title: From gene to harvest: insights into upstream process development for the GMP production of a monoclonal antibody in transgenic tobacco plants.
abstract: The EU Sixth Framework Programme Integrated Project 'Pharma-Planta' developed an approved manufacturing process for recombinant plant-made pharmaceutical proteins (PMPs) using the human HIV-neutralizing monoclonal antibody 2G12 as a case study. In contrast to the well-established Chinese hamster ovary platform, which has been used for the production of therapeutic antibodies for nearly 30 years, only draft regulations were initially available covering the production of recombinant proteins in transgenic tobacco plants. Whereas recombinant proteins produced in animal cells are secreted into the culture medium during fermentation in bioreactors, intact plants grown under nonsterile conditions in a glasshouse environment provide various 'plant-specific' regulatory and technical challenges for the development of a process suitable for the acquisition of a manufacturing licence for clinical phase I trials. During upstream process development, several generic steps were addressed (e.g. plant transformation and screening, seed bank generation, genetic stability, host plant uniformity) as well as product-specific aspects (e.g. product quantity). This report summarizes the efforts undertaken to analyse and define the procedures for the GMP/GACP-compliant upstream production of 2G12 in transgenic tobacco plants from gene to harvest, including the design of expression constructs, plant transformation, the generation of production lines, master and working seed banks and the detailed investigation of cultivation and harvesting parameters and their impact on biomass, product yield and intra/interbatch variability. The resulting procedures were successfully translated into a prototypic manufacturing process that has been approved by the German competent authority.
authors.affiliation: Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany.
authors.forename: Markus
authors.identifier: http://orcid.org/0000-0002-7380-9228
authors.initials: M
authors.lastname: Sack
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Rademacher
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Spiegel
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Boes
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Hellwig
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Juergen
authors.initials: J
authors.lastname: Drossard
authors.affiliation: Department of Applied Genetics and Cell Biology (IAGZ), University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Eva
authors.initials: E
authors.lastname: Stoger
authors.affiliation: Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany.|Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics*; D018533:Biomass; D000080908:Broadly Neutralizing Antibodies; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000378:metabolism; D015483:HIV Antibodies; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000378:metabolism; D030821:Plants, Genetically Modified; D014026:Nicotiana / Q000235:genetics*
chemical_list: C529157:2G12 monoclonal antibody; D000911:Antibodies, Monoclonal; D000080908:Broadly Neutralizing Antibodies; D015483:HIV Antibodies; D008164:Luminescent Proteins; C413663:fluorescent protein 583
keywords: 2G12; GMP/GACP; HIV; glasshouse; manufacturing licence; plant-made pharmaceuticals; transgenic tobacco; upstream process development