Molecular Farming in Tobacco Hairy Roots by Triggering the Secretion of a Pharmaceutical Antibody

Recombinantpharmaceuticalproteinsexpressed 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 wasenhanced30-foldbytheadditionof14g/LKNO3,19mg/L 1-naphthaleneacetic acid and 1.5g/L of the stabilizing agent polyvinylpyrrolidone. Analysis of hairy root cross sections revealed that the optimized medium induced lateral root formationandmorphologicalchangesintheinnercortexand 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 5.9mg 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 predomi- nance of a specific glycoform. Tobacco hairy root cultures therefore offer a practical solution for the production of homogeneous pharmaceutical antibodies in containment. Biotechnol. Bioeng. 2014;111: 336-346. 2013 Wiley Periodicals, Inc.