Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco

Rhizosecretion is an attractive technology for the production of recombinant proteins from transgenic plants. However, to date, yields of plant-derived recombinant pharmaceuticals by this method have been too low for commercial viability. Studies conducted focused on three transgenic plant lines grown in hydroponic culture medium, two expressing monoclonal antibodies Guy's 13 and 4E10 and one expressing a small microbicide polypeptide cyanovirin-N. Rhizosecretion rates increased significantly by the addition of the plant growth regulator alpha-naphthalene acetic acid. The maximum rhizosecretion rates achieved were 58 microg/g root dry weight/24 h for Guy's 13, 10.43 microg/g root dry weight/24 h for 4E10, and 766 microg/g root dry weight/24 h for cyanovirin-N, the highest figures so far reported for a full-length antibody and a recombinant protein, respectively. The plant growth regulators indole-butyric acid, 6-benzylaminopurine, and kinetin were also demonstrated to increase rhizosecretion of Guy's 13. The effect of the growth regulators differed, as alpha-naphthalene acetic acid and indole-butyric acid increased the root dry weight of hydroponic plants, whereas the cytokinins benzylaminopurine and kinetin increased rhizosecretion without affecting root mass. A comparative glycosylation analysis between MAb Guy's 13 purified from either hydroponic culture medium or from leaf extracts demonstrated a similar pattern of glycosylation comprising high mannose to complex glycoforms. Analysis of the hydroponic culture medium at harvest revealed significantly lower and less complex levels of proteolytic enzymes, in comparison with leaf extracts, which translated to a higher proportion of intact Guy's 13 IgG in relation to other IgG products. Hydroponic medium could be added directly to a chromatography column for affinity purification, allowing simple and rapid production of high purity Guy's 13 antibody. In addition to the attractiveness of controlled cultivation within a contained environment for pharmaceutical-producing plants, this study demonstrates advantages with respect to the quality and downstream purification of recombinant proteins.