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Glycosylation of the spike protein is critical for the protein\u2019s functions in viral attachment and host cell entry. For scalable and cost-effective production of the spike protein, expression system-driven divergence in glycosylation patterns on recombinant spike proteins needs to be fully understood. This study assessed the <jats:italic>N<\/jats:italic>-glycosylation profiles of a full-length trimeric spike protein expressed in either Human Embryonic Kidney (HEK Expi293F) or Chinese Hamster Ovary (CHO-S) cells. Glycopeptide analysis was performed using a tandem mass spectrometry workflow and BioPharma <jats:inline-formula>\n              <jats:alternatives>\n                <jats:tex-math>$${\\hbox {Finder}}^{\\textrm{TM}}$$<\/jats:tex-math>\n                <mml:math xmlns:mml=\"http:\/\/www.w3.org\/1998\/Math\/MathML\">\n                  <mml:msup>\n                    <mml:mrow>\n                      <mml:mtext>Finder<\/mml:mtext>\n                    <\/mml:mrow>\n                    <mml:mtext>TM<\/mml:mtext>\n                  <\/mml:msup>\n                <\/mml:math>\n              <\/jats:alternatives>\n            <\/jats:inline-formula> incorporating HEK and CHO glycan databases for protein characterisation. The results outline important differences in the variety and types of <jats:italic>N<\/jats:italic>-glycan generated by the two cell lines across the 22 known <jats:italic>N<\/jats:italic>-glycosylation sites of the spike protein. A notable increase in terminal sialylation, as well as the presence of the potentially immunogenic <jats:italic>N<\/jats:italic>-glycolylneuraminic acid at a functionally key <jats:italic>N<\/jats:italic>-glycosylation site, was observed in the CHO-S derived spike protein. With the potential for the relatively vast and more complex CHO glycan repertoire (182 glycans relative to 39 human glycans) to produce functional implications with CHO-S expressed spike protein, this study adds valuable knowledge to aid Quality by Design approaches and enable Multi Attribute Monitoring of specific <jats:italic>N<\/jats:italic>-glycosylation sites for proteoform analyses. This can further inform antigen development with future variants in order to devise updated diagnostic tests and therapeutic vaccine designs.<\/jats:p>","DOI":"10.1007\/s12033-024-01288-2","type":"journal-article","created":{"date-parts":[[2024,10,1]],"date-time":"2024-10-01T02:01:43Z","timestamp":1727748103000},"page":"3737-3752","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Glycoprofile Comparison of the SARS-CoV-2 Spike Proteins Expressed in CHO and HEK Cell Lines"],"prefix":"10.1007","volume":"67","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-4771-1618","authenticated-orcid":false,"given":"Helen L.","family":"Wright","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Caroline","family":"Evans","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Philip J.","family":"Jackson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David C.","family":"James","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kang Lan","family":"Tee","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tuck Seng","family":"Wong","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mark J.","family":"Dickman","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jagroop","family":"Pandhal","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2024,10,1]]},"reference":[{"key":"1288_CR1","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2022.835750","author":"C Song","year":"2022","unstructured":"Song, C., Li, Z., Li, C., Huang, M., Liu, J., Fang, Q., Cao, Z., Zhang, L., Gao, P., Nie, W., Luo, X., Kang, J., Xie, S., Lyu, J., & Zhu, X. 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