{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:16:47Z","timestamp":1760145407427,"version":"build-2065373602"},"reference-count":58,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T00:00:00Z","timestamp":1759968000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ph.D. scholarship from Capes","award":["001"],"award-info":[{"award-number":["001"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibodies"],"abstract":"<jats:p>Background: Recombinant monoclonal antibodies (mAbs) represent the fastest-growing sector of the biopharmaceutical industry, with their efficient expression being a key technological factor for scalability. Objectives: In this study we compared the performance of two bicistronic vectors, which alternate the positions of the light and heavy chain coding genes, employing a wild-type Encephalomyocarditis virus (EMCV) IRES functional element to drive expression of the second gene. Methods: Using two neutralizing anti-SARS-CoV-2 IgG1 antibodies as model molecules, we conducted transient transfections in the commercially available ExpiCHOTM platform. Following protein A affinity purification and quantification, vectors positioning the light chain as the first cistron consistently yielded higher expression levels than those with the heavy chain upstream. To confirm the quality attributes of the mAbs, we applied a comprehensive analytical workflow, including SDS-PAGE and Western blot for molecular mass and purity, circular dichroism for secondary structure, intrinsic tryptophan fluorescence for tertiary structure, and SEC-HPLC for quaternary structure and aggregate detection. Additionally, we assessed binding affinity to the target using spot blot and surface plasmon resonance, analyzed N-glycosylation profiles by HILIC-HPLC and mass spectrometry, and examined molecular structure by transmission electron microscopy. Results and Conclusions: Together, these results provide insight into the impact of gene positioning within bicistronic vectors on mAb expression efficiency and quality, supporting optimization strategies for scalable recombinant antibody production.<\/jats:p>","DOI":"10.3390\/antib14040086","type":"journal-article","created":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:50:16Z","timestamp":1760107816000},"page":"86","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Structural and Functional Characterization of Anti-SARS-CoV-2 Spike Monoclonal Antibodies Produced via Bicistronic Expression in CHO Cells"],"prefix":"10.3390","volume":"14","author":[{"given":"Federico Francisco","family":"Marsili","sequence":"first","affiliation":[{"name":"Cell Culture Engineering Laboratory, COPPE, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-596, RJ, Brazil"},{"name":"Biochemistry Program, Chemistry Institute, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-909, RJ, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fernanda Bittencourt","family":"de Aquino","sequence":"additional","affiliation":[{"name":"Cell Culture Engineering Laboratory, COPPE, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-596, RJ, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0948-5484","authenticated-orcid":false,"given":"Hiam Rodrigo da Silva","family":"Arruda","sequence":"additional","affiliation":[{"name":"Leopoldo de Meis Institute of Medical Biochemistry, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-902, RJ, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mayra Amorim","family":"Marques","sequence":"additional","affiliation":[{"name":"Faculty of Pharmacy, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-902, RJ, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Katia Maria dos Santos","family":"Cabral","sequence":"additional","affiliation":[{"name":"Protein Advanced Biochemistry, National Center for Structural Biology and Bioimaging (CENABIO), Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-902, RJ, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4921-8185","authenticated-orcid":false,"given":"Marcius da Silva","family":"Almeida","sequence":"additional","affiliation":[{"name":"Protein Advanced Biochemistry, National Center for Structural Biology and Bioimaging (CENABIO), Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-902, RJ, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0063-5888","authenticated-orcid":false,"given":"Guilherme Augusto Piedade","family":"de Oliveira","sequence":"additional","affiliation":[{"name":"Leopoldo de Meis Institute of Medical Biochemistry, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-902, RJ, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1946-7191","authenticated-orcid":false,"given":"Andrea Queiroz","family":"Maranh\u00e3o","sequence":"additional","affiliation":[{"name":"Department of Cellular Biology, Institute of Biological Sciences, University of Brasilia (UNB), Brasilia 70910-900, DF, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2530-752X","authenticated-orcid":false,"given":"Renato Sampaio","family":"Carvalho","sequence":"additional","affiliation":[{"name":"Laboratory of Pain and Inflammatory Pharmacology, Institute of Biomedical Sciences, Health Science Centre, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-577, RJ, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5904-410X","authenticated-orcid":false,"given":"Leda dos Reis","family":"Castilho","sequence":"additional","affiliation":[{"name":"Cell Culture Engineering Laboratory, COPPE, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-596, RJ, Brazil"},{"name":"Biochemistry Program, Chemistry Institute, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-909, RJ, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"110376","DOI":"10.1016\/j.intimp.2023.110376","article-title":"Production of monoclonal antibodies for therapeutic purposes: A review","volume":"120","year":"2023","journal-title":"Int. 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