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ORIGINAL RESEARCH article

Front. Plant Sci.
Sec. Plant Biotechnology
Volume 15 - 2024 | doi: 10.3389/fpls.2024.1531710
This article is part of the Research Topic Plant Molecular Farming for Biopharmaceutical Production and Beyond View all 4 articles

Improving the N-glycosylation occupancy of plant-produced IgG1 by engineering the amino acid environment at Asn297

Provisionally accepted
Kathrin Göritzer Kathrin Göritzer 1*Valentina Ruocco Valentina Ruocco 1Ulrike Vavra Ulrike Vavra 1Shiva Izadi Shiva Izadi 1Omayra C Bolaños-Martínez Omayra C Bolaños-Martínez 1Thareeya Phetphoung Thareeya Phetphoung 2Nuttapat Pisuttinusart Nuttapat Pisuttinusart 2Waranyoo Phoolcharoen Waranyoo Phoolcharoen 2Richard Strasser Richard Strasser 1
  • 1 Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria
  • 2 Department of Pharmacognosy and Pharmaceutical Botany, Chulalongkorn University, Bangkok, Bangkok, Thailand

The final, formatted version of the article will be published soon.

    Monoclonal antibodies are crucial recombinant biopharmaceuticals, with N-glycosylation at Asn297 essential for their functionality. Plants are increasingly used for antibody production, achieving high expression levels and enabling glycoengineering to produce homogenous human-like N-glycan structures. However, plant-produced human IgG1 often shows significant underglycosylation with potential adverse effects for immune functions and stability. This study addressed this limitation of the widely used plant-based expression platform Nicotiana benthamiana by employing protein engineering to enhance N-glycosylation occupancy in plant-produced IgG1. This was achieved through an amino acid mutation near the conserved glycosylation site in the CH2 domain of the heavy chain. The transient expression of trastuzumab and SARS-CoV-2 neutralizing IgG1 antibody COVA2-15 in N. benthamiana, with mutations such as Y300L, resulted in a notable improvement in glycosylation occupancy. While the structural integrity and monodispersity of the IgG1 variant remained unaltered, an improvement in thermal stability was observed. Furthermore, functional assays showed that antigen binding and human hFcRn interaction were unaffected, while FcγRIIIa binding affinity increased. These findings demonstrate the potential of protein-engineering to enhance the quality and functionality of plant-produced IgG1 antibodies, making them comparable to mammalian-produced counterparts.

    Keywords: monoclonal antibodies, Nicotiana benthamiana, IgG1, N-glycosylation, Glycosylation efficiency

    Received: 20 Nov 2024; Accepted: 20 Dec 2024.

    Copyright: © 2024 Göritzer, Ruocco, Vavra, Izadi, Bolaños-Martínez, Phetphoung, Pisuttinusart, Phoolcharoen and Strasser. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Kathrin Göritzer, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria

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