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Virus\u2010like particles (VLPs) are a highly immunogenic, safe, and robust approach that can be used to base several vaccine candidates on. Particularly, HIV\u20101 Gag VLPs is a flexible system comprising a Gag core surrounded by a lipid bilayer that can be modified to present diverse types of membrane proteins or antigens against several diseases, like influenza, dengue, West Nile virus, or human papillomavirus, where it has been proven successful. The size distribution and structural characteristics of produced VLPs vary depending on the cell line used to produce them. In this study, we established an analytical method of characterization for the Gag protein core and clarified the current variability of Gag stoichiometry in HIV\u20101 VLPs depending on the cell\u2010based production platform, directly determining the number of Gag molecules per VLP in each case. Three Gag peptides have been validated to quantify the number of monomers using parallel reaction monitoring, an accurate and fast, mass\u2010spectrometry\u2010based method that can be used to assess the quality of the produced Gag VLPs regardless of the cell line used. An average of 3617\u2009\u00b1\u200917 monomers per VLP was obtained for HEK293, substantially varying between platforms, including mammalian and insect cells. This offers a key advantage in quantification and quality control methods to characterize VLP production at a large scale to accelerate new recombinant vaccine production technologies.<\/jats:p>","DOI":"10.1002\/bit.27786","type":"journal-article","created":{"date-parts":[[2021,4,12]],"date-time":"2021-04-12T08:00:05Z","timestamp":1618214405000},"page":"2660-2675","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":31,"title":["Characterization of HIV\u20101 virus\u2010like particles and determination of Gag stoichiometry for different production platforms"],"prefix":"10.1002","volume":"118","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9993-6332","authenticated-orcid":false,"given":"Jes\u00fas","family":"Lavado\u2010Garc\u00eda","sequence":"first","affiliation":[{"name":"Grup d'Enginyeria Cel\u00b7lular i Bioprocessos, Department of Chemical, Biological and Environmental Engineering, Escola d'Enginyeria Universitat 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