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.

Production of a tumour‐targeting antibody with a human‐compatible glycosylation profile in N. benthamiana hairy root cultures

“…This amount is slightly lower but comparable to that of N. tabacum clone 6 (35% of total antibody), indicating that there is no major difference in trafficking ability of the two mAb H10 glyco‐variants. Significant variations in the amount of secreted antibody in three different N. tabacum HR clones expressing mAb M12 were reported in a previous work with levels ranging from 25 to 57% of the total antibody yield . Microscopy analysis of roots after induction revealed a peculiar phenotype with the formation of swollen root tips, root hair proliferation and elongation.…”

Engineering ‘designer’ glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline‐O‐glycosylation process in plants

“…The engineered (SP4) 18 or (SP) 32 module substantially enhanced the secretion of EGFP. Secreted protein yields achieved in this study represented one of the highest among tobacco hairy root cultures, where protein yields of less 5.0 mg/L were typically reported (Georgiev et al ., ; Hakkinen et al ., ; Pham et al ., ). However, compared with the BY‐2 cell culture expressing (SP) 32 ‐EGFP (up to 131 mg/L) (Zhang et al ., 2016b), the secreted protein yield obtained in this study was still low, which might be attributed to the diffusion limited through the apoplastic pathway to the tissue exterior.…”

“…Similar findings were reported by Hakkinen et al . () who compared the expression of a recombinant antibody M12 in different plant expression platforms (leaves, hairy roots, BY‐2 cells and moss), and found that BY‐2 cell suspension culture produced the highest overall secreted antibody yields.…”

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“…Some genome re-engineering and epigenetic approaches can have tremendous potential through the suppression or decrease of protease activities. For example, RNA interference is capable of shifting the metabolism of a given host plant and can repress the silencers that inhibit the expression of transiently transformed genes (Arzola, Chen, Rattanaporn, Maclean, & McDonald, 2011;Hakkinen et al, 2013;Rigano, De Guzman, Walmsley, Frusciante, & Barone, 2013;Tremblay, Diao, Huner, Jevnikar, & Ma, 2011).…”