title: Optimization of BY-2 cell suspension culture medium for the production of a human antibody using a combination of fractional factorial designs and the response surface method.
abstract: We have developed a strategy for the optimization of plant cell suspension culture media using a combination of fractional factorial designs (FFDs) and response surface methodology (RSM). This sequential approach was applied to transformed tobacco BY-2 cells secreting a human antibody (M12) into the culture medium, in an effort to maximize yields. We found that the nutrients KNO₃, NH₄NO₃ and CaCl₂ and the hormones 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP) had the most significant impact on antibody accumulation. The factorial screening revealed strong interactions within the nutrients group (KNO₃, NH₄NO₃ and CaCl₂) and also individually between 2,4-D and three other components (KNO₃, NH₄NO₃ and BAP). The RSM design resulted in a fivefold increase in the antibody concentration after 5 days and a twofold reduction in the packed cell volume (PCV). Longer cultivation in the optimized medium led to the further accumulation of antibody M12 in the culture medium (up to 107 μg/mL, day 10). Because the packed cell volume was reduced in the optimized medium, this enhanced the overall yield by 20-fold (day 7) and 31-fold (day 10) compared to the conventional MS medium.
authors.affiliation: Department Plant Biotechnology, Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Aachen, Germany.
authors.forename: Nikolay
authors.initials: N
authors.lastname: Vasilev
authors.forename: Ulrike
authors.initials: U
authors.lastname: Grömping
authors.forename: Anja
authors.initials: A
authors.lastname: Lipperts
authors.forename: Nicole
authors.initials: N
authors.lastname: Raven
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.forename: Stefan
authors.initials: S
authors.lastname: Schillberg
mesh_terms: D000906:Antibodies / Q000235:genetics*; D018929:Cell Culture Techniques; D002460:Cell Line; D003470:Culture Media / Q000737:chemistry*; D006801:Humans; D009751:Nutritional Requirements; D030821:Plants, Genetically Modified / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism
chemical_list: D000906:Antibodies; D003470:Culture Media; D011994:Recombinant Proteins
keywords: medium optimization; molecular farming; monoclonal antibodies; statistical experimental designs; tobacco cell cultures; transgenic plant cells