Moss bioreactors producing improved biopharmaceuticals - Plant ...

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Moss bioreactors producing improved biopharmaceuticals
Eva L Decker and Ralf Reski
Plants may serve as superior production systems for complex
recombinant pharmaceuticals. Current strategies for improving
plant-based systems include the development of large-scale
production facilities as well as the optimisation of protein
modifications. While post-translational modifications of plant
proteins generally resemble those of mammalian proteins,
certain plant-specific protein-linked sugars are immunogenic in
humans, a fact that restricts the use of plants in
biopharmaceutical production so far. The moss Physcomitrella
patens was developed as a contained tissue culture system for
recombinant protein production in photo-bioreactors. By
targeted gene replacements, moss strains were created with
non-immunogenic humanised glycan patterns. These were
proven to be superior to currently used mammalian cell lines in
producing antibodies with enhanced effectiveness.
Addresses
Plant Biotechnology, Faculty of Biology, University of Freiburg,
Schaenzlestr. 1, D-79104 Freiburg, Germany
Corresponding author: Reski, Ralf (ralf.reski@biologie.uni-freiburg.de)
Current Opinion in Biotechnology 2007, 18:393–398
This review comes from a themed issue on
Expression technologies
Edited by Hansjoerg Hauser and Martin Fussenegger
Available online 14th September 2007
0958-1669/$ – see front matter
# 2007 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2007.07.012
Introduction
Current recombinant pharmaceuticals are produced
mainly in microbial (e.g. Escherichia coli, Saccharomyces
cerevisiae) or mammalian systems (mainly Chinese Hamster
Ovary cells). While microbial systems are superior in terms
of ease of handling and high product yields, mammalian
cell lines are favoured for the production of complex
biopharmaceuticals that need proper folding and/or correct
post-translational modifications [1,2 ]. Plant-based systems
combine advantages of both production systems:
As higher eukaryotes, plants synthesise complex multimeric
proteins with post-translational modifications closely
resembling mammalian modifications. In addition, production
in plants eliminates the risk of product contamination
by human pathogens possibly hidden in mammalian
cell lines or in their complex organic production media [3].
While agriculture offers easy and nearly unlimited adaptation
of production scale combined with low-cost production,
non-standardised soil and weather prohibit good
manufacturing practice (GMP) conditions – indispensable
for pharmaceutical production – for field-grown plants [4].
Some of these limitations can be overcome by contained
growth in greenhouses.
In contrast, in vitro plant tissue cultures can be grown in
precisely controlled environments suitable for GMP conditions
[5]. In addition, such systems offer a cheaper
downstream processing, especially when the biopharmaceutical
is secreted from the cells. Furthermore, host cells
secreting the product to the medium can be grown
continuously, minimising the need for batch cultures
and huge master cell banks.
Efficient transformation protocols were established for a
variety of plants [6]. Subsequently, several were tested as
production hosts, leading to plant-derived pharmaceuticals
that have been submitted for clinical trials. Only
recently the first plant-derived pharmaceutical, a veterinary
vaccine produced in plant cell culture, received
approval for market release [7]. However, the development
of large-scale production facilities is not yet realised
in many cases and most plant systems are limited in their
product spectrum for glycosylated proteins.
Human blood proteins such as antibodies, growth factors,
cytokines, and hormones are prime candidates for complex
biopharmaceuticals and most are N-glycosylated at
an Asparagine. This glycosylation may be important for
activity, stability or immunogenicity [2], for example it
may influence affinity and effectiveness of antibodies
[8,9]. While plants and mammals glycosilate their
proteins in a quite similar fashion, some sugar moieties
are plant-specific and thus immunogenic [10 ,11].
A few years ago, a non-seed plant, the moss Physcomitrella
patens, was established and commercialised as a production
host for complex biopharmaceuticals as it is
exceptionally amenable for precise genetic modifications,
for example to modify glycosylation pathways, and
allows low-cost cultivation in photo-bioreactors [12],
(http://www.greenovation.com/). Here we summarise
recent progress in establishing moss as a safe and efficient
production system for recombinant biopharmaceuticals.
Moss cultivation
Physcomitrella can be grown throughout its complete
life cycle under contained conditions in vitro. Haploid
spores germinate giving rise to a filamentous tissue, the
protonema. On these tissues leafy gametophores may
develop, closely resembling the morphology of seed
plants (Figure 1a). Sex organs occur only under inductive
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394 Expression technologies
conditions, giving rise to the diploid sporophyte with
persistent spores. While sexual reproduction occurs predominantly
when growing moss on agar plates (Figure 1b)
and requires inductive conditions, culture in liquid media
such as flasks and bioreactors (Figure 1c,d) favours vegetative
growth. Techniques have been established to
maintain Physcomitrella predominantly in the actively
growing filamentous form. In addition, for long-term
storage of moss strains, efficient cryo-protocols were
established [13] and realised in the International Moss
Stock Centre (http://www.moss-stock-center.org/).
Some in vitro cultures of seed plants rely on de-differentiated
cells and thus harbour inherently the risk of
genetic instability. In contrast, intact moss plants (protonema)
are grown in simple media of inorganic salts with
airborne CO 2 as sole carbon source. Consequently,
genetic instabilities were never observed in Physcomitrella
in vitro cultures. Cell differentiation and plant growth can
be tightly controlled by distinct parameters [14]. On a
small scale, moss plants are grown in agitated glass flasks.
On a scale up to 15 L, growth takes place in highly
controllable photo-bioreactors, either in stirred glass tanks
(Figure 1c) or in a modular, fully scalable glass tubular
reactor (Figure 1d) [15–18]. A 30-L tubular moss bioreactor
was established and parameters for high-density
and uniform moss cultures determined for batch [17 ]as
well as continuous cultivation [16]. Currently, a modular
100-L photo-bioreactor is being established for the production
of complex biopharmaceuticals according to
GMP standards (http://www.greenovation.com/).
Increasing product yields
Well-established protocols for the generation of stable
transgenic moss strains [13,19] were modified to establish
a transient expression system that allows proof of principle
within days [20]. Recently, the Physcomitrella genome was
fully sequenced (http://www.cosmoss.org/) as the fourth
plant genome after Arabidopsis, rice and poplar. From that,
a comprehensive phylogenetic database for plant transcription
factors was established [21], facilitating the design of
highly effective Physcomitrella production strains. The
strengths of various heterologous promoters from plants
and animals were quantified in Physcomitrella and compared
to endogenous promoters [22], including moss tubulin
and actin promoters [23,24], thus generating a set of
vectors activating gene expression in Physcomitrella within
three orders of magnitude. In addition, several promoters
were identified to be inducible, for example by the plant
hormone indole acetic acid (IAA, an auxin) [25,26] orby
moderate heat shock [27], in Physcomitrella.
Short N-terminal peptide extensions may navigate
proteins from the cytosol to specific organelles. For most
post-translational modifications, such as N-glycosylation,
a passage through the endoplasmic reticulum and the
Golgi-Apparatus is required. At the same time, this is the
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preferred route for the vast majority of extra-cellular
proteins. In various studies such signal peptides were
evaluated for functionality in Physcomitrella. Even though
signal peptides from human sequences were functional in
moss, most plant-derived signal peptides were more efficient
in targeting recombinant proteins to the secretory
system [24,28]. In addition, signal peptides for intracellular
storage of proteins in chloroplasts, mitochondria or the
central vacuole were identified and characterised via reporter
fusions with fluorescent proteins [29–34].
Protein recovery was markedly increased by addition of
stabilising agents (polyvinylpyrrolidone (PVP), human
serum albumin (HSA)) to the moss medium [35 ]. However,
at higher concentrations these agents lead to foam
formation and thus interfered negatively with the cultivation
process as well as with downstream processing.
Moreover, adding commercial HSA would introduce a
putative source of product contamination. These challenges
were elegantly overcome by co-expressing recombinant
HSA and the recombinant biopharmaceutical in
one production strain [35 ]. These strains produced only
tiny amounts of recombinant HSA, far too low to be
beneficial when added to control strains. Although the
precise mechanism by which HSA stabilises recombinant
proteins in the medium is unknown, it is plausible to
assume that the co-production of HSA and a second
protein in one plant facilitates their interactions already
during their joint passage through the endoplasmic reticulum.
The implementation of cross-flow filtration in the tubular
bioreactor module allowed a robust and flexible perfusion
of the suspension. Thus, secreted recombinant human
VEGF (vascular endothelial growth factor) was harvested
and concentrated via continuous product separation [16].
The secretory moss expression system was also used for
transient protein production allowing quick feasibility
studies of expression cassettes, transgenic moss strains,
or new products [23,24,28,33,36 ,37]. This transient system
was optimised for expression of recombinant human
VEGF up to 10 mg/mL [20].
Engineering product quality
All non-human expression hosts synthesise recombinant
proteins with slight differences to their human counterpart.
While folding and assembly of multimeric proteins is
essentially the same in all higher eukaryotes (e.g. plants
and mammals), post-translational modifications are
species-specific. This is one of the reasons why mammalian
cell lines such as Chinese Hamster Ovary (CHO)
cells are currently the system of choice. Therefore, plant
systems should be adaptable to human glycosylation to
become a realistic alternative to CHO cells. An appropriate
production host is genetically well-characterised
and amenable for genetic modifications. The Physcomitrella
genome has recently been sequenced and large
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Figure 1
amounts of EST data comprise more than 95% of the
transcribed moss genes [38–40]. Codon usage in moss
allows expression of human genes without previous codon
adaptation [39]. The genetically most interesting feature
of Physcomitrella is the high degree of homologous recombination
in its nuclear DNA, greatly facilitating precise
and base-specific targeted gene knockouts, a possibility
still not given for any other plant.
Parallel to the exploitation of the moss transcriptome,
protein N-glycosylation was characterised and shown to
exhibit the same structures as in higher plants [41,42]. In
addition, the glycosyltransferase genes responsible for
key steps of complex-type glycosylation were identified
and isolated from the moss genome [41].
The most critical differences between plant and human
glycosylation are two plant-specific sugar moieties, a xylose
connected to the core mannose residue by beta1,2-linkage
and a fucose, alpha1,3-linked to the proximal N-acetylglucosamine
(GlcNAc) residue of the core glycan (Figure 2).
While xylose is unknown in human glycans, the proximal
fucose residue in human glycans is linked in a different way
(alpha1,6). Both plant-specific sugars are immunogenic.
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Moss bioreactors Decker and Reski 395
In vitro cultivation of a moss, Physcomitrella patens for production of biopharmaceuticals. (a) Life cycle of mosses with haploid (1n) and diploid
(2n) stages. R!: meiosis (b) Storage of transgenic moss lines on multi-well agar plates. (c, d) In vitro propagation of moss protonema in stirred
glass-tank and tubular photobioreactors, respectively. Photographs courtesy of Andreas Schaaf (b) and Clemens Posten (TU Karlsruhe; d).
About one-quarter of individuals with allergies developed
antibodies of the allergy-relevant IgE class which specifically
recognise xylose or fucose-containing complex-type
glycans. However, the clinical relevance of carbohydratespecific
antibodies is still questionable [10 ,43]. Moss
knockout strains were created which lacked the enzymes
responsible for xylosylation and fucosylation, beta1,2-xylosyltransferase
and alpha1,3-fucosyltransferase, respectively
[37]. The resulting double knockout strains were
completely devoid of the allergenic sugar residues and
were employed to synthesise several products of pharmaceutical
value, including human VEGF [37], IgG class
antibodies [44,45], and erythropoietin [46].
Further ‘humanisation’ of plant N-glycans comprised
expression of a human beta1,4-galactosyltransferase,
responsible for linking terminal galactose to mammalian
N-glycans [47]. In addition, the engineering of plant
genomes in order to attach sialyl residues to the ends
of sugar chains was realised recently [48 ]. The humanlike
galactosylation was also realised by ‘knockin’ of the
human galactosyltransferase gene in the xylosyltransferase
and fucosyltransferase locus, respectively, from
double knockout moss strains [36 ,44].
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396 Expression technologies
Figure 2
Typical structures of antibody N-glycosylation. The sugar chains are
linked to the protein via the proximal GlcNAc residue (right).
Unmodified moss glycan containing terminal galactose and fucose
residues (top). Typical bisecting human antibody glycan (middle).
Glyco-optimised moss N-glycan providing antibodies with improved
ADCC (bottom). F: fucose; X: xylose. Sugars or linkages that may
result in immunogenicity or low effector function are marked in red.
Sialic acid residues are linked to many human blood
proteins and enhance their half-life in the circulation
[49]. However, monoclonal antibodies – probably the
most interesting and largest group of biotech drugs –
bear one conserved glycosylation site with an attached
N-glycan, and are only rarely sialylated. Furthermore,
these occasionally occurring sialic acid residues seem to
have no obvious function. Therefore, plant-based antibody
production with appropriate glycosylation is a realisable
task which was recently fulfilled in Physcomitrella
[44].
Moreover, by glyco-engineering via targeted gene knockouts,
the quality of plant-produced antibodies could even
be superior to that of CHO cells. The pharmacological
efficiency of some antibodies produced in mammalian
systems is rather disappointing due to weak antibodydependent
cellular cytotoxicity (ADCC), an important
effector function of antibodies. ADCC is mediated by
receptor (FcgammaRIII) binding of IgG antibodies. This
IgG-receptor binding affinity is increased in antibodies
lacking the human core fucose residue linked to the IgG
N-glycan [8,9]. Recombinant antibodies with increased
ADCC were produced in two glyco-engineered plants,
the aquatic Lemna minor [50 ] and in the moss (P. patens)
bioreactor [45,51 ]. The recombinant IgG1 antibody from
glyco-optimised moss had a 40-fold enhanced ADCC
activity and thus was convincingly superior to the parental
antibody produced in mammalian cells [45,51 ]. To significantly
enhance ADCC of CHO-derived antibodies, a
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second transgene is needed, to modify the native glycosylation
of CHO-derived proteins. One of the most promising
examples here is the regulated expression of
beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)
in CHO cells [52].
Conclusions
Moss bioreactors offer a safe and efficient scalable system
to produce complex modified recombinant pharmaceuticals
under GMP conditions. Genome engineering and
characterisation of transgenic strains is facilitated by
ample genomic resources. Optimisation of culture conditions
and genetic engineering of production lines via
targeted gene replacement helped to enhance product
yields and safety. Improved antibody function via glycan
optimisation makes the system advantageous compared
to current mammalian-based production systems.
Acknowledgements
Research in our lab is supported by the German Federal Ministry of
Education and Research (BMBF grants 0312624 and 0313852), the German
Academic Exchange Service (DAAD), and the Wissenschaftliche
Gesellschaft of the University of Freiburg.
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