Comparison and critical analysis of robotized technology for monoclonal antibody high-throughput production

Authors

  • Pieranna Chiarella,

    Corresponding author
    1. Laboratory of Molecular Medicine and Biotechnology, CIR, University of Rome Campus Bio-medico, Via Alvaro del Portillo 21, 00128 Rome, Italy
    • Laboratory of Molecular Medicine and Biotechnology, CIR, University of Rome Campus Bio-medico, Via Alvaro del Portillo 21, 00128 Rome, Italy
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  • Melanie Leuener,

    1. Monoclonal Antibody Core Facility, Mouse Biology Programme, European Molecular Biology Laboratory, Via Ramarini 32, 00015 Monterotondo Scalo, Italy
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  • Christian Fasci,

    1. Monoclonal Antibody Core Facility, Mouse Biology Programme, European Molecular Biology Laboratory, Via Ramarini 32, 00015 Monterotondo Scalo, Italy
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  • Ario de Marco,

    1. Cogentech, IFOM-IEO Campus, Via Adamello 16, 20139 Milan, Italy
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  • Maria Paola Santini,

    1. Harefield Heart Science Centre, Imperial College London, Middlesex UB9 6JH, England, UK
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  • Vito M. Fazio,

    1. Laboratory of Molecular Medicine and Biotechnology, CIR, University of Rome Campus Bio-medico, Via Alvaro del Portillo 21, 00128 Rome, Italy
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  • Alan M. Sawyer

    Corresponding author
    1. Monoclonal Antibody Core Facility, Mouse Biology Programme, European Molecular Biology Laboratory, Via Ramarini 32, 00015 Monterotondo Scalo, Italy
    • Monoclonal Antibody Core Facility, Mouse Biology Programme, European Molecular Biology Laboratory, Via Ramarini 32, 00015 Monterotondo Scalo, Italy
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Abstract

We have previously demonstrated how to transform the conventional method of hybridoma production and screening into a fast, high-throughput technology. Nevertheless, there were still open questions related to automated procedures and immunization protocols that we address now by comparing the hybridoma production work-flow in automated and manually executed processes. In addition, since the animals' antibody responses to single or multiple antigen challenge affect monoclonal antibody throughput, different immunization and fusion strategies were tested. Specifically, the results obtained with multiplexing (multiple target antigens injected into a single animal) and single antigen immunization followed by splenocyte pooling immediately before fusion were compared with conventional methods. The results presented here demonstrate that the optimal protocol consists of automated somatic-cell fusion and hybridoma dilution followed by manual plating of hybridoma cells. Additionally, more specific and productive hybridoma clones were obtained with multiplexed immunization in a single animal with respect to the splenocyte pooling from single antigen immunized animals. However, in terms of overall antibody yield, the conventional method consisting of single immunization for each single animal assured ten times more specific hybridoma cell lines than the strategy based on the multiple antigen immunization followed by separate fusion step. In conclusion, the most productive approach for recovering a large number of suitable antibodies relies on single antigen immunization followed by automated fusion and dilution steps and manual plating. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011

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