Optimisation of PD-FcY veterinary antigen secretion from Nicotiana benthamiana hairy roots and purification from the culture medium

Hairy roots (HR) are a promising plant tissue-based platform for the production of biopharmaceuticals as the secretion of recombinant proteins in the culture medium may simplify the purification and recovery steps. In this study, Nicotiana benthamiana HR cultures were generated to produce the secretory version of an infectious bursal disease virus chimeric antigen (PD-FcY) of interest for the formulation of poultry vaccines, with the aim of optimizing production and purification. Recombinant protein accumulation kinetics in the medium after induction with the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), whose effects were still poorly characterized, was firstly investigated using a HR clone expressing the red fluorescent protein. The results demonstrated that HR cultures induced once and grown 21 days efficiently secrete the protein and, even after replacement of the medium with a hormone-free counterpart, maintain the same secretion rate for several days. This protocol applied to HR expressing PD-FcY resulted in similar accumulation kinetics and allowed to obtain protein recovery concentrations of ~ 4 mg/l. A downstream processing procedure consisting in recombinant product concentration through ultrafiltration followed by two sequential chromatographic steps led to final yields of 0.8 mg purified PD-FcY/l of culture. The HR-derived product showed a similar glycosylation pattern compared to the counterpart obtained from agroinfiltrated N. benthamiana, but higher quality in terms of purity and protein degradation, and was also shown to retain full antigenic potential. In conclusion, the proposed expression and purification strategy holds promises for the development of an innovative platform to produce low-cost subunit vaccines.