title: High-yield production of a human monoclonal IgG by rhizosecretion in hydroponic tobacco cultures.
abstract: Rhizosecretion of recombinant pharmaceuticals from in vitro hydroponic transgenic plant cultures is a simple, low cost, reproducible and controllable production method. Here, we demonstrate the application and adaptation of this manufacturing platform to a human antivitronectin IgG1 monoclonal antibody (mAb) called M12. The rationale for specific growth medium additives was established by phenotypic analysis of root structure and by LC-ESI-MS/MS profiling of the total protein content profile of the hydroponic medium. Through a combination of optimization approaches, mAb yields in hydroponic medium reached 46 μg/mL in 1 week, the highest figure reported for a recombinant mAb in a plant secretion-based system to date. The rhizosecretome was determined to contain 104 proteins, with the mAb heavy and light chains the most abundant. This enabled evaluation of a simple, scalable extraction and purification protocol and demonstration that only minimal processing was necessary prior to protein A affinity chromatography. MALDI-TOF MS revealed that purified mAb contained predominantly complex-type plant N-glycans, in three major glycoforms. The binding of M12 purified from hydroponic medium to vitronectin was comparable to its Chinese hamster ovary (CHO)-derived counterpart. This study demonstrates that in vitro hydroponic cultivation coupled with recombinant protein rhizosecretion can be a practical, low-cost production platform for monoclonal antibodies.
authors.affiliation: The Hotung Molecular Immunology Unit, Institute for Infection and Immunity, St. George's University of London, London, UK.
authors.forename: Luisa M
authors.initials: LM
authors.lastname: Madeira
authors.affiliation: The Hotung Molecular Immunology Unit, Institute for Infection and Immunity, St. George's University of London, London, UK.
authors.forename: Tim H
authors.initials: TH
authors.lastname: Szeto
authors.affiliation: Plant Research International, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Maurice
authors.initials: M
authors.lastname: Henquet
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Aachen, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Raven
authors.affiliation: Department of Biological and Medical Sciences - Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, UK.
authors.forename: John
authors.initials: J
authors.lastname: Runions
authors.affiliation: Brunel Institute for Bioengineering, Brunel University, London, UK.
authors.forename: Jon
authors.initials: J
authors.lastname: Huddleston
authors.affiliation: Brunel Institute for Bioengineering, Brunel University, London, UK.
authors.forename: Ian
authors.initials: I
authors.lastname: Garrard
authors.affiliation: The Hotung Molecular Immunology Unit, Institute for Infection and Immunity, St. George's University of London, London, UK.
authors.forename: Pascal M W
authors.initials: PM
authors.lastname: Drake
authors.affiliation: The Hotung Molecular Immunology Unit, Institute for Infection and Immunity, St. George's University of London, London, UK.
authors.forename: Julian K-C
authors.initials: JK
authors.lastname: Ma
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D018527:Hydroponics / Q000379:methods*; D007074:Immunoglobulin G / Q000096:biosynthesis*; D007210:Indoleacetic Acids / Q000494:pharmacology; D009566:Nitrates / Q000494:pharmacology; D010641:Phenotype; D018517:Plant Roots / Q000187:drug effects / Q000378:metabolism*; D030821:Plants, Genetically Modified; D014026:Nicotiana / Q000187:drug effects / Q000235:genetics*; D019096:Vitronectin / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007210:Indoleacetic Acids; D009566:Nitrates; D019096:Vitronectin
keywords: genetically modified plants; monoclonal antibody; recombinant antibody production; rhizosecretion