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However, the vaccine manufacturer prefers its transportation in frozen condition. Data regarding the stability of the vaccine after exposure to repeated freezing processes have not been explored yet. In the present study, different batches were exposed to 3 repeated freezing\/thawing cycles. An orthogonal testing using various techniques was employed to assess any induced changes in the properties of the vaccine in comparison with control samples. Physicochemical properties, including appearance, pH, and molecular size distribution by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were evaluated. Anion-exchange chromatography (AEC) was used for the determination of virus particles, and DNA to-protein ratio to study the biochemical attributes of the vaccine. Furthermore, the immunological properties were evaluated employing <jats:italic>in-vivo<\/jats:italic> test to compare the results obtained by the testing protocol to the vaccine efficiency in animals. Results revealed that the physicochemical attributes of the vaccine were not affected by the applied freeze\u2013thaw cycles. Although the virus particles measured for thawed samples by AEC were below the specified limits of 0.7 \u2013 1.3\u2009\u00d7\u200910<jats:sup>11<\/jats:sup> VP\/mL, the values of DNA to protein ratio remained unaffected (1.1 \u2013 1.5), and there was no reduction in the <jats:italic>in vivo<\/jats:italic>\u00a0potency of the tested samples. Statistical analysis of the results of the quantitative tests implied that the vaccine could withstand up to three successive freezing and thawing cycles during transportation without significant loss in its structural integrity and biological potency.\u00a0<\/jats:p>\n          <jats:p>\n            <jats:bold>Graphical Abstract<\/jats:bold>\n          <\/jats:p>","DOI":"10.1208\/s12249-025-03220-6","type":"journal-article","created":{"date-parts":[[2025,9,10]],"date-time":"2025-09-10T19:12:17Z","timestamp":1757531537000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Integrated Analytical Techniques to Investigate the Effect of the Freezing\/Thawing Cycles on the Non-replicating Recombinant Chimpanzee Adenovirus Viral Vector COVID-19 Vaccine"],"prefix":"10.1208","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9075-5631","authenticated-orcid":false,"given":"Moushira M.","family":"Mostafa","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7058-2649","authenticated-orcid":false,"given":"Nourhan H.","family":"AbdelAllah","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0918-4662","authenticated-orcid":false,"given":"Eman S.","family":"Elzanfaly","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0323-2512","authenticated-orcid":false,"given":"Ghada A.","family":"Sedik","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2025,9,10]]},"reference":[{"issue":"7798","key":"3220_CR1","doi-asserted-by":"publisher","first-page":"270","DOI":"10.1038\/s41586-020-2012-7","volume":"579","author":"P Zhou","year":"2020","unstructured":"Zhou P, Yang X-L, Wang X-G, Hu B, Zhang L, Zhang W, et al. 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