help to detect SF vaccines. A significant increase in glucose after seven days of exposure to elevated temperatures of 37 and 45°C was observed for COMIRNATY™, COVISHIELD™ and Nimenrix™. For Nimenrix™, although the increase was significant, the increase in glucose was only 1.1- and 1.2-fold for 37 and 45°C whereas for the other vaccines the fold change was considerably higher (2.8- to 9.6-fold for 37°C and 6.3- to 27.6 fold for 45°C; Table S1). For Rabipur™, another lyophilised vaccine, no significant increase in glucose was observed. Lyophilised vaccines have been shown to have good stability at 37°C for up to one month 19 . The lower increase in glucose for Nimenrix™ and no increase in glucose for Rabipur™ is due to these vaccines being lyophilised since water is needed for sucrose hydrolysis and with such a small fold change for Nimenrix™ it may be challenging to use glucose assays to detect its exposure to heat. No significant increase in glucose was observed also for Rotarix™ and Ticovac™ (Fig. 1 ) suggesting that sucrose degradation depends on vaccine formulation. 19 Rotarix™ is a highly viscous liquid including a thickening agent disodium adipate. 27 Unlike all the other injectable vaccines tested, Rotarix™ is given orally and contains sucrose to improve taste. The high viscosity of the vaccine and the extremely high level of sucrose compared to injectable vaccines (Table 1 ) could be the reason why no significant increase in glucose was observed. Ticovac™ contains human serum albumin that functions as sheer protection and thermal stability enhancer. 28 The presence of this excipient may have helped to reduce the thermal degradation of sucrose. No significant change in glucose levels was observed for all vaccines when exposed to freeze-thaw cycles and stored at ambient room temperature of 20°C for seven days. However, damage from accidental freezing can result in potency loss for freeze-sensitive vaccines 31 and this could not be readily inspected using our glucose assay. A drop in infectivity has been shown for a ChAdOx1 vaccine exposed to 22°C from 35 days onwards and for a ChAdOx2 vaccine from 180 days onwards. 19 These vaccines had the same excipients as COVISHIELD™ and the same sucrose concentration of 75 mg/ml. For all vaccines tested in our study, we did not see any change in glucose after 7 days at RT (20°C). However, we have not tested if glucose increases after 35 days at this ambient temperature since it is unlikely that vaccines would be left for so long at RT in the supply chain. Levels of glucose were measured in sucrose samples made up in water and stored at both 4°C and 45°C (Figure S1). Sucrose solutions were made up at various concentrations from 20 to 715 mg/ml since they cover the sucrose concentrations commonly used in vaccines, e.g. Bexsero (20 mg/ml), Spikevax (87 mg/ml), RotaTeq (540 mg/ml) and Rotarix (715 mg/ml). The sucrose solutions stored at 4°C had very low levels of glucose (below 10 µM). However, for all solutions stored at 45°C, there was a large increase in glucose of around 100-fold (Figure S1) unlike the vaccine data with relatively small increases in glucose (Fig. 1 ). This indicates that all concentrations of sucrose commonly used in vaccines could show an increase in glucose with heat exposure. However, we either observed a relatively lower increase in glucose of 1.2 to 27.6-fold (Table S1, Fig. 1 A-D) or no significant increase at all (Fig. 1 E-G). The sucrose solutions made up in water did not have any other excipients and therefore the lower or no increase in glucose observed for the vaccines must be due to other excipients which are reducing sucrose hydrolysis (Table 1 ). Sucrose degradation could be detected as early as 12 hours post-incubation when Bexsero™, COVISHIELD™ and COMIRNATY™ vaccines were exposed to 37 and 45°C (Fig. 2 .). In the case of COVISHIELD™, a significant change in glucose (p < 0.01) was observed after only 3 hours of incubation at these temperatures (Fig. 2 C) with glucose concentration changes of 18.5 and 32.4 µM for 37°C vs 4°C and 45°C vs 4°C, respectively. The bioluminescent glucose assay was sensitive enough to detect these small changes. The assay is capable of quantifying glucose levels down to 0.0031 µM, which is the lowest standard concentration used in the assay. Although a plate reader was required to read the bioluminescence, the assay is straightforward and most medicine regulators and vaccine manufacturers would have this device since they are already used for vaccine potency assays. While both potency assays and the bioluminescent glucose assay use a plate reader, the glucose assay is lower cost, faster and easier to carry out. To avoid the use of a plate reader and to decrease costs further, we investigated the use of a rapid and simpler colorimetric glucose assay. This assay could also be used with a plate reader but only requires absorbance measurements instead of luminescence and such spectrophotometers are more widely available globally and of lower cost. Most importantly, the changes in colour intensity seen with the heat-exposed vaccines and comparing this to the pink colour of the glucose standards, it was possible to determine the concentration of glucose semi-quantitatively and visually without the need for any device (Fig. 3 A). The method simply involves mixing vaccine with a reagent, incubating at 37°C for 30 minutes and any colour change to pink would indicate that the vaccine was exposed to heat. The reagents in this kit could be used along with a reference of colours expected for correctly stored (colourless) and heat-exposed (pink) vaccines. This is the first rapid and device-free assay which correlates with vaccine potency and can significantly aid LMICs where instrumentation is lacking. Although the sensitivity of this assay is lower than the bioluminescent assay, it can in the same way successfully determine vaccines exposed to both 37°C and 45°C. The colorimetric glucose assay used in this study was manufactured by Cell Biolabs who distribute this kit worldwide including to over a dozen LMICs such as Nigeria, India, Pakistan and Egypt, which have average high temperatures exceeding 40°C during summer months. A biochemical analyser was repurposed to detect analytes in vaccine excipients. This analyser can measure glucose in clinical samples (along with 7 other analytes), is very low cost (below £10 to measure all 8 analytes) and is widely available in most diagnostic laboratories worldwide. Although the instrument did not have the same level of sensitivity compared to the bioluminescent or colorimetric assays, it has the advantage of confirming many analytes contained in the genuine vaccine and can be used to evaluate vaccine authenticity (Table 2 and Supplementary Table 2). While the biochemical analyser was unable to detect glucose in heat exposed Bexsero and COMIRNATY™, it was able to successfully detect glucose in heat-exposed COVISHIELD™ (Table 2 ). This was expected since the bioluminescent assay showed higher levels of glucose after heat exposure for COVISHIELD™ (Fig. 1 C), which was within the working range of the biochemical analyser, compared to Bexsero (Fig. 1 A) and COMIRNATY™ (Fig. 1 B). Also, the concentration of sucrose in COVISHIELD™ is 3.75 times higher than both Bexsero and COMIRNATY™ (Table 1 ). It is possible that the biochemical analyser may be able to detect glucose in vaccines with a similar sucrose concentration as COVISHIELD™. The manufacturer of this vaccine, Serum Institute of India, has recently manufactured the R21 malaria vaccine which contains sucrose although its concentration is unknown. It is possible that a biochemical analyser could detect heat exposure for this R21 malaria vaccine, and other sucrose-containing vaccines, assuming that the sucrose concentration are similar. Table 2 Concentrations of glucose measured in vaccine samples using a biochemical analyser after exposure to different altered temperature conditions. Values in bold indicate if the analytes could be detected and quantified showing their mean concentrations (± standard deviation) of eight measurements. All other values with the less than symbol (<) were below the limit of quantitation and the lower limit of quantitation is shown. Vaccine Glucose concentration (mM) 4°C 3× FT RT 37°C 45°C Bexsero * < 0.06 < 0.06 < 0.06 < 0.06 < 0.06 COMIRNATY < 0.06 < 0.06 < 0.06 < 0.06 < 0.06 COVISHIELD < 0.06 < 0.06 < 0.06 0.06 ± 0.01 0.14 ± 0.01 * Calculation based on six measurements due to the limited sample; 3× FT, three cycles of freeze-thaw; RT, room temperature (20 ± 1°C). The other analytes detected and quantified by the biochemical analyser helped to determine an analyte fingerprint for the vaccines and we have shown how this is a low-cost way to distinguish genuine vaccines from falsified surrogates 29 . The analytes identified and quantified for the three vaccines act as a fingerprint to confirm authenticity (Supplementary Table 2). All vaccines tested contain sodium chloride and as expected, both sodium and chloride ions were detected. Although Bexsero™ does not contain potassium, a positive result was seen possibly due to interference from other excipients which is not a problem since the result was consistent among all runs. Such false positives may occur since the analyser is intended for urine samples instead of vaccines which have a very different sample matrix. COMIRNATY™ tested positive for protein even though no protein is expected in this vaccine. As we have discussed separately, an explanation for this false positive in mRNA vaccines could be due to benzethonium chloride used for the protein method which interacts with lipids in the vaccine and disrupts the lipid nanoparticles 29 . Magnesium chloride is present in COVISHIELD™ and as expected magnesium was detected. The Oxford COVID-19 ChAdOx1-S vaccine has the same excipient list as COVISHIELD™ and we have shown that magnesium ions could also be detected in this vaccine 29 . Since sucrose is composed of both glucose and fructose, levels of fructose could have also been detected instead of glucose. However, since glucose assays are far more common, more affordable and the biochemical analyser is unable to measure fructose, we decided to only focus on detecting glucose. The approaches described may also be expanded to detect degradation in lactose-containing liquid medicines 24 , as lactose breaks down into glucose and galactose when heated. Galactose assays are also available but have the same disadvantages as fructose assays. Previous work assessing adenovirus-vectored vaccines exposed to temperatures ranging from 4 to 45°C using an in vitro infectivity assay reported a rapid loss of infectivity at 30 and 45°C in less than 10 days of exposure. Only a small decrease in infectivity was observed at ambient 22°C and no loss of infectivity was seen over five freeze-thaw cycles. 19 Therefore, while the glucose assays were not able to detect vaccines which had been exposed to freeze-thaw cycles, the potency may have been unaffected, as observed in our potency correlation data for the hAd5 vaccine (Fig. 4 ). A rapid initial loss of infectivity has also been observed for a measles vaccine when only exposed to temperatures above ambient. 32 Furthermore, a case report assessing the stability of an adenovirus-vectored ChAdOx1-S vaccine stored at ambient 21°C for 18 hours before vaccination reported the same safety profile and efficacy as the vaccines stored at the recommended temperature 17 . This maintained efficacy at ambient is in line with our observed no significant increase in glucose at ambient. We incubated the vaccine at ambient for a much longer period of 7 days (Fig. 1 ). However, for ambient exposure over the same 18 hours, the increase in glucose is likely to be minimal or not significant based on our data for exposure to 37°C (at least for Bexsero™ and COMIRNATY™; Fig. 2 A and Fig. 2 B). Elevated temperatures have been shown to degrade an in-house manufactured mRNA vaccine 33 . A decrease in mRNA integrity and increase in fragmentation were observed after four days of incubation at 37, 45, and 60°C. 33 At this same 4 day (96 hours) timepoint, we were able to successfully detect a significant increase in glucose for the COMIRNATY™ mRNA vaccine at both 37 and 45°C (Fig. 2 B). This suggests that our novel approach using glucose assays could be used to detect heat-exposed mRNA vaccines which have reduced mRNA integrity and increased fragmentation. The stability of mRNA, as of percentage of intact RNA, correlates with in vitro potency as both were rapidly decreased when exposed to increasing temperature from 25 to 45°C. 34 All mRNA vaccine manufacturers use sucrose and similar excipients (lipids and salts) and therefore the glucose assays are likely to work for all mRNA vaccines. Collectively, the pattern of sucrose degradation within 7 days in this study correlates with the results of vaccine stability assays. We propose that the detection of glucose could potentially be an endogenous indicator of temperature exposure for some sucrose-containing vaccines and thus of cold chain failure. Our proposed use of the colorimetric glucose assay reagents is the fastest and lowest cost assay since result are obtained after only 30 minutes. To our knowledge, this is the first device-free test which can detect heat-exposed vaccines and correlates with vaccine potency making it ideal in resource-limited LMICs. This assay simply involves mixing vaccine with a reagent and visualising any colour change to pink would indicate heat-exposed vaccines. An oven is required to incubate the mixed sample and ovens are present in laboratories of all medicine regulators. However, even an oven may not be needed in countries where the ambient temperature is high enough to show the colour change to pink. Our lower cost and more rapid assays could be used by medicine regulators, vaccine manufacturers and inspectors as an initial screening test to help prevent substandard vaccines from being used. Methods Vaccine samples Five non-COVID-19 vaccines and two COVID-19 vaccines were used in this study (Table 1 ). Non-COVID vaccines were purchased from the Oxford University Hospitals Pharmacy, Oxford, United Kingdom. All vaccines were in-date and tested before expiry. The COVISHIELD™ (Serum Insitute of India) and COMIRNATY™ (Pfizer Ltd) (30 µg for adults and 10 µg for children) COVID-19 vaccines were received from the Serum Institute of India, Pvt. Ltd. and the National Health Service England (NHS England), respectively. Vaccines were stored at the recommended 2–8°C storage temperature. COMIRNATY™ COVID-19 vaccines were immediately stored in a refrigerator at 2–8°C when received and used within one month of receipt according to the manufacturer’s recommendations after the frozen vaccine is thawed. Vials and syringes were kept on wet ice prior to the tests. A human Adeno5-based vaccine (hAd5 KC5) was generated by single-round infection with an MOI of 1 on a hyperflask containing approximately 1E + 08 human embryonic kidney HEK 293 cells. Cells were harvested 48 hours post-infection and pelleted at 200xg for 30 minutes. Cell pellets were lysed in Cell Lysis Buffer (10 mM Tris, 135 mM sodium chloride, 1 mM magnesium chloride) and freeze/thawed three times. The cell lysate was treated with 250 Units/mL of Benzonase after first thaw and incubated for 30 minutes at room temperature. The viral vector was then purified by double caesium chloride ultracentrifugation, and dialysed three times in Formulation Buffer A438 (10 mM histidine, 7.5% sucrose, 35 mM sodium chloride, 1 mM magnesium chloride, 0.1% polysorbate 80, 0.1 mM ethylenediaminetetraacetic acid, 0.5% (v/v) ethanol, pH 6.6). Experimental settings Different techniques were used to measure glucose concentrations in the seven vaccines exposed to different temperature conditions for 7 days. After establishing which vaccines gave rise to measurable glucose levels upon heat treatment after a week, we looked at the sucrose degradation kinetics at 10 different time points over 7 days under the two temperature conditions (37 and 45°C) that produced significant glucose changes in the previous tests, to determine when sucrose degradation could be first detected by the techniques under evaluation. For one vaccine (hAd5 KC5) we conducted a potency assay in parallel, in order to look at the correlation between glucose levels and potency loss due to heat-exposure. Vaccine samples exposed to different temperature conditions Vaccine samples were grouped and each group was exposed to one of the following temperature conditions in the dark: (i) stored at 4°C within the recommended 2–8°C storage condition, (ii) stored at ambient room temperature (RT, recorded as 20 ± 1°C) for 7 days, (iii) stored in an incubator oven set at 37°C for 7 days, (iv) stored in an incubator oven set at 45°C for 7 days, and (v) exposed to three freeze-thaw cycles of 24 hrs freezing at -70°C and 1 hr thawing at 4°C per cycle. All samples were immediately stored at 4°C after completing the incubation period. In the case of vaccines which had a separate vial of lyophilised powder and a syringe for the solvent (Nimenrix™ and Rabipur™), both the vial and syringe were exposed to the five conditions without prior mixing/reconstitution to resemble a real-life situation of how the vaccine could be exposed to altered temperature on the supply chain. The powdered vaccines were reconstituted with their solvent just prior to analysis. A degradation test on hAd5 KC5 was performed by incubating glass vials containing 350 µL of the vaccine at RT (recorded as 20° ± 2°C), 4°C, 37°C and 45°C for 7 days. Vials were also exposed to the same freeze-thaw cycles as described above or stored at -80°C (as a reference control). Each condition was tested in triplicate and tested using both the colorimetric and potency assays. Sucrose in water exposed to an elevated temperature Sucrose was made up in water at the following concentrations: 20, 87, 200, 350, 540 and 715 mg/ml. An aliquot of each sucrose sample was stored at 4°C while another aliquot was incubated at 45°C. These aliquots were 5 ml in volume and stored in the dark at these temperatures for 7 days. All samples were immediately stored at 4°C after completing the incubation period. Glucose levels were measured in each sample in duplicate using the bioluminescent glucose assay. Time course for sucrose degradation after heat exposure Vaccine samples were aliquoted (120 µL) into 20 microcentrifuge tubes which were separated into two groups (37 and 45°C). The tubes in each group were exposed to 37 and 45°C for 3, 6, 12, 24 (1 day), 48 (2 days), 72 (3 days), 96 (4 days), 120 (5 days), 144 (6 days), and 168 (7 days) hours. After completing each incubation time, the tubes were stored at 4°C. Aliquots of the vaccines were also prepared in the same manner and stored at 4°C as the 2–8°C controls. Following the incubation period, 50 µL of the vaccine samples from each aliquot was assayed, in duplicate, for its glucose concentration. Bioluminescent glucose assay The concentration of glucose, as a product of sucrose hydrolysis, was measured using the Glucose-Glo Assay (Promega, Madison, WI, USA) according to the manufacturer’s protocol. In principle, glucose in the vaccine sample was oxidised by glucose dehydrogenase concomitant with the reduction of NAD + to NADH. In the presence of NADH, a reductase enzyme was used to catalyse the reduction of pro-luciferin to luciferin. The intensity of the light generated was proportional to the amount of glucose and was used to determine its concentration in the vaccine sample. Vaccine samples (50 µL) and 0–50 µM glucose standards (50 µL) were transferred to wells of a white-bottom 96-well plate (Corning, NY, USA). The reagent buffer was included as a negative control (buffer only) for determining the assay background. Glucose detection reagent (50 µL; a mix of luciferin detection solution, reductase, reductase substrate, glucose dehydrogenase, and NAD) was added to the sample and the plate was shaken for 60 seconds. The plate was then incubated at room temperature for 60 minutes. Luminescence was read using a plate-reading luminometer (CLARIOstar, BMG Labtech, Germany). The glucose standard curve was generated using a 4-parameter logistic model using the BMG MARS Data Analysis Software (BMG Labtech) and used to calculate the concentration of glucose in the vaccine samples. Colorimetric glucose assay The glucose concentrations in COMIRNATY™ vaccine samples were measured using a colorimetric glucose assay kit (Cell Biolabs, San Diego, CA, USA), according to the manufacturer’s protocol. A reaction mix consisting of a colorimetric probe, horseradish peroxidase (HRP), and glucose oxidase was prepared in 1× assay buffer. Glucose standards (50 µL) and vaccine samples (50 µL) were mixed with 50 µL of the reaction mix and prepared in triplicate in a 96-well microtiter plate (Corning). The mixture was incubated for 30 minutes at 37°C protected from light. Glucose in the vaccine sample was oxidized by glucose oxidase into D-gluconic acid and hydrogen peroxide. The generated hydrogen peroxide was detected with a colorimetric probe in a reaction catalysed by HRP. The plate was analysed visually by eye and a photograph of the plate was taken using a mobile phone camera. The plate was also read with a microplate reader (CLARIOstar) at 540 nm. The readouts were blanked to the assay buffer without any glucose standard and Milli-Q water (Merck, Germany) was used as a negative control. The known concentrations of glucose were used to generate a standard curve to calculate the amount of glucose in the sample. A 4-parameter logistic model within the BMG MARS Data Analysis Software (BMG Labtech) was used to calculate the concentration of glucose in the vaccine samples. Potency determination for heat-exposed vaccines Vaccine potency was determined by infectivity assay and expressed as infectious units (IFU) per mL. Briefly, HEK 293 cells were seeded in 96-well plates at 5.8 × 10 4 cells/well and subsequently infected with serial dilutions of vaccine test samples. At 48 hrs post-infection, cells were fixed with methanol, blocked with 1% BSA, and stained with a mouse monoclonal anti-Hexon antibody (Abcam B025/AD51). Following the incubation with the secondary antibody, Rabbit polyclonal anti-mouse IgG - H&L, HRP-conjugated (Abcam ab6728), positive cells were visualised with DAB staining and enumerated using NyOne (Synentec). Biochemical analyser The Abbott Architect c16000 analyser (Abbott Laboratories, Maidenhead, UK) was used to analyse the vaccine samples according to our described method. 29 The heat-exposed Bexsero™, COMIRNATY™, and COVISHIELD™ vaccine samples (after seven days of exposure to degradation conditions) along with their correctly stored controls were measured with eight separate runs on the instrument. The method optimised for urine specimens was used and the following eight analytes were measured: calcium, chloride, glucose, magnesium, phosphate, potassium, protein and sodium. Statistical analysis Ordinary one-way ANOVA with Dunnett’s tests was used to compare glucose levels between vaccine samples stored at the recommended 2–8°C and the other temperature-altered conditions. Statistical analysis was performed using GraphPad Prism v.10.1.2 (GraphPad Software, Boston, MA, USA). A p -value less than 0.05 was considered statistically significant. Declarations Author contribution B.G, N.Z, P.N.N, C.C, P.M and J.M designed the study. B.Y.A, A.M, M.B, J.B, T.J, T.B, and B.G performed the experiments and/or data analysis. B.Y.A prepared the original draft. All authors contributed to the writing, reviewing, and editing the manuscript. S.R.C, K.D.P, S.G, R.K, and A.M provided the COVISHIELD™ vaccine. P.N.N, N.Z, J.M and P.M were involved in funding acquisition. B.G, N.Z, P.N.N, C.C, J.M and P.M were involved in project management. All authors read and approved the final manuscript. Acknowledgements We are grateful to two anonymous donor families and the Oak Foundation who provided dedicated funds to the University of Oxford to support this research. We thank the Serum Institute of India for providing the COVISHIELD™ vaccine samples and are grateful to the WHO for facilitating the transfer of these vaccines. We are very grateful to NHS England for providing COMIRNATY™ vaccine samples. We thank the WHO for their grant support to the Rutherford Appleton Laboratory (Ref. 2021/1170671-0). Furthermore, we would like to thank Susanna Dunachie, Sarah Gilbert, Adrian Hill, Andrew Pollard, Cathrin Hauk, Kerlijn Van Assche, Raymond A. Dwek, Karl Tulip, Paul Stickings, Nicola Rose, Paul Matejtschuk, Rory Care, Pernette Bourdillon Esteve, Anita Sands, Michelle Taylor-Siddons, Ali Amini, Simon Draper, Audrey Dubot-Peres, Islip Surgery and the Oxford University Hospitals NHS Foundation Trust for their support for this project and expert advice. MD, KVA, CC and PNN are supported by the Wellcome Trust (222506/Z/21/Z). BYA is funded by the Indonesian Education Scholarship (Beasiswa Pendidikan Indonesia) from the Ministry of Higher Education, Science and Technology of the Republic of Indonesia (Kemendiktisaintek) within a funding scheme from Indonesia Endowment Fund for Education (LPDP). BG was partly supported by the Oxford Glycobiology Endowment and is currently supported by the Medical and Life Sciences Translational Fund from the Translational Research Office of the Medical Sciences Division at the University of Oxford. This work was funded in part, by the Wellcome Trust [220211/Z/20/Z, 222506/Z/21/Z and 202935/Z/16/Z]. For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. The funder played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript. Competing interests P.M declares consultancy for Agilent Technologies, of which R.S is an employee. All other authors have no competing intesrests. The authors alone are responsible for the views expressed in this and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated. 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All other authors have no competing intesrests. The authors alone are responsible for the views expressed in this and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated. Supplementary Files SupportingFigureandTables.docx Figure S1 and Tables S1 and S2 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Error bars show the standard deviations from two measurements for each temperature point. 3× FT, three freeze-thaw cycles; RT, room temperature. Ordinary one-way ANOVA with Dunnett’s multiple comparisons tests. ns, not significant; *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.005; ****p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6839799/v1/f72fb8155e335f4c4c6b6965.jpg"},{"id":84384452,"identity":"8013d9c2-4ef3-4b02-bdf6-e6a32c21ca30","added_by":"auto","created_at":"2025-06-11 09:46:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":733534,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysing the thermal degradation of sucrose over 7 days for (A) Bexsero™, (B) COVISHIELD™, and (C) COMIRNATY™ at 37 °C (blue line) and 45 °C (red line), compared to the recommended storage temperature of 2-8 °C (green line) using the bioluminescent glucose assay. Error bars show the standard deviations from two measurements. Ordinary two-way ANOVA with Dunnett’s multiple comparison test. ns, not significant; **p\u0026lt;0.01; ***p\u0026lt;0.005; ****p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6839799/v1/1cd1f55ec9db9e04070f04eb.jpg"},{"id":84385227,"identity":"e82172f2-25b4-4b88-b523-392116aeb414","added_by":"auto","created_at":"2025-06-11 09:54:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1458073,"visible":true,"origin":"","legend":"\u003cp\u003eA colorimetric glucose assay was used to measure levels of glucose in 30 µg adult and 10 µg children’s COMIRNATY™ vaccines after exposure to different temperature conditions. (A) Photograph of the plate showing an increase in glucose at 37 °C and a further increase at 45 °C. The difference in colour intensity for 37 °C and 45 °C compared to 2-8°C storage can be observed by eye without the need for a plate reader. (B) The intensity of the colour change was also analysed using a spectrophotometer at 540 nm and the concentrations of glucose in the vaccine samples were determined based on the glucose standards. The levels of glucose in temperature-altered conditions were compared to the recommended 2-8 °C storage temperature. Error bars show the standard deviations from three measurements for each temperature point. 3× FT, three freeze-thaw cycles; RT, room temperature. Ordinary one-way ANOVA with Dunnett’s multiple comparisons tests. ns, not significant; ****p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6839799/v1/0f911cfb1fa7336c0ec7e623.jpg"},{"id":84384451,"identity":"ce31aa00-e82d-464f-9046-2cff9a29ed40","added_by":"auto","created_at":"2025-06-11 09:46:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":585943,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of the impact of degradation conditions on vaccine potency indicated by infectivity and the resultant glucose levels. (A) Correlation between vaccine potency and glucose levels, (B) Pearson correlation between vaccine potency and glucose levels.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6839799/v1/be7a6dfb543f3d93a98f739a.jpg"},{"id":84736134,"identity":"4e38a05a-ac8a-426b-9142-c7fb043eb58c","added_by":"auto","created_at":"2025-06-16 18:27:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4494491,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6839799/v1/6e6e61e4-f513-4fd7-87e5-f3a0cda228e7.pdf"},{"id":84384454,"identity":"f4d53fb6-604c-4c61-92d4-bf4b292a11b4","added_by":"auto","created_at":"2025-06-11 09:46:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":38692,"visible":true,"origin":"","legend":"Figure S1 and Tables S1 and S2","description":"","filename":"SupportingFigureandTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6839799/v1/d3b4b7f4fa002bf26f8486bc.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nP.M declares consultancy for Agilent Technologies, of which R.S is an employee. All other authors have no competing intesrests. The authors alone are responsible for the views expressed in this and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated.","formattedTitle":"Ensuring vaccine cold chain integrity: A rapid and low-cost test for identifying heat-exposed sucrose-containing vaccines","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe surge of substandard and falsified (SF) medicines and vaccines coupled with the lack of accessible and affordable methods for their screening and detection in supply chains is a significant problem.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e One in 10 medical products are reported to be either substandard or falsified.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Substandard products are genuine products that fail to meet either quality standards or specifications, or both. In contrast, falsified products are where the products\u0026rsquo; identity, composition or source is deliberately and fraudulently misrepresented by criminals.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e SF medical products have become significant health threats and potentially lead to higher rates of illness and death, as well as eroding public trust in the form of hesitancy as they are unsafe and ineffective.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOur Vaccine Identity Evaluation (VIE) consortium has been evaluating novel techniques for detecting SF vaccines in supply chains.\u003csup\u003e \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e \u003c/sup\u003e We have successfully used spatially offset Raman spectroscopy (SORS)\u003csup\u003e \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e \u003c/sup\u003e, rapid diagnostic tests\u003csup\u003e \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e \u003c/sup\u003e, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS)\u003csup\u003e \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e \u003c/sup\u003e to differentiate genuine vaccines from falsified vaccine surrogates. However, complex devices or tests are required to determine substandard vaccines due to heat exposure. The World Health Organisation (WHO) estimated that over half of vaccines are wasted globally each year due to temperature control failures, logistics challenges, and shipment-related issues.\u003csup\u003e \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e \u003c/sup\u003e Environmental factors, such as temperature and light, should be examined in vaccine stability studies\u003csup\u003e \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e \u003c/sup\u003e with heat exposure being the greatest problem since light exposure can be more easily avoided by the packaging of vaccine vials and syringes. Cold chain is a crucial aspect of protecting vaccines from deterioration, as improper temperature storage could affect vaccine stability and cause harmful effects of reduced efficacy and changes in the safety profile.\u003csup\u003e \u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e \u003c/sup\u003e Maintaining the cold chain is deemed expensive and contributes substantially to costs with the pharmaceutical cold chain logistics market size of US \u003cspan\u003e$\u003c/span\u003e18.61\u0026nbsp;billion in 2024 and expected to reach \u003cspan\u003e$\u003c/span\u003e27.11\u0026nbsp;billion by 2033.\u003csup\u003e \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e \u003c/sup\u003e Successful strategies are needed to generate stable and efficacious dosage forms; these include efforts to stabilise the antigen, selection of adjuvants, and advanced analytical methods to monitor vaccine stability.\u003csup\u003e \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e \u003c/sup\u003e \u003c/p\u003e \u003cp\u003ePotency assays for vaccines and medical products rely on biological assays in the form of \u003cem\u003ein vivo\u003c/em\u003e (animal immunogenicity tests) or \u003cem\u003ein vitro\u003c/em\u003e (cell-based or antibody-binding-based) assays.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Vaccine potency is an indicator of vaccine stability and is a regulatory requirement for release into the market.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e However, these assays are complex, expensive, time-consuming and can be variable with the need for specialist laboratory instruments and trained personnel.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Vaccine vial monitors (VVM) are thermochromic labels on vaccine vials which change in colour when exposed to elevated temperatures.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e However, many vaccines do not have VVM attached and there are no simple, rapid and low-cost methods to screen for substandard heat-exposed vaccines in supply chains and evaluate the accuracy of VVM. Furthermore, VVM do not show whether a vaccine has deteriorated and only indicates if a vaccine has been cumulatively exposed to elevated temperature.\u003c/p\u003e \u003cp\u003eSugars, such as sucrose and lactose, are common ingredients in approved vaccines where they are used as stabilisers from natural sources\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e due to their antigen-stabilising and immunogenicity-maintaining properties.\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e When an aqueous solution of the disaccharide sucrose is heated, it breaks down into its monosaccharides glucose and fructose.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Here we demonstrate three novel, simple, and sensitive approaches to detect heat-exposed sucrose-containing vaccines based on the detection of the glucose formed as a product of degraded sucrose molecules: 1) a quantitative bioluminescent glucose assay, 2) a semi-quantitative colorimetric glucose assay which involves mixing the vaccine with a reagent and then observing a colour change by eye, and 3) a quantitative biochemical analyser which is available in most hospitals worldwide was used to determine eight analytes, including the levels of glucose in heat-exposed vaccines. These quantitative and semi-quantitative glucose assays are not intended to replace the standard vaccine potency assay and are only presented as surrogatecost approaches. These assays can be used as predictors of unstable heat-exposed vaccines within supply chains in accordance with the WHO\u0026rsquo;s Prevent, Detect, and Respond strategy. Of most importance is the colorimetric assay since it does not require a plate reader or other spectrophotometer, is of very low-cost and therefore could be easily deployed in low- and middle-income countries which may not have access to such instrumentation.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGlucose levels in sucrose-containing vaccines after exposure to different temperature conditions\u003c/h2\u003e \u003cp\u003eUsing the bioluminescent assay, glucose concentrations were measured as an indicator of thermal degradation of sucrose after the vaccines were exposed to different temperatures for seven days and compared to vaccines correctly stored at 2\u0026ndash;8\u0026deg;C as recommended by the manufacturer. Among seven vaccines tested, three vaccines (Bexsero\u0026trade;, COMIRNATY\u0026trade; and COVISHIELD\u0026trade;) showed a clear and statistically significant increase in glucose concentration after exposure to elevated temperatures of 37 and 45\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. A-C). A smaller increase was observed for Nimenrix\u0026trade; but was significant at both 37 and 45\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The increase was recorded as 4.5-, 9.6-, 2.8-, and 1.1- fold in Bexsero\u0026trade;, COMIRNATY\u0026trade;, COVISHIELD\u0026trade;, and Nimenrix\u0026trade; respectively, after seven days of incubation at 37\u0026deg;C compared to the vaccine samples correctly stored at 2\u0026ndash;8\u0026deg;C. After incubation at 45\u0026deg;C, a higher increase in glucose was observed compared to incubation at 37\u0026deg;C, with a 9.2-, 27.6-, and 6.3-fold change in Bexsero\u0026trade;, COMIRNATY\u0026trade;, and COVISHIELD\u0026trade;, respectively (Supplementary Table S1) compared to the 2\u0026ndash;8\u0026deg;C storage condition. However, no significant increase in glucose after heat exposure was observed for Rabipur\u0026trade;, Rotarix\u0026trade;, and Ticovac\u0026trade; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-G). Exposure to three freeze-thaw cycles and room temperature conditions after seven days did not result in significant changes in glucose levels in any of the vaccines tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnalysis of thermal degradation of sucrose over time using the bioluminescent glucose assay\u003c/h3\u003e\n\u003cp\u003eAfter establishing which vaccines gave rise to measurable glucose levels upon heat treatment after a week, we proceeded to measure those vaccines (Bexsero\u0026trade;, COMIRNATY\u0026trade;, and COVISHIELD\u0026trade;) over a seven-day time-course to determine when sucrose degradation could be first detected by the bioluminescent glucose assay. Thermal degradation of sucrose in all three vaccines could be observed by increasing levels of glucose when exposed to 37 and 45\u0026deg;C. Significant increases in glucose levels were detected after 12 hours for Bexsero\u0026trade; and COMIRNATY\u0026trade;, and 3 hours for COVISHIELD\u0026trade; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eColorimetric glucose assay to detect glucose levels in sucrose-containing vaccines\u003c/h3\u003e\n\u003cp\u003eA simpler colorimetric glucose assay was used to determine the glucose levels in COMIRNATY vaccines for both the 30 \u0026micro;g adult and 10 \u0026micro;g children\u0026rsquo;s vaccines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The development of colour intensity along with the increasing level of glucose could be observed visually by eye (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The assay could significantly detect vaccine vials exposed to the elevated temperatures 37 and 45\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). A similar increase in glucose was observed in both 30 \u0026micro;g adult and 10 \u0026micro;g children's vaccines which are manufactured with the same amount of sucrose in both vaccines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSucrose-containing vaccines used in the study. Data were retrieved from the Electronic Medicine Compendium (EMC), available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.medicines.org.uk/emc\u003c/span\u003e\u003cspan address=\"https://www.medicines.org.uk/emc\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e and other sources in the public domain.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVaccine trade name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDescription and usage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eManufacturer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePharmaceutical form\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExcipients including sucrose concentration in bold\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRotarix\u0026trade;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLive attenuated rotavirus vaccine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGlaxoSmithKline (GSK)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral suspension in squeezable tube, clear and colourless viscous liquid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSucrose (\u003cb\u003e715 mg/mL\u003c/b\u003e), disodium Adipate, Dulbecco's Modified Eagle Medium/DMEM (containing phenylalanine, sodium, glucose, and other substances), sterile water\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNimenrix\u0026trade;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMeningococcal groups A, C, W-135 and Y conjugate vaccine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePfizer Ltd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePowder and solvent for solution for injection in pre-filled syringe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePowder\u003c/em\u003e:\u003c/p\u003e \u003cp\u003eSucrose (\u003cb\u003e56 mg/ml\u003c/b\u003e after reconstitution), trometamol\u003c/p\u003e \u003cp\u003e\u003cem\u003eSolvent\u003c/em\u003e:\u003c/p\u003e \u003cp\u003eSodium chloride, water for injection\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRabipur\u0026trade;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabies vaccine (inactivated, strain Flury LEP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBavarian Nordic A/S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePowder and solvent for solution for injection in pre-filled syringe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePowder\u003c/em\u003e:\u003c/p\u003e \u003cp\u003eTrometamol, sodium chloride, disodium edetate, potassium-L-glutamate, polygeline, sucrose (\u003cb\u003e60 mg/mL\u003c/b\u003e after reconstitution\u003csup\u003ea\u003c/sup\u003e)\u003c/p\u003e \u003cp\u003e\u003cem\u003eSolvent\u003c/em\u003e:\u003c/p\u003e \u003cp\u003eWater for injection\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBexsero\u0026trade;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMeningococcal group-B vaccine (rDNA, component, adsorbed)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGSK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSuspension for injection in pre-filled syringe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSodium chloride, histidine, sucrose (\u003cb\u003e20 mg/mL\u003c/b\u003e), water for injections, aluminium hydroxide (hydrated)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTicoVac\u0026trade; Junior\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTick-Borne Encephalitis Vaccine (whole virus, inactivated)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePfizer Ltd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSuspension for injection in a pre-filled syringe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHuman albumin, sodium chloride, disodium phosphate-dihydrate, potassium dihydrogenphosphate, water for Injection, sucrose (expected to be \u003cb\u003e\u0026le;\u0026thinsp;30 mg/mL\u003c/b\u003e as for TicoVac\u0026trade; adult dose), aluminium hydroxide, hydrated.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCOVISHIELD\u0026trade;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCOVID-19 vaccine; Recombinant, replication-deficient chimpanzee adenovirus vector encoding the SARS-CoV-2 Spike (S) glycoprotein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSerum Institute of India\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSolution for injection, colourless to slightly brown, clear to slightly opaque and particle-free with a pH of 6.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eL-Histidine, L-Histidine hydrochloride monohydrate, magnesium chloride hexahydrate, polysorbate 80, ethanol, sodium chloride, disodium edetate dihydrate (EDTA), sucrose (\u003cb\u003e75 mg/mL\u003c/b\u003e)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCOMIRNATY\u0026trade;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCOVID-19 vaccine; mRNA vaccine (nucleoside modified): 10 micrograms per dose (children 5 to 11 years) and 30 micrograms per dose (ages 12 years and older)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePfizer Ltd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConcentrate for dispersion for injection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC); cholesterol, trometamol; trometamol hydrochloride; water for injections; sucrose (\u003cb\u003e20 mg/mL\u003c/b\u003e\u003csup\u003e35\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003ea\u003c/sup\u003einformation obtained from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.medsafe.govt.nz/profs/datasheet/r/rabipurinj.pdf\u003c/span\u003e\u003cspan address=\"https://www.medsafe.govt.nz/profs/datasheet/r/rabipurinj.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003eb\u003c/sup\u003einformation obtained from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.seruminstitute.com/health_faq_covishield.php\u003c/span\u003e\u003cspan address=\"https://www.seruminstitute.com/health_faq_covishield.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eCorrelation of potency with glucose for heat-exposed vaccines\u003c/h3\u003e\n\u003cp\u003eTo assess whether glucose levels could correlate with vaccine potency, a human Adenovirus 5-based vaccine formulated in a buffer containing 7.5% sucrose was exposed to various temperature-altered conditions. Storage at 4\u0026deg;C, room temperature, or freeze/thaw (F/T) three times had no impact on viral potency compared to the reference sample stored at -80\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Conversely, the detected glucose levels showed no variation compared to the reference sample. Exposure of samples to higher temperatures (37\u0026deg;C and 45\u0026deg;C) resulted in reduced levels of potency, with a 2-log decrease for 37\u0026deg;C incubation and a 3-log reduction for the 45\u0026deg;C storage. Glucose levels from those samples were elevated, between 5- and 11-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Furthermore, the correlation analysis confirmed that degradation-mediated glucose levels correlate significantly with vector infectivity, confirming that glucose is potentially a good predictor for vaccine potency (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eGlucose assays using a biochemical analyser\u003c/h3\u003e\n\u003cp\u003eUsing the biochemical analyser, glucose could be successfully detected in COVISHIELD\u0026trade; exposed to 37\u0026deg;C (at the lower limit of quantitation 60 \u0026micro;M) and 45\u0026deg;C (140 \u0026micro;M). Both of these measured glucose concentrations were similar to the levels detected by bioluminescent glucose assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Glucose could also be detected at even higher levels in COVISHIELD\u0026trade; samples exposed to 100\u0026deg;C (data not shown since vaccines would never be exposed to such a high temperature on the supply chain). However, glucose could not be detected and quantified in Bexsero\u0026trade; and COMIRNATY\u0026trade; samples.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlmost all vaccines currently need to be either refrigerated or frozen during storage and shipment. This is an essential requirement to maintain their potencies which could be reduced or lost if vaccines are exposed to high ambient temperature which in many countries can exceed 45\u0026deg;C.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Potency assays are difficult to carry out rapidly in the supply chain since they require sophisticated laboratory instruments and sometimes reagents manufactured by the vaccine manufacturer which would further increase costs. VVM can help to determine heat exposure, but they are not always used on vaccine vials and syringes. All seven vaccines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) used in this study did not have VVM highlighting the importance of tests on the vaccine itself. Furthermore, VVMs only indicate if vaccine is likely to have been degraded with no correlation to potency. In contrast, we show that the glucose assay has good correlation to potency highlighting that our reported rapid test adds value to even products which already have VVM.\u003c/p\u003e \u003cp\u003eThere is a need for faster and easier methods which could be used without the need for devices. Such rapid tests would help to initially screen for substandard vaccines that have been exposed to elevated temperature, before being sent for reference potency assays, facilitating timely and more cost-effective detection.\u003c/p\u003e \u003cp\u003eSugars, such as sucrose and lactose, are often used as stabilisers in injectable vaccines but sucrose has also been used in oral vaccines to improve taste. We have demonstrated that the surrogate measurement of glucose, as a product of sucrose degradation, could be used as a predictor of vaccines exposed to elevated temperatures. This novel approach was found to successfully work for most of the vaccines tested. Importantly, we have shown that the increase in glucose correlates significantly with vector infectivity, confirming that this novel marker for heat-exposed sucrose-containing vaccines is a good predictor for vaccine potency (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe have recently shown that SORS and MALDI-ToF MS are unable to detect if a vaccine, which contained sucrose, was exposed to heat.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e SORS, MALDI-ToF MS and rapid diagnostic tests can aid in determining falsified vaccines whereas the glucose assays can help in identifying substandard heat-exposed sucrose-containing vaccines, highlighting that these techniques in combination can help to detect SF vaccines.\u003c/p\u003e \u003cp\u003eA significant increase in glucose after seven days of exposure to elevated temperatures of 37 and 45\u0026deg;C was observed for COMIRNATY\u0026trade;, COVISHIELD\u0026trade; and Nimenrix\u0026trade;. For Nimenrix\u0026trade;, although the increase was significant, the increase in glucose was only 1.1- and 1.2-fold for 37 and 45\u0026deg;C whereas for the other vaccines the fold change was considerably higher (2.8- to 9.6-fold for 37\u0026deg;C and 6.3- to 27.6 fold for 45\u0026deg;C; Table S1). For Rabipur\u0026trade;, another lyophilised vaccine, no significant increase in glucose was observed. Lyophilised vaccines have been shown to have good stability at 37\u0026deg;C for up to one month\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The lower increase in glucose for Nimenrix\u0026trade; and no increase in glucose for Rabipur\u0026trade; is due to these vaccines being lyophilised since water is needed for sucrose hydrolysis and with such a small fold change for Nimenrix\u0026trade; it may be challenging to use glucose assays to detect its exposure to heat.\u003c/p\u003e \u003cp\u003eNo significant increase in glucose was observed also for Rotarix\u0026trade; and Ticovac\u0026trade; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) suggesting that sucrose degradation depends on vaccine formulation.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Rotarix\u0026trade; is a highly viscous liquid including a thickening agent disodium adipate.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Unlike all the other injectable vaccines tested, Rotarix\u0026trade; is given orally and contains sucrose to improve taste. The high viscosity of the vaccine and the extremely high level of sucrose compared to injectable vaccines (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) could be the reason why no significant increase in glucose was observed. Ticovac\u0026trade; contains human serum albumin that functions as sheer protection and thermal stability enhancer.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e The presence of this excipient may have helped to reduce the thermal degradation of sucrose. No significant change in glucose levels was observed for all vaccines when exposed to freeze-thaw cycles and stored at ambient room temperature of 20\u0026deg;C for seven days. However, damage from accidental freezing can result in potency loss for freeze-sensitive vaccines\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e and this could not be readily inspected using our glucose assay. A drop in infectivity has been shown for a ChAdOx1 vaccine exposed to 22\u0026deg;C from 35 days onwards and for a ChAdOx2 vaccine from 180 days onwards.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e These vaccines had the same excipients as COVISHIELD\u0026trade; and the same sucrose concentration of 75 mg/ml. For all vaccines tested in our study, we did not see any change in glucose after 7 days at RT (20\u0026deg;C). However, we have not tested if glucose increases after 35 days at this ambient temperature since it is unlikely that vaccines would be left for so long at RT in the supply chain.\u003c/p\u003e \u003cp\u003eLevels of glucose were measured in sucrose samples made up in water and stored at both 4\u0026deg;C and 45\u0026deg;C (Figure S1). Sucrose solutions were made up at various concentrations from 20 to 715 mg/ml since they cover the sucrose concentrations commonly used in vaccines, e.g. Bexsero (20 mg/ml), Spikevax (87 mg/ml), RotaTeq (540 mg/ml) and Rotarix (715 mg/ml). The sucrose solutions stored at 4\u0026deg;C had very low levels of glucose (below 10 \u0026micro;M). However, for all solutions stored at 45\u0026deg;C, there was a large increase in glucose of around 100-fold (Figure S1) unlike the vaccine data with relatively small increases in glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This indicates that all concentrations of sucrose commonly used in vaccines could show an increase in glucose with heat exposure. However, we either observed a relatively lower increase in glucose of 1.2 to 27.6-fold (Table S1, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D) or no significant increase at all (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-G). The sucrose solutions made up in water did not have any other excipients and therefore the lower or no increase in glucose observed for the vaccines must be due to other excipients which are reducing sucrose hydrolysis (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSucrose degradation could be detected as early as 12 hours post-incubation when Bexsero\u0026trade;, COVISHIELD\u0026trade; and COMIRNATY\u0026trade; vaccines were exposed to 37 and 45\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.). In the case of COVISHIELD\u0026trade;, a significant change in glucose (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) was observed after only 3 hours of incubation at these temperatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) with glucose concentration changes of 18.5 and 32.4 \u0026micro;M for 37\u0026deg;C vs 4\u0026deg;C and 45\u0026deg;C vs 4\u0026deg;C, respectively. The bioluminescent glucose assay was sensitive enough to detect these small changes. The assay is capable of quantifying glucose levels down to 0.0031 \u0026micro;M, which is the lowest standard concentration used in the assay. Although a plate reader was required to read the bioluminescence, the assay is straightforward and most medicine regulators and vaccine manufacturers would have this device since they are already used for vaccine potency assays. While both potency assays and the bioluminescent glucose assay use a plate reader, the glucose assay is lower cost, faster and easier to carry out.\u003c/p\u003e \u003cp\u003eTo avoid the use of a plate reader and to decrease costs further, we investigated the use of a rapid and simpler colorimetric glucose assay. This assay could also be used with a plate reader but only requires absorbance measurements instead of luminescence and such spectrophotometers are more widely available globally and of lower cost. Most importantly, the changes in colour intensity seen with the heat-exposed vaccines and comparing this to the pink colour of the glucose standards, it was possible to determine the concentration of glucose semi-quantitatively and visually without the need for any device (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The method simply involves mixing vaccine with a reagent, incubating at 37\u0026deg;C for 30 minutes and any colour change to pink would indicate that the vaccine was exposed to heat. The reagents in this kit could be used along with a reference of colours expected for correctly stored (colourless) and heat-exposed (pink) vaccines. This is the first rapid and device-free assay which correlates with vaccine potency and can significantly aid LMICs where instrumentation is lacking. Although the sensitivity of this assay is lower than the bioluminescent assay, it can in the same way successfully determine vaccines exposed to both 37\u0026deg;C and 45\u0026deg;C. The colorimetric glucose assay used in this study was manufactured by Cell Biolabs who distribute this kit worldwide including to over a dozen LMICs such as Nigeria, India, Pakistan and Egypt, which have average high temperatures exceeding 40\u0026deg;C during summer months.\u003c/p\u003e \u003cp\u003eA biochemical analyser was repurposed to detect analytes in vaccine excipients. This analyser can measure glucose in clinical samples (along with 7 other analytes), is very low cost (below \u0026pound;10 to measure all 8 analytes) and is widely available in most diagnostic laboratories worldwide. Although the instrument did not have the same level of sensitivity compared to the bioluminescent or colorimetric assays, it has the advantage of confirming many analytes contained in the genuine vaccine and can be used to evaluate vaccine authenticity (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Supplementary Table\u0026nbsp;2). While the biochemical analyser was unable to detect glucose in heat exposed Bexsero and COMIRNATY\u0026trade;, it was able to successfully detect glucose in heat-exposed COVISHIELD\u0026trade; (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This was expected since the bioluminescent assay showed higher levels of glucose after heat exposure for COVISHIELD\u0026trade; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), which was within the working range of the biochemical analyser, compared to Bexsero (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and COMIRNATY\u0026trade; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Also, the concentration of sucrose in COVISHIELD\u0026trade; is 3.75 times higher than both Bexsero and COMIRNATY\u0026trade; (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It is possible that the biochemical analyser may be able to detect glucose in vaccines with a similar sucrose concentration as COVISHIELD\u0026trade;. The manufacturer of this vaccine, Serum Institute of India, has recently manufactured the R21 malaria vaccine which contains sucrose although its concentration is unknown. It is possible that a biochemical analyser could detect heat exposure for this R21 malaria vaccine, and other sucrose-containing vaccines, assuming that the sucrose concentration are similar.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eConcentrations of glucose measured in vaccine samples using a biochemical analyser after exposure to different altered temperature conditions. Values in bold indicate if the analytes could be detected and quantified showing their mean concentrations (\u0026plusmn;\u0026thinsp;standard deviation) of eight measurements. All other values with the less than symbol (\u0026lt;) were below the limit of quantitation and the lower limit of quantitation is shown.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVaccine\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eGlucose concentration (mM)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u0026times; FT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e45\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBexsero\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCOMIRNATY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCOVISHIELD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e*\u003c/sup\u003eCalculation based on six measurements due to the limited sample; 3\u0026times; FT, three cycles of freeze-thaw; RT, room temperature (20\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe other analytes detected and quantified by the biochemical analyser helped to determine an analyte fingerprint for the vaccines and we have shown how this is a low-cost way to distinguish genuine vaccines from falsified surrogates\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The analytes identified and quantified for the three vaccines act as a fingerprint to confirm authenticity (Supplementary Table\u0026nbsp;2). All vaccines tested contain sodium chloride and as expected, both sodium and chloride ions were detected. Although Bexsero\u0026trade; does not contain potassium, a positive result was seen possibly due to interference from other excipients which is not a problem since the result was consistent among all runs. Such false positives may occur since the analyser is intended for urine samples instead of vaccines which have a very different sample matrix. COMIRNATY\u0026trade; tested positive for protein even though no protein is expected in this vaccine. As we have discussed separately, an explanation for this false positive in mRNA vaccines could be due to benzethonium chloride used for the protein method which interacts with lipids in the vaccine and disrupts the lipid nanoparticles\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Magnesium chloride is present in COVISHIELD\u0026trade; and as expected magnesium was detected. The Oxford COVID-19 ChAdOx1-S vaccine has the same excipient list as COVISHIELD\u0026trade; and we have shown that magnesium ions could also be detected in this vaccine\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSince sucrose is composed of both glucose and fructose, levels of fructose could have also been detected instead of glucose. However, since glucose assays are far more common, more affordable and the biochemical analyser is unable to measure fructose, we decided to only focus on detecting glucose. The approaches described may also be expanded to detect degradation in lactose-containing liquid medicines\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, as lactose breaks down into glucose and galactose when heated. Galactose assays are also available but have the same disadvantages as fructose assays.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ePrevious work assessing adenovirus-vectored vaccines exposed to temperatures ranging from 4 to 45\u0026deg;C using an \u003cem\u003ein vitro\u003c/em\u003e infectivity assay reported a rapid loss of infectivity at 30 and 45\u0026deg;C in less than 10 days of exposure. Only a small decrease in infectivity was observed at ambient 22\u0026deg;C and no loss of infectivity was seen over five freeze-thaw cycles.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Therefore, while the glucose assays were not able to detect vaccines which had been exposed to freeze-thaw cycles, the potency may have been unaffected, as observed in our potency correlation data for the hAd5 vaccine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A rapid initial loss of infectivity has also been observed for a measles vaccine when only exposed to temperatures above ambient.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Furthermore, a case report assessing the stability of an adenovirus-vectored ChAdOx1-S vaccine stored at ambient 21\u0026deg;C for 18 hours before vaccination reported the same safety profile and efficacy as the vaccines stored at the recommended temperature\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. This maintained efficacy at ambient is in line with our observed no significant increase in glucose at ambient. We incubated the vaccine at ambient for a much longer period of 7 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, for ambient exposure over the same 18 hours, the increase in glucose is likely to be minimal or not significant based on our data for exposure to 37\u0026deg;C (at least for Bexsero\u0026trade; and COMIRNATY\u0026trade;; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eElevated temperatures have been shown to degrade an in-house manufactured mRNA vaccine\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. A decrease in mRNA integrity and increase in fragmentation were observed after four days of incubation at 37, 45, and 60\u0026deg;C.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e At this same 4 day (96 hours) timepoint, we were able to successfully detect a significant increase in glucose for the COMIRNATY\u0026trade; mRNA vaccine at both 37 and 45\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This suggests that our novel approach using glucose assays could be used to detect heat-exposed mRNA vaccines which have reduced mRNA integrity and increased fragmentation. The stability of mRNA, as of percentage of intact RNA, correlates with \u003cem\u003ein vitro\u003c/em\u003e potency as both were rapidly decreased when exposed to increasing temperature from 25 to 45\u0026deg;C.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e All mRNA vaccine manufacturers use sucrose and similar excipients (lipids and salts) and therefore the glucose assays are likely to work for all mRNA vaccines.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eCollectively, the pattern of sucrose degradation within 7 days in this study correlates with the results of vaccine stability assays. We propose that the detection of glucose could potentially be an endogenous indicator of temperature exposure for some sucrose-containing vaccines and thus of cold chain failure. Our proposed use of the colorimetric glucose assay reagents is the fastest and lowest cost assay since result are obtained after only 30 minutes. To our knowledge, this is the first device-free test which can detect heat-exposed vaccines and correlates with vaccine potency making it ideal in resource-limited LMICs. This assay simply involves mixing vaccine with a reagent and visualising any colour change to pink would indicate heat-exposed vaccines. An oven is required to incubate the mixed sample and ovens are present in laboratories of all medicine regulators. However, even an oven may not be needed in countries where the ambient temperature is high enough to show the colour change to pink. Our lower cost and more rapid assays could be used by medicine regulators, vaccine manufacturers and inspectors as an initial screening test to help prevent substandard vaccines from being used.\u003c/p\u003e "},{"header":"Methods","content":"\u003ch3\u003eVaccine samples\u003c/h3\u003e\n\u003cp\u003eFive non-COVID-19 vaccines and two COVID-19 vaccines were used in this study (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Non-COVID vaccines were purchased from the Oxford University Hospitals Pharmacy, Oxford, United Kingdom. All vaccines were in-date and tested before expiry. The COVISHIELD\u0026trade; (Serum Insitute of India) and COMIRNATY\u0026trade; (Pfizer Ltd) (30 \u0026micro;g for adults and 10 \u0026micro;g for children) COVID-19 vaccines were received from the Serum Institute of India, Pvt. Ltd. and the National Health Service England (NHS England), respectively. Vaccines were stored at the recommended 2\u0026ndash;8\u0026deg;C storage temperature. COMIRNATY\u0026trade; COVID-19 vaccines were immediately stored in a refrigerator at 2\u0026ndash;8\u0026deg;C when received and used within one month of receipt according to the manufacturer\u0026rsquo;s recommendations after the frozen vaccine is thawed. Vials and syringes were kept on wet ice prior to the tests.\u003c/p\u003e \u003cp\u003eA human Adeno5-based vaccine (hAd5 KC5) was generated by single-round infection with an MOI of 1 on a hyperflask containing approximately 1E\u0026thinsp;+\u0026thinsp;08 human embryonic kidney HEK 293 cells. Cells were harvested 48 hours post-infection and pelleted at 200xg for 30 minutes. Cell pellets were lysed in Cell Lysis Buffer (10 mM Tris, 135 mM sodium chloride, 1 mM magnesium chloride) and freeze/thawed three times. The cell lysate was treated with 250 Units/mL of Benzonase after first thaw and incubated for 30 minutes at room temperature. The viral vector was then purified by double caesium chloride ultracentrifugation, and dialysed three times in Formulation Buffer A438 (10 mM histidine, 7.5% sucrose, 35 mM sodium chloride, 1 mM magnesium chloride, 0.1% polysorbate 80, 0.1 mM ethylenediaminetetraacetic acid, 0.5% (v/v) ethanol, pH 6.6).\u003c/p\u003e\n\u003ch3\u003eExperimental settings\u003c/h3\u003e\n\u003cp\u003eDifferent techniques were used to measure glucose concentrations in the seven vaccines exposed to different temperature conditions for 7 days. After establishing which vaccines gave rise to measurable glucose levels upon heat treatment after a week, we looked at the sucrose degradation kinetics at 10 different time points over 7 days under the two temperature conditions (37 and 45\u0026deg;C) that produced significant glucose changes in the previous tests, to determine when sucrose degradation could be first detected by the techniques under evaluation.\u003c/p\u003e \u003cp\u003eFor one vaccine (hAd5 KC5) we conducted a potency assay in parallel, in order to look at the correlation between glucose levels and potency loss due to heat-exposure.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eVaccine samples exposed to different temperature conditions\u003c/h2\u003e \u003cp\u003eVaccine samples were grouped and each group was exposed to one of the following temperature conditions in the dark: (i) stored at 4\u0026deg;C within the recommended 2\u0026ndash;8\u0026deg;C storage condition, (ii) stored at ambient room temperature (RT, recorded as 20\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) for 7 days, (iii) stored in an incubator oven set at 37\u0026deg;C for 7 days, (iv) stored in an incubator oven set at 45\u0026deg;C for 7 days, and (v) exposed to three freeze-thaw cycles of 24 hrs freezing at -70\u0026deg;C and 1 hr thawing at 4\u0026deg;C per cycle. All samples were immediately stored at 4\u0026deg;C after completing the incubation period. In the case of vaccines which had a separate vial of lyophilised powder and a syringe for the solvent (Nimenrix\u0026trade; and Rabipur\u0026trade;), both the vial and syringe were exposed to the five conditions without prior mixing/reconstitution to resemble a real-life situation of how the vaccine could be exposed to altered temperature on the supply chain. The powdered vaccines were reconstituted with their solvent just prior to analysis.\u003c/p\u003e \u003cp\u003eA degradation test on hAd5 KC5 was performed by incubating glass vials containing 350 \u0026micro;L of the vaccine at RT (recorded as 20\u0026deg; \u0026plusmn; 2\u0026deg;C), 4\u0026deg;C, 37\u0026deg;C and 45\u0026deg;C for 7 days. Vials were also exposed to the same freeze-thaw cycles as described above or stored at -80\u0026deg;C (as a reference control). Each condition was tested in triplicate and tested using both the colorimetric and potency assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSucrose in water exposed to an elevated temperature\u003c/h2\u003e \u003cp\u003eSucrose was made up in water at the following concentrations: 20, 87, 200, 350, 540 and 715 mg/ml. An aliquot of each sucrose sample was stored at 4\u0026deg;C while another aliquot was incubated at 45\u0026deg;C. These aliquots were 5 ml in volume and stored in the dark at these temperatures for 7 days. All samples were immediately stored at 4\u0026deg;C after completing the incubation period. Glucose levels were measured in each sample in duplicate using the bioluminescent glucose assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTime course for sucrose degradation after heat exposure\u003c/h2\u003e \u003cp\u003eVaccine samples were aliquoted (120 \u0026micro;L) into 20 microcentrifuge tubes which were separated into two groups (37 and 45\u0026deg;C). The tubes in each group were exposed to 37 and 45\u0026deg;C for 3, 6, 12, 24 (1 day), 48 (2 days), 72 (3 days), 96 (4 days), 120 (5 days), 144 (6 days), and 168 (7 days) hours. After completing each incubation time, the tubes were stored at 4\u0026deg;C. Aliquots of the vaccines were also prepared in the same manner and stored at 4\u0026deg;C as the 2\u0026ndash;8\u0026deg;C controls. Following the incubation period, 50 \u0026micro;L of the vaccine samples from each aliquot was assayed, in duplicate, for its glucose concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBioluminescent glucose assay\u003c/h2\u003e \u003cp\u003eThe concentration of glucose, as a product of sucrose hydrolysis, was measured using the Glucose-Glo Assay (Promega, Madison, WI, USA) according to the manufacturer\u0026rsquo;s protocol. In principle, glucose in the vaccine sample was oxidised by glucose dehydrogenase concomitant with the reduction of NAD\u003csup\u003e+\u003c/sup\u003e to NADH. In the presence of NADH, a reductase enzyme was used to catalyse the reduction of pro-luciferin to luciferin. The intensity of the light generated was proportional to the amount of glucose and was used to determine its concentration in the vaccine sample.\u003c/p\u003e \u003cp\u003eVaccine samples (50 \u0026micro;L) and 0\u0026ndash;50 \u0026micro;M glucose standards (50 \u0026micro;L) were transferred to wells of a white-bottom 96-well plate (Corning, NY, USA). The reagent buffer was included as a negative control (buffer only) for determining the assay background. Glucose detection reagent (50 \u0026micro;L; a mix of luciferin detection solution, reductase, reductase substrate, glucose dehydrogenase, and NAD) was added to the sample and the plate was shaken for 60 seconds. The plate was then incubated at room temperature for 60 minutes. Luminescence was read using a plate-reading luminometer (CLARIOstar, BMG Labtech, Germany). The glucose standard curve was generated using a 4-parameter logistic model using the BMG MARS Data Analysis Software (BMG Labtech) and used to calculate the concentration of glucose in the vaccine samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eColorimetric glucose assay\u003c/h2\u003e \u003cp\u003eThe glucose concentrations in COMIRNATY\u0026trade; vaccine samples were measured using a colorimetric glucose assay kit (Cell Biolabs, San Diego, CA, USA), according to the manufacturer\u0026rsquo;s protocol. A reaction mix consisting of a colorimetric probe, horseradish peroxidase (HRP), and glucose oxidase was prepared in 1\u0026times; assay buffer. Glucose standards (50 \u0026micro;L) and vaccine samples (50 \u0026micro;L) were mixed with 50 \u0026micro;L of the reaction mix and prepared in triplicate in a 96-well microtiter plate (Corning). The mixture was incubated for 30 minutes at 37\u0026deg;C protected from light. Glucose in the vaccine sample was oxidized by glucose oxidase into D-gluconic acid and hydrogen peroxide. The generated hydrogen peroxide was detected with a colorimetric probe in a reaction catalysed by HRP. The plate was analysed visually by eye and a photograph of the plate was taken using a mobile phone camera. The plate was also read with a microplate reader (CLARIOstar) at 540 nm. The readouts were blanked to the assay buffer without any glucose standard and Milli-Q water (Merck, Germany) was used as a negative control. The known concentrations of glucose were used to generate a standard curve to calculate the amount of glucose in the sample. A 4-parameter logistic model within the BMG MARS Data Analysis Software (BMG Labtech) was used to calculate the concentration of glucose in the vaccine samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePotency determination for heat-exposed vaccines\u003c/h2\u003e \u003cp\u003eVaccine potency was determined by infectivity assay and expressed as infectious units (IFU) per mL. Briefly, HEK 293 cells were seeded in 96-well plates at 5.8 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well and subsequently infected with serial dilutions of vaccine test samples. At 48 hrs post-infection, cells were fixed with methanol, blocked with 1% BSA, and stained with a mouse monoclonal anti-Hexon antibody (Abcam B025/AD51). Following the incubation with the secondary antibody, Rabbit polyclonal anti-mouse IgG - H\u0026amp;L, HRP-conjugated (Abcam ab6728), positive cells were visualised with DAB staining and enumerated using NyOne (Synentec).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical analyser\u003c/h2\u003e \u003cp\u003eThe Abbott Architect c16000 analyser (Abbott Laboratories, Maidenhead, UK) was used to analyse the vaccine samples according to our described method.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e The heat-exposed Bexsero\u0026trade;, COMIRNATY\u0026trade;, and COVISHIELD\u0026trade; vaccine samples (after seven days of exposure to degradation conditions) along with their correctly stored controls were measured with eight separate runs on the instrument. The method optimised for urine specimens was used and the following eight analytes were measured: calcium, chloride, glucose, magnesium, phosphate, potassium, protein and sodium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eOrdinary one-way ANOVA with Dunnett\u0026rsquo;s tests was used to compare glucose levels between vaccine samples stored at the recommended 2\u0026ndash;8\u0026deg;C and the other temperature-altered conditions. Statistical analysis was performed using GraphPad Prism v.10.1.2 (GraphPad Software, Boston, MA, USA). A \u003cem\u003ep\u003c/em\u003e-value less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor contribution\u003c/h2\u003e \u003cp\u003eB.G, N.Z, P.N.N, C.C, P.M and J.M designed the study. B.Y.A, A.M, M.B, J.B, T.J, T.B, and B.G performed the experiments and/or data analysis. B.Y.A prepared the original draft. All authors contributed to the writing, reviewing, and editing the manuscript. S.R.C, K.D.P, S.G, R.K, and A.M provided the COVISHIELD\u0026trade; vaccine. P.N.N, N.Z, J.M and P.M were involved in funding acquisition. B.G, N.Z, P.N.N, C.C, J.M and P.M were involved in project management. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe are grateful to two anonymous donor families and the Oak Foundation who provided dedicated funds to the University of Oxford to support this research. We thank the Serum Institute of India for providing the COVISHIELD\u0026trade; vaccine samples and are grateful to the WHO for facilitating the transfer of these vaccines. We are very grateful to NHS England for providing COMIRNATY\u0026trade; vaccine samples. We thank the WHO for their grant support to the Rutherford Appleton Laboratory (Ref. 2021/1170671-0). Furthermore, we would like to thank Susanna Dunachie, Sarah Gilbert, Adrian Hill, Andrew Pollard, Cathrin Hauk, Kerlijn Van Assche, Raymond A. Dwek, Karl Tulip, Paul Stickings, Nicola Rose, Paul Matejtschuk, Rory Care, Pernette Bourdillon Esteve, Anita Sands, Michelle Taylor-Siddons, Ali Amini, Simon Draper, Audrey Dubot-Peres, Islip Surgery and the Oxford University Hospitals NHS Foundation Trust for their support for this project and expert advice. MD, KVA, CC and PNN are supported by the Wellcome Trust (222506/Z/21/Z). BYA is funded by the Indonesian Education Scholarship (Beasiswa Pendidikan Indonesia) from the Ministry of Higher Education, Science and Technology of the Republic of Indonesia (Kemendiktisaintek) within a funding scheme from Indonesia Endowment Fund for Education (LPDP). BG was partly supported by the Oxford Glycobiology Endowment and is currently supported by the Medical and Life Sciences Translational Fund from the Translational Research Office of the Medical Sciences Division at the University of Oxford.\u003c/p\u003e \u003cp\u003eThis work was funded in part, by the Wellcome Trust [220211/Z/20/Z, 222506/Z/21/Z and 202935/Z/16/Z]. For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. The funder played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript.\u003c/p\u003e \u003cp\u003eCompeting interests\u003c/p\u003e \u003cp\u003eP.M declares consultancy for Agilent Technologies, of which R.S is an employee. All other authors have no competing intesrests. The authors alone are responsible for the views expressed in this and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMedicine Quality Research Group, University of Oxford (2022) Medical Product Quality Report \u0026ndash; COVID-19 Issues. 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Hum Vaccines Immunother 17, 4112\u0026ndash;4114\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6839799/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6839799/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMaintaining 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 \u0026lsquo;out of specification\u0026rsquo;. 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\u0026deg;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\u0026deg;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\u0026rsquo;s Prevent, Detect, and Respond strategy.\u003c/p\u003e","manuscriptTitle":"Ensuring vaccine cold chain integrity: A rapid and low-cost test for identifying heat-exposed sucrose-containing vaccines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-11 09:46:33","doi":"10.21203/rs.3.rs-6839799/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9fb064b9-516f-11e9-9e20-12b504df345a","owner":[],"postedDate":"June 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49820881,"name":"Biological sciences/Biochemistry"},{"id":49820882,"name":"Biological sciences/Immunology/Vaccines"},{"id":49820883,"name":"Biological sciences/Microbiology/Vaccines"},{"id":49820884,"name":"Health sciences/Health care/Public health"}],"tags":[],"updatedAt":"2025-06-18T19:41:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-11 09:46:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6839799","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6839799","identity":"rs-6839799","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}