Improving yield of a recombinant biologic in a Brassica hairy root manufacturing process.

Hairy root systems have proven to be a viable alternative for recombinant protein production. For recalcitrant proteins, maximizing the productivity of hairy root cultures is essential. The aim of this study was to optimize a Brassica rapa rapa hairy root process for secretion of alpha- l-iduronidase (IDUA), a biologic of medical value. The process was first optimized with hairy roots expressing eGFP. For the biomass optimization, the highest biomass yields were achieved in modified Gamborg B5 culture medium. For the secretion induction, the optimized secretion media was obtained with additives (1.5 g/l PVP + 1 mg/l 2,4- d + 20.5 g/l KNO3 ) resulting in 3.4 fold eGFP secretion when compared to the non-induced control. These optimized conditions were applied to the IDUA-expressing hairy root clone, confirming that the highest yields of secreted IDUA occurred when using the defined additive combination. The functionality of the IDUA protein, secreted and intracellular, was confirmed with an enzymatic activity assay. A > 150-fold increase of the IDUA activity was observed using an optimized secretion medium, compared with a non-induced medium. We have proven that our B. rapa rapa hairy root system can be harnessed to secrete recalcitrant proteins, illustrating the high potential of hairy roots in plant molecular farming.

Hairy Root Cultures-A Versatile Tool With Multiple Applications.

Hairy roots derived from the infection of a plant by Rhizobium rhizogenes (previously referred to as Agrobacterium rhizogenes) bacteria, can be obtained from a wide variety of plants and allow the production of highly diverse molecules. Hairy roots are able to produce and secrete complex active glycoproteins from a large spectrum of organisms. They are also adequate to express plant natural biosynthesis pathways required to produce specialized metabolites and can benefit from the new genetic tools available to facilitate an optimized production of tailor-made molecules. This adaptability has positioned hairy root platforms as major biotechnological tools. Researchers and industries have contributed to their advancement, which represents new alternatives from classical systems to produce complex molecules. Now these expression systems are ready to be used by different industries like pharmaceutical, cosmetics, and food sectors due to the development of fully controlled large-scale bioreactors. This review aims to describe the evolution of hairy root generation and culture methods and to highlight the possibilities offered by hairy roots in terms of feasibility and perspectives.

Brassica rapa hairy root based expression system leads to the production of highly homogenous and reproducible profiles of recombinant human alpha-L-iduronidase.

The Brassica rapa hairy root based expression platform, a turnip hairy root based expression system, is able to produce human complex glycoproteins such as the alpha-L-iduronidase (IDUA) with an activity similar to the one produced by Chinese Hamster Ovary (CHO) cells. In this article, a particular attention has been paid to the N- and O-glycosylation that characterize the alpha-L-iduronidase produced using this hairy root based system. This analysis showed that the recombinant protein is characterized by highly homogeneous post translational profiles enabling a strong batch to batch reproducibility. Indeed, on each of the 6 N-glycosylation sites of the IDUA, a single N-glycan composed of a core Man3 GlcNAc2 carrying one beta(1,2)-xylose and one alpha(1,3)-fucose epitope (M3XFGN2) was identified, highlighting the high homogeneity of the production system. Hydroxylation of proline residues and arabinosylation were identified during O-glycosylation analysis, still with a remarkable reproducibility. This platform is thus positioned as an effective and consistent expression system for the production of human complex therapeutic proteins.