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Volume 14
Issue 4
10.3390/antib14040086
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Article

Structural and Functional Characterization of Anti-SARS-CoV-2 Spike Monoclonal Antibodies Produced via Bicistronic Expression in CHO Cells

by
Federico Francisco Marsili
1,2,
Fernanda Bittencourt de Aquino
1,
Hiam Rodrigo da Silva Arruda
3,
Mayra Amorim Marques
4,
Katia Maria dos Santos Cabral
5,
Marcius da Silva Almeida
5,
Guilherme Augusto Piedade de Oliveira
3,
Andrea Queiroz Maranhão
6,
Renato Sampaio Carvalho
7 and
Leda dos Reis Castilho
1,2,*
1
Cell Culture Engineering Laboratory, COPPE, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-596, RJ, Brazil
2
Biochemistry Program, Chemistry Institute, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-909, RJ, Brazil
3
Leopoldo de Meis Institute of Medical Biochemistry, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-902, RJ, Brazil
4
Faculty of Pharmacy, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-902, RJ, Brazil
5
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
6
Department of Cellular Biology, Institute of Biological Sciences, University of Brasilia (UNB), Brasilia 70910-900, DF, Brazil
7
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
*
Author to whom correspondence should be addressed.
Antibodies 2025, 14(4), 86; https://doi.org/10.3390/antib14040086
Submission received: 19 July 2025 / Revised: 25 September 2025 / Accepted: 26 September 2025 / Published: 9 October 2025
(This article belongs to the Special Issue A Festschrift Celebrating Dr. Dimiter Stanchev Dimitrov: Antibodies, Innovation, and Impact on Infectious Disease and Cancer Research)
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Abstract

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.
Keywords: monoclonal antibodies (mAbs); bicistronic expression vectors; IRES element; CHO cells; protein characterization monoclonal antibodies (mAbs); bicistronic expression vectors; IRES element; CHO cells; protein characterization
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MDPI and ACS Style

Marsili, F.F.; de Aquino, F.B.; Arruda, H.R.d.S.; Marques, M.A.; Cabral, K.M.d.S.; Almeida, M.d.S.; de Oliveira, G.A.P.; Maranhão, A.Q.; Carvalho, R.S.; Castilho, L.d.R. Structural and Functional Characterization of Anti-SARS-CoV-2 Spike Monoclonal Antibodies Produced via Bicistronic Expression in CHO Cells. Antibodies 2025, 14, 86. https://doi.org/10.3390/antib14040086

AMA Style

Marsili FF, de Aquino FB, Arruda HRdS, Marques MA, Cabral KMdS, Almeida MdS, de Oliveira GAP, Maranhão AQ, Carvalho RS, Castilho LdR. Structural and Functional Characterization of Anti-SARS-CoV-2 Spike Monoclonal Antibodies Produced via Bicistronic Expression in CHO Cells. Antibodies. 2025; 14(4):86. https://doi.org/10.3390/antib14040086

Chicago/Turabian Style

Marsili, Federico Francisco, Fernanda Bittencourt de Aquino, Hiam Rodrigo da Silva Arruda, Mayra Amorim Marques, Katia Maria dos Santos Cabral, Marcius da Silva Almeida, Guilherme Augusto Piedade de Oliveira, Andrea Queiroz Maranhão, Renato Sampaio Carvalho, and Leda dos Reis Castilho. 2025. "Structural and Functional Characterization of Anti-SARS-CoV-2 Spike Monoclonal Antibodies Produced via Bicistronic Expression in CHO Cells" Antibodies 14, no. 4: 86. https://doi.org/10.3390/antib14040086

APA Style

Marsili, F. F., de Aquino, F. B., Arruda, H. R. d. S., Marques, M. A., Cabral, K. M. d. S., Almeida, M. d. S., de Oliveira, G. A. P., Maranhão, A. Q., Carvalho, R. S., & Castilho, L. d. R. (2025). Structural and Functional Characterization of Anti-SARS-CoV-2 Spike Monoclonal Antibodies Produced via Bicistronic Expression in CHO Cells. Antibodies, 14(4), 86. https://doi.org/10.3390/antib14040086

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