Biosafety Aspects of Hybridoma Technology: Nature of Risks and Approaches to Their Management
DOI:
https://doi.org/10.20535/ibb.2025.9.2.320712Keywords:
hybridoma technology, monoclonal antibodies, biosafety level, contamination risksAbstract
This study investigates the biosafety aspects of hybridoma technology, focusing on the identification and management of associated risks. Monoclonal antibodies, essential tools in immunology, biotechnology, and medicine, are primarily produced through hybridoma technology. This process involves fusing B lymphocytes from immunized animals with myeloma cells to create hybridomas, which are then cultured to produce specific antibodies. The research highlights significant contamination risks, particularly from rodent-borne viruses and other pathogens, during both in vivo and in vitro cultivation. It systematically analyzes existing strategies for identifying and mitigating these risks at various stages of monoclonal antibody production, including hybridoma identification, cell fusion, and antibody purification. The study underscores the importance of stringent biosafety protocols and optimized purification methodologies to ensure the production of high-quality, contaminant-free monoclonal antibodies. Additionally, it emphasizes the necessity of comprehensive risk assessments and the implementation of advanced contamination control systems in laboratories. The conclusions drawn from this study provide valuable insights into enhancing the safety and efficacy of monoclonal antibody production. By addressing these biosafety concerns, the research supports the widespread application of monoclonal antibodies in scientific and medical fields, ensuring their reliability and effectiveness in various diagnostic and therapeutic contexts.
References
Abba Moussa D, Vazquez M, Chable-Bessia C, Roux-Portalez V, Tamagnini E, Pedotti M, et al. Discovery of a pan anti-SARS-CoV-2 monoclonal antibody with highly efficient infected cell killing capacity for novel immunotherapeutic approaches. Emerging Microbes & Infections. 2025;14(1):2432345. DOI: 10.1080/22221751.2024.2432345
Kokojka F, Marks RS. Rapid and specific screening of monoclonal antibodies in hybridoma supernatants by an enzyme-based dipstick immunoassay. Talanta. 2025;284:127240. DOI: 10.1016/j.talanta.2024.127240
Galkin OYu, Gorshunov YuV, Besarab OB, Ivanova OM. Development and characterization of highly informative ELISA for the detection of IgG and IgA antibodies to Сhlamydia trachomatis. The Ukrainian Biochemical Journal. 2018;90(3):70-83. DOI: 10.15407/ubj90.03.070
Andersen RA. Preface. In: Algal Culturing Techniques. Elsevier Academic Press, 2005, pp. IX-X. DOI: 10.1016/b978-012088426-1/50001-9
Khudetskyy I, Antonova-Rafi J. Human influence and changes in nature on biological security (overview of the problem). Fitoterapia. 2023;(2):26-34. DOI: 10.32782/2522-9680-2023-2-37
Liu HF, Ma J, Winter C, Bayer R. Recovery and purification process development for monoclonal antibody production. mAbs. 2010;2(5):480-99. DOI: 10.4161/mabs.2.5.12645
Tassinari E, Danielli L, Marchetti A, Rosellini M, Ricci C, Piazza P, et al. State of the art of adjuvant immunotherapy in urothelial cancer: New developments and upcoming changes. Human Vaccines & Immunotherapeutics. 2025;21(1):2440165. DOI: 10.1080/21645515.2024.2440165
Galkin OYu, Shirobokov VP, Grigorenko AA, Dugan OM, Lutsenko TM, Komar AG. Biotechnological foundations of the creation of means of serological diagnostics of infectious and non-infectious diseases: Monograph. Kyiv: NTUU "KPI", 2015.
Oliinyk OS, Palyvoda KO, Lugovskaya NE, Kolibo DV, Lugovskoy EV, Komisarenko SV. Recombinant single chain variable fragment antibodies (scFv) against Pro(144)-Leu(155) fragment of human protein C. The Ukrainian Biochemical Journal. 2015;87(2):88-94. DOI: 10.15407/ubj87.02.088
Galkin OY, Besarab AB, Lutsenko TN. Characteristics of enzyme-linked immunosorbent assay for detection of IgG antibodies specific to Сhlamydia trachomatis heat shock protein (HSP-60). The Ukrainian Biochemical Journal. 2017;89(1):22-30. DOI: 10.15407/ubj89.01.022
Zhang Y, Cheng H, Yu P, Wang S, Dong H, Lu S, et al. High-throughput single-cell analysis reveals Omp38-specific monoclonal antibodies that protect against Acinetobacter baumannii infection. Emerging Microbes & Infections. 2025;14(1):2437243. DOI: 10.1080/22221751.2024.2437243
Liu M, Ren T, Zhang L, Li P, Zhong Z, Zhou L, et al. Development of a monoclonal antibody specifically recognizing a linear epitope on the E1 protein of Getah virus. Virology. 2025;602:110315. DOI: 10.1016/j.virol.2024.110315
Xiao J, Yu S, Jiang K, He X, Bi L, Zhao P, et al. Identification of linear B cell epitopes on the leukotoxin protein of Fusobacterium necrophorum. Anaerobe. 2024;90:102884. DOI: 10.1016/j.anaerobe.2024.102884
Mitra S, Tomar PC. Hybridoma technology; advancements, clinical significance, and future aspects. Journal of Genetic Engineering and Biotechnology. 2021;19(1):159. DOI: 10.1186/s43141-021-00264-6
Galkin O, Dekhtyarenko N, Gorchakov V, Dugan O. Medical biotechnology. Supervised medical biotechnology: Atlas for students of the direction of training 6.0514 "Biotechnology". Kyiv: NTUU "KPI", 2011, 36 p.
Fratz-Berilla EJ, Angart P, Graham RJ, Powers DN, Mohammad A, Kohnhorst C, et al. Impacts on product quality attributes of monoclonal antibodies produced in CHO cell bioreactor cultures during intentional mycoplasma contamination events. Biotechnology and Bioengineering. 2020;117(9):2802-15. DOI: 10.1002/bit.27436
Li F, Vijayasankaran N, Shen AY, Kiss R, Amanullah A. Cell culture processes for monoclonal antibody production. mAbs. 2010;2(5):466-79. DOI: 10.4161/mabs.2.5.12720
Daya J, Cusick V, Mattila J. Characterization of ionic strength for X‐MuLV inactivation by low pH treatment for monoclonal antibody purification. Biotechnology and Bioengineering. 2023;120(6):1605-13. DOI: 10.1002/bit.28379
Adjuvant Use and Monoclonal Antibody Production. Institutional Animal Care and Use Committee. Available from: https://research.wayne.edu/iacuc/adjuvantuseandmonoclonalantibodyproduction
McCormick-Ell J, Connell N. Laboratory safety, biosecurity, and responsible animal use. ILAR Journal. 2019;60(1):24-33. DOI: 10.1093/ilar/ilz012
Barone PW, Wiebe ME, Leung JC, Hussein ITM, Keumurian FJ, Bouressa J, et al. Viral contamination in biologic manufacture and implications for emerging therapies. Nature Biotechnology. 2020;38(5):563-72. DOI: 10.1038/s41587-020-0507-2
Pan X, de Souza MO, Figueiras FM, Huang A, Banach BB, Wolfe JR, et al. Optimized single-cell gates for yeast display screening. Protein Engineering, Design and Selection. 2025;38:gzae018. DOI: 10.1093/protein/gzae018
Sakaguchi A, Nakajima C, Sawano A, Tanaka Y, Kurihara Y. Rapid and reliable hybridoma screening method that is suitable for production of functional structure-recognizing monoclonal antibody. Journal of Bioscience and Bioengineering. 2021;131(6):696-702. DOI: 10.1016/j.jbiosc.2021.02.006
Mortezagholi S, Maghsood F, Shojaeian S, Shokri F, Amiri MM, Ghorbani A, et al. Production and characterization of a panel of anti-mouse placenta-specific protein 1 (plac1) monoclonal antibodies. Analytical Biochemistry. 2025;696:115682. DOI: 10.1016/j.ab.2024.115682
Arkhypova M, Deriabin O, Trokhymchuk T, Starosyla D, Atamaniuk V, Zavelevich M, et al. The influence of flavonoid compositions on Nrf2 transcription factor expression in case of infections triggered by influenza a virus and transmissible gastroenteritis coronavirus. Innovative Biosystems and Bioengineering. 2023;7(4):48-56. DOI: 10.20535/ibb.2023.7.4.290103
Antibody hybridoma core - Fusion and screening. Mayo Clinic. Available from: https://www.mayo.edu/research/core-resources/antibody-hybridoma-core/services/fusion-screening
Tomita M, Tsumoto K. Hybridoma technologies for antibody production. Immunotherapy. 2011;3(3):371-80. DOI: 10.2217/imt.11.4
Muhsin A, Rangel R, Vien L, Bover L. Monoclonal antibodies generation: Updates and protocols on hybridoma technology. In: Cancer Immunoprevention: Methods and Protocols, vol. 2435. New York, NY: Springer US, 2022, pp. 73-93. DOI: 10.1007/978-1-0716-2014-4_6
Bols NC, Scharer JM, Phillips HA, Moo-Young M. Media for hybridoma growth and monoclonal antibody production. Biotechnology Advances. 1988;6(2):169-82. DOI: 10.1016/0734-9750(88)90003-1
O'Brien SA, Hu W-S. Cell culture bioprocessing - the road taken and the path forward. Current Opinion in Chemical Engineering. 2020;30:128-34. DOI: 10.1016/j.coche.2020.100663
Bergin A, Carvell J, Butler M. Applications of bio-capacitance to cell culture manufacturing. Biotechnology Advances. 2022;61:108048. DOI: 10.1016/j.biotechadv.2022.108048
Parks DR, Bryan VM, Oi VT, Herzenberg LA. Antigen-specific identification and cloning of hybridomas with a fluorescence-activated cell sorter. Proceedings of the National Academy of Sciences. 1979;76(4):1962-66. DOI: 10.1073/pnas.76.4.1962
Xu Y, Wang G, Xu M. Biohazard levels and biosafety protection for Mycobacterium tuberculosis strains with different virulence. Biosafety and Health. 2020;2(3):135-41. DOI: 10.1016/j.bsheal.2020.04.001
Wang F, Liu LS, Li P, Lau CH, Leung HM, Chin YR, et al. Cellular uptake, tissue penetration, biodistribution, and biosafety of threose nucleic acids: assessing in vitro and in vivo delivery. Materials Today Bio. 2022;15:100299. DOI: 10.1016/j.mtbio.2022.100299
Mizuno M, Matsuda J, Watanabe K, Shimizu N, Sekiya I. Effect of disinfectants and manual wiping for processing the cell product changeover in a biosafety cabinet. Regenerative Therapy. 2023;22:169-75. DOI: 10.1016/j.reth.2023.01.009
Féraudet-Tarisse C, Vaisanen-Tunkelrott M-L, Moreau K, Lamourette P, Créminon C, Volland H. Pathogen-free screening of bacteria-specific hybridomas for selecting high-quality monoclonal antibodies against pathogen bacteria as illustrated for Legionella pneumophila. Journal of Immunological Methods. 2013;391(1-2):81-94. DOI: 10.1016/j.jim.2013.02.012
Soley A, Fontova A, Gálvez J, Sarró E, Lecina M, Bragós R, et al. Development of a simple disposable six minibioreactor system for suspension mammalian cell culture. Process Biochemistry. 2012;47(4):597-605. DOI: 10.1016/j.procbio.2011.12.022
Moraes JZ, Hamaguchi B, Braggion C, Speciale ER, Cesar FBV, da Silva Soares GdF, et al. Hybridoma technology: is it still useful? Current Research in Immunology. 2021;2:32-40. DOI: 10.1016/j.crimmu.2021.03.002
Doerner A, Rhiel L, Zielonka S, Kolmar H. Therapeutic antibody engineering by high efficiency cell screening. FEBS Letters. 2014;588(2):278-87. DOI: 10.1016/j.febslet.2013.11.025
Parray HA, Shukla S, Samal S, Shrivastava T, Ahmed S, Sharma C, Kumar R. Hybridoma technology a versatile method for isolation of monoclonal antibodies, its applicability across species, limitations, advancement and future perspectives. International Immunopharmacology. 2020;85:106639. DOI: 10.1016/j.intimp.2020.106639
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2025 The Author(s)
This work is licensed under a Creative Commons Attribution 4.0 International License.
The ownership of copyright remains with the Authors.
Authors may use their own material in other publications provided that the Journal is acknowledged as the original place of publication and National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute” as the Publisher.
Authors are reminded that it is their responsibility to comply with copyright laws. It is essential to ensure that no part of the text or illustrations have appeared or are due to appear in other publications, without prior permission from the copyright holder.
IBB articles are published under Creative Commons licence:- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under CC BY 4.0 that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work.
