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.