title: Animal component-free Agrobacterium tumefaciens cultivation media for better GMP-compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana.
abstract: Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large-scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal-derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP). We replaced all the animal-derived components in yeast extract broth (YEB) cultivation medium with soybean peptone, and then used a design-of-experiments approach to optimize the medium composition, increasing the biomass yield while maintaining high levels of transient expression in subsequent infiltration experiments. The resulting plant peptone Agrobacterium medium (PAM) achieved a two-fold increase in OD600compared to YEB medium during a 4-L batch fermentation lasting 18 h. Furthermore, the yields of the monoclonal antibody 2G12 and the fluorescent protein DsRed were maintained when the cells were cultivated in PAM rather than YEB. We have thus demonstrated a simple, efficient and scalable method for medium optimization that reduces process time and costs. The final optimized medium for the cultivation of A. tumefaciens completely lacks animal-derived components, thus facilitating the GMP-compliant large-scale transient expression of recombinant proteins in plants.
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Marcel
authors.initials: M
authors.lastname: Houdelet
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Galinski
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Holland
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Wenzel
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.|Institute for Phytopathology and Applied Zoology, Phytopathology Department, Justus-Liebig University Giessen, Giessen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schillberg
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.|Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany.
authors.forename: Johannes Felix
authors.initials: JF
authors.lastname: Buyel
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D018929:Cell Culture Techniques; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D030821:Plants, Genetically Modified; D014176:Protein Biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D014026:Nicotiana / Q000235:genetics* / Q000254:growth & development
chemical_list: D016895:Culture Media, Serum-Free; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C413663:fluorescent protein 583
keywords: Biopharmaceutical proteins; Cultivation medium optimization; Design of experiments; Molecular farming; Transient protein expression