title: Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody.
abstract: Recombinant pharmaceutical proteins expressed in hairy root cultures can be secreted into the medium to improve product homogeneity and to facilitate purification, although this may result in significant degradation if the protein is inherently unstable or particularly susceptible to proteases. To address these challenges, we used a design of experiments approach to develop an optimized induction protocol for the cultivation of tobacco hairy roots secreting the full-size monoclonal antibody M12. The antibody yield was enhanced 30-fold by the addition of 14 g/L KNO3 , 19 mg/L 1-naphthaleneacetic acid and 1.5 g/L of the stabilizing agent polyvinylpyrrolidone. Analysis of hairy root cross sections revealed that the optimized medium induced lateral root formation and morphological changes in the inner cortex and pericycle cells, indicating that the improved productivity was at least partially based on the enhanced efficiency of antibody secretion. We found that 57% of the antibody was secreted, yielding 5.9 mg of product per liter of induction medium. Both the secreted and intracellular forms of the antibody could be isolated by protein A affinity chromatography and their functionality was confirmed using vitronectin-binding assays. Glycan analysis revealed three major plant complex-type glycans on both forms of the antibody, although the secreted form was more homogeneous due to the predominance of a specific glycoform. Tobacco hairy root cultures therefore offer a practical solution for the production of homogeneous pharmaceutical antibodies in containment.
authors.affiliation: VTT Technical Research Centre of Finland, P.O. Box 1000, 02044, VTT, Finland.
authors.forename: Suvi T
authors.initials: ST
authors.lastname: Häkkinen
authors.forename: Nicole
authors.initials: N
authors.lastname: Raven
authors.forename: Maurice
authors.initials: M
authors.lastname: Henquet
authors.forename: Marja-Leena
authors.initials: ML
authors.lastname: Laukkanen
authors.forename: Tibor
authors.initials: T
authors.lastname: Anderlei
authors.forename: Juha-Pekka
authors.initials: JP
authors.lastname: Pitkänen
authors.forename: Richard M
authors.initials: RM
authors.lastname: Twyman
authors.forename: Dirk
authors.initials: D
authors.lastname: Bosch
authors.forename: Kirsi-Marja
authors.initials: KM
authors.lastname: Oksman-Caldentey
authors.forename: Stefan
authors.initials: S
authors.lastname: Schillberg
authors.forename: Anneli
authors.initials: A
authors.lastname: Ritala
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D006031:Glycosylation; D058634:Molecular Farming / Q000379:methods*; D018517:Plant Roots / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000032:analysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013678:Technology, Pharmaceutical / Q000379:methods*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D000906:Antibodies; D003470:Culture Media; D011134:Polysaccharides; D011994:Recombinant Proteins
keywords: antibody; hairy roots; molecular farming; secretion; tobacco