الفهرس 
Study of the thermostability and efficacy of selected antisera and vaccines in the Egyptian markat
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Abstract
Cold chain process is a system that ensures the maintenance of the products at the recommended temperature till administration to the consumer. It is a system in which biological products as antisera and vaccines are transported, stored, and distributed to ensure the maintenance of their potency. Applying the cold chain system indiscriminately on all products added unnecessary costs and causes resource wastes, where some products are stable out of the restricted temperature
Recently, the World Health Organization permitted controlled temperature chain (CTC) as an innovative approach during vaccine storage. CTC allows excursion of the product into ambient temperature not exceeding 40°C under proper monitoring for a limited period of time prior to administration. In this study, the possible applicability of CTC and the effect of freezing for selected antisera and vaccines was evaluated.
Bivalent oral polio vaccine (OPV), hepatitis B vaccine (HepB vaccine; monovalent and combined) and antisera (lyophilized and liquid scorpion-antivenom and liquid snake-antivenom) were tested. Samples were stored at accelerated (35 ± 5°C) and freezing (−25 ± 5 °C) conditions for 24 h, one week and one month in addition to recommended storage condition (2–8 °C), except OPV samples that were tested at accelerated and refrigerated (2–8 °C) conditions compared to recommended storage conditions (−25 ± 5 °C). All testing was done on three and five batches for antisera and vaccines, respectively. Except OPV samples, where eight samples were tested from each batch; seven samples were tested from each batch.
The potency of antisera samples was evaluated via indirect Enzyme linked immunosorbent assay (ELISA) using the specified venoms according to the Egyptian National Regulatory Authority (ENRA) recommendations. The lyophilized scorpion-antivenom showed higher stability than the liquid form, where the lyophilized samples maintained a non-significant difference in potency when stored at accelerated condition for a month. The liquid scorpion-antivenom samples maintained their potency under accelerated condition for a week only. For the liquid snake-antivenom samples, storage at accelerated conditions for one week did not significantly affect the potency (p < 0.05). However, freezing significantly reduced the potency of all samples under all test conditions in case of liquid scorpion-antivenom samples against all tested venoms, and in case of liquid snake-antivenom samples tested against Naja haje venom (p < 0.05). Freezing of snake-antivenom samples for one week reduced the potency against all tested strains. But the lyophilized scorpion antivenom maintained their potency for 24 h under the freezing condition.
The protein content of tested antisera samples was evaluated via biuret assay and protein composition was visualized using SDS-PAGE. Both tests revealed non-significant difference in components and composition of samples proteins stored under different conditions.
The potency of oral polio vaccine (OPV) was tested using the cell culture titration method. OPV samples stored in refrigerator for a month and at accelerated condition for 48 h maintained their potency within the acceptable range recommended by the WHO.
The potency of hepatitis B (HepB) vaccine was tested via sandwich ELISA using HBsAg one Version ULTRA, DIA.PRO Kit. The monovalent and combined (penta) HepB vaccine samples maintained their potency within the acceptable limit under the accelerated conditions for a month and a week, respectively.
The VVM was evaluated for all the vaccine vials under accelerated testing condition in case of HepB vaccine and under both refrigerator and accelerated conditions in case of OPV. The VVMs successfully predicted the loss of potency in samples stored under different conditions at temperatures higher than that recommended for vaccines’ storage. The VVM of the OPV samples stored at 2–8 °C for 24 h, week and a month and at accelerated condition at 48 h displayed sample acceptance for use. While other OPV samples stored at accelerated condition for a week and a month displayed sample discard sign. Monovalent HepB vaccine samples’ VVM revealed an accepted condition for use when stored at accelerated condition (35 ± 5 °C) for 24 h and a week. But, for the combined HepB vaccine, the samples’ VVM revealed acceptance for use only when stored for 24 h at accelerated condition.
HepB vaccine was subjected to aluminium-content assay via back titration. No significant difference was recorded in aluminium content of samples stored under accelerated or freezing conditions compared to those stored under recommended conditions.
Adjuvant containing vaccine (HepB vaccine) were tested for possible effect of freezing. Shake test was performed on frozen vaccine samples and the results were compared to results of samples stored under normal storage conditions. Frozen HepB vaccines either monovalent or combined showed faster sedimentation in shake test. Microscopical examination of HepB vaccine samples stored under different storages conditions confirmed the adverse effect of freezing on adjuvant containing vaccine which appeared as aggregates.
In conclusion, reevaluation of storage conditions of the tested products is urgently required; this can reduce storage costs and improves their availability by managing their storage at temperatures outside the traditional cold chain for a limited period under monitored and controlled conditions. Further studies on other types of antisera and vaccines are still required to confirm their suitability for use under CTC.
Freezing and freeze damage of adjuvant containing vaccines and antisera represent real risks to the effectiveness of these products. Vaccines and antisera should be protected from freezing by strengthening the cold chain system or, alternatively, developing freeze-stable formulations.