INTRODUCTION
Over the last three decades, transgenic plants have been established as an alternative system to produce recombinant proteins. At the moment of writing, there are 27 studies for plant-derived biopharmaceuticals at different stages of clinical trials in the world (U.S. National Library of Medicine, 2022). An example of an already-in-the-market and FDA-approved plant-manufactured biopharmaceutical is the recombinant glucocerebrosidase, a therapeutic for Gaucher disease from Protalix BioTherapeutics(Fox, 2012). Currently, the most interesting example of plant biopharmaceuticals is the well-advanced (Phase 3 Clinical Trials) recombinant coronavirus-like particle COVID-19 vaccine from Medicago(Medicago, 2021).
Plant systems for recombinant protein production offer unique advantages such as cost-effective mass production, absence of inherent human or animal pathogens and high possibility of glycoengineering. Nevertheless, the associated environmental factors and social acceptance factors limit the implementation of transgenic plant cultivation particularly in open fields (A. M. Shelton et al., 2002; Lucht, 2015). Hairy root cultures integrate both the intrinsic advantages of plant-based protein synthesis together with a production in confinement. Hairy root cultures have been widely studied and used for production of high value plant secondary metabolites and recombinant proteins (Häkkinen et al., 2014; Vasilev et al., 2014; Ele Ekouna et al., 2017a; Cardon et al., 2019). Hairy roots also offer genotypic and phenotypic stability and, more importantly, possibility to secrete the expressed proteins (Gutierrez-Valdes et al., 2020; Halder et al., 2019). Optimizing the protein secretion in a given hairy root culture system, can be advantageous particularly for purification of the target proteins (Madeira et al., 2016).
After hairy root culture has been created with molecular farming and synthetic biology tools, the two main approaches for optimization of a hairy root culture system are the biomass production capacity and the target protein secretion. Different kinds and combinations of reagents can be used during the liquid culture. For instance, to ensure better nitrogen availability for the tissue [e.g. using KNO3) (Rini Vijayan and Raghu, 2020)], to allow wall permeabilization for secretion of target proteins that would otherwise remain bound to the biomass [e.g. using DMSO (Wongsamuth and Doran, 1997)], to protect the integrity of secreted proteins [e.g. using BSA, PVP, PEG) (Alvarez, 2014)], or, to minimize the cell lysis by regulating the osmotic pressure in the medium [e.g. using mannitol (Halder et al., 2019)]. Statistical modeling can be helpful to design proper experiments that need to include several culture reagents at the same time (Häkkinen et al., 2014; Häkkinen et al., 2018). Ultimately, a well-rounded optimization should offer synergistic effects of the used compounds in the culture to ensure high yields of good-quality target protein.
The therapeutic Alpha-L-iduronidase (IDUA), laronidase from Genzyme, is a recombinant form of the human IDUA that is produced by recombinant DNA technology using mammalian Chinese Hamster Ovary (CHO) cell culture (He et al., 2012). The plant-based analogue of IDUA produced in transgenicBrassica rapa hairy roots has demonstrated to have reproducible and highly homogeneous glycosylation profiles, as well as similar affinity and specific activity when compared to the one produced by CHO cells (Cardon et al., 2019). IDUA is clinically important as an enzyme replacement pharmaceutical for the treatment of mucopolysaccharidosis type I (MPS I), a progressive lysosomal storage disorder. IDUA (EC 3.2.1.176) is a secreted (71kDa) lysosomal enzyme that presents a signal peptide (1M-23A, released in its final secretion form), and six potential N-glycosylation sites as well as hydroxylation. Optimizing a hairy root system that already consistently produces functional plant-based IDUA such as the one described by Cardon et al. (2019), represented an opportunity to evaluate if it can be further harnessed to generate higher recombinant protein yields and/or ease the downstream processing.
The aim of this study was to optimize a hairy root process for secretion of α-L-iduronidase (IDUA). The process was first optimized with hairy roots expressing eGFP (Green Fluorescent Protein). As a production host we used Brassica rapa rapa hairy roots, which are currently used in commercial production purposes and which have shown to possess high recombinant protein production capacity (Huet et al., 2014). Our optimization approach intended to identify a range of culture medium additives that, when used alone or in combination, would increase the productivity of the process for “hard-to-produce” recombinant proteins. In addition, we wanted to evaluate if the eGFP secretion optimized conditions would also result in high secretion of the actual target protein, IDUA, a biologic of medical value.