Bioprocessing
Protein Expression Systems:
Ringing in the New
By Felipe Monteclaro
at Percivia LLC
Despite a general preference for the predictability of established systems,
alternative protein expression systems provide higher yields, are more
cost-effective and enable much faster cell line development, scale-up and
processing – all of which will have a direct bearing on speed to market.
Protein expression systems are widely used in
biotechnology and medicine for the efficient and
economic production of therapeutic proteins from a
potentially limitless source. Since the time that insulin
was first expressed as a recombinant protein
therapeutic, the use of E. coli and subsequently
mammalian expression systems has become standard
practice for the expression of biopharmaceuticals. More
recently, alternative protein expression systems have
started to be developed with the aim of providing
improved functional properties, higher product quality
and – in some cases – cost reduction in manufacturing.
CONVENTIONAL SYSTEMS
The conventional expression systems used today for
protein therapeutics have the main advantage of
regulatory acceptance and are derived primarily from
mammalian cells, E. coli and – to a lesser extent – yeast,
and also more recently even from insect cells and
transgenic animals. The majority of early work on the
expression of simple therapeutic proteins – such as
insulin, growth hormone and interferon – was carried out
in relatively simple expression systems such as E. coli,
where proper folding or extensive post-translational
processing was not required for a protein to be
biologically active. Due to this limitation, the
biotechnology industry directed its efforts towards using
more complex eukaryotic expression systems. Despite the
long list of mammalian cell lines available for the
expression of therapeutic proteins, the predominant
choice has been myeloma and CHO cells due to their
easy access and acceptable regulatory standards (1) –
beginning with the approval of recombinant tPA
(Activase) in 1987, erythropoietin in 1989, and antibody
products such as ReoPro in 1994 and Herceptin in 1998
(2). While there are also scattered examples of
therapeutics expressed in yeast and insect cells, demand
for the production of complex therapeutic proteins still
rests heavily on the use of mammalian expression systems.
ALTERNATIVE EXPRESSION SYSTEMS
Alternative expression systems are currently available that
have shown the ability to express currently marketed protein
therapeutics. These technologies – namely human cells,
transgenic animals, plant cells and unicellular algae, and in
vitro (cell-free) systems – are all claimed to be unique and
may circumvent infringements on current method-based
patents, as well as having the potential to meet clinical
demand. Especially appealing to the biotech industry is the
promise of high protein yields as well as a lower upfront
investment required to implement these technologies.
Human Cells
Various human cell lines have been used to produce
approved therapeutic proteins; these include
HEK293 (Xigris), HT-1080 (Dynepo,
Replagal, Elaprase) and Namalwa cells
(non-recombinant Sumiferon). However,
very few cell lines are available that are
non-virally transformed, non-tumour
derived and immortalised to generate
Figure 1: XD process PER.C6 cell concentration profiles
including CHO cells, NS0 and SP2/0
– produce proteins that contain
the terminal galactose-α-1,3-galactose
(α-Gal) antigen, known to induce
anaphylactic response (5). These
proteins are considered by the human
host to be foreign, as are the residual
host proteins carried over from
purification in non-human cell
lines. The potential for reduced
immunogenicity and enhanced efficacy
heightens the appeal of human cell
lines to produce human protein
therapeutics economically without any
sacrifice of quality or patient safety.
250
Cell concentration (E6 cells/mL)
200
150
100
50
0
0
5
10
15
20
Time (days)
Medium A
stable high producer cells. Such cells
include CAP cells (Cevec) and
PER.C6®cells (Crucell/Percivia) –
Medium C
PER.C6 cells have a well documented
history and evidence of safety testing
(3), and have been extensively developed
for the production of vaccines and recombinant proteins,
such as monoclonal antibodies and blood factors.
Immortalisation of the PER.C6 cell line was achieved by
expressing the E1A and E1B genes that confer the
necessary robustness for use in highly intensive
manufacturing processes (see Figure 1). Using a
high density technology (XDTM, DSM) gives a high level
of production, at 27 grams per litre for IgGs; this is
obtained by pushing the cell density in the bioreactor to
extremely high levels and thereby achieving maximum
volumetric productivity in a short time (see Figure 2, page
48). These yields far exceed the productivities achieved in
other cell lines and are competitive with conventional
expression systems.
Medium B
Human cells, being the native production host, can add
great value to the therapeutic properties and safety of the
complex proteins used in human therapeutics. These
complex proteins are adorned with post-translational
modifications (PTMs) that may be required for
enhanced activity and pharmacokinetic properties, and a
potential reduction in any untoward immunological
responses. By contrast, the same proteins when processed
in non-human cells may elicit immunogenic responses as
a consequence of the addition of N-glycolylneuraminic
acid-containing oligosaccharides, as reported in
rodent cells (4). Several manufacturing cell lines –
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Transgenic Animals
The technology of transgenic drugproducing animals shows great
potential in the production of
biotherapeutics, and can arguably be more cost-effective
than conventional protein-production engines. For
example, Rhucin (C1 esterase inhibitor) – a therapeutic
protein for the treatment of hereditary angioedema – is
being produced in transgenic rabbits by Pharming and is
currently in clinical trials. The rabbits have been
engineered using a bovine milk-specific promoter
sequence (alpha-S1 casein) to drive high-level expression
of the C1 esterase inhibitor protein in mammary glands
and subsequent collection in the milk. In terms of yield,
whereas highly optimised cell cultures that can typically
generate 0.2 to 1 gram of protein per litre of culture
medium, each transgenic rabbit is able to produce
between 10 to 12 grams of protein per litre of milk.
Individual rabbits are able to produce up to 10 litres of
milk a year, and therefore a facility that can house 500
rabbits has the capacity to generate nearly 5kg of the
unpurified recombinant protein per year. To achieve
similar production levels in a traditional bioreactor, a
20,000-liter vessel would be required with a host
expression level of two grams per litre.
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The firm GTC Biotherapeutics has developed a
transgenic goat that produces recombinant human
antithrombin III (Atryn) – an anticoagulant for the
treatment of thrombotic disorders. With an annual yield
of 800 litres, over one kilogram of recombinant protein
can be produced per lactating animal; by comparison, it
would take an average of 30,000 donors to generate an
equivalent amount from plasma.
As with all animal systems, transgenic technology has
the potential to transmit human pathogens and strict
Innovations in Pharmaceutical Technology
Figure 2: XD process titer
for the production
therapeutic proteins.
30
Product concentration (g/L)
25
20
15
10
5
0
0
5
10
15
20
Time (days)
Medium A
control measures are put in place to
monitor safety against pathogens, with
robust processes in place for the
Medium C
removal and inactivation of viruses
and also prions. The expression of
therapeutic proteins in these lactating
animals provides significant production and cost
advantages, but these may ultimately be outweighed by
reduced efficacy due to rapid clearance as a result of
inappropriate PTMs.
Medium B
Plants
The production of therapeutic proteins in plants holds
substantial promise, particularly with green algal
chloroplast as a production platform. The eukaryotic
green algae Chlamydomonas reinhardtii is capable of
synthesising and assembling a full-length IgG1
human monoclonal antibody (mAb) in transgenic
chloroplasts, and the algal-expressed anti-anthrax
antibody (83K7C) has been shown to have similar
binding characteristics to the CHO-expressed
antibody, although it appears to lack glycosylation as
expected (6). Aglycosyated antibodies should lack the
ability to fix complement or recruit killer cells, and
would offer a unique type of antibody where the
bypassing antibody dependent cell-mediated cytoxicity
is desirable. The antibody 83K7C has been shown to
bind to the toxin (PA83) and to neutralise it in cellbased assays as well as to protect rats from anthrax
toxicity in vivo (7). Expression of some recombinant
proteins in plant chloroplasts has reached very
high levels – well above 10 per cent of the total protein
– illustrating the potential of algae as a platform
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of
human
It is important to note several benefits
of producing human-like recombinant
therapeutic proteins in plant expression
systems. Unlike mammalian cells, plant
cells do not harbour human pathogens
nor do they produce endotoxins like
some microbial systems. Plant cell
cultures grow in simple synthetic media
that are free of animal-derived
components – including animal viruses
– and so do not require upstream
and downstream viral inactivation
processes. Transformed chloroplasts
have the potential for high expression
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of recombinant protein for biologics
production in plants with minimal risk,
since chloroplasts are not transmitted
through pollen – thereby avoiding the escape of genes
into the environment and transfer to conventional crops.
Plant-based biopharmaceutical companies have taken
advantage of the capability of plants to produce
human-like proteins. Protalix uses a patented cell
system to express human genes in engineered carrot
and tobacco cells that are grown on an industrial scale
in a low-cost disposable bioreactor system, producing
kilograms of purified proteins. A similar production
capacity using mammalian cells could well be costprohibitive. Protalix claims that its engineered
plant cells produce glycosylation patterns more
closely resembling human proteins and provide
similar post-translational modifications as occur in
human cells – a distinct advantage over microbial
systems. Using this plant expression system for
their lead product Uplyso (B-glucoceramidase), the
plant-produced recombinant protein has shown
to be equivalent to the mammalian cell-based
product. In a 31 patient Phase III trial, Uplyso has
met its primary and secondary endpoints while
developing non-neutralising antibodies in six per cent
of the patients (8).
Biolex, on the other hand, uses a small aquatic water
plant, Lemna, for protein production in disposable
plastic bags and medium consisting of water and
inorganic salts; during the process, the biomass of Lemna
can double in 36 hours. This rapid scale-up can facilitate
development of Biolex’s key product, a glyco-optimised
rituximab (anti-CD20) with significantly enhanced
antibody-dependent cellular cytotoxicity (ADCC),
Innovations in Pharmaceutical Technology
decreased complement-dependent cytotoxicity (CDC)
and similar apoptotic activity (9).
Acknowledgement
The author would like to thank Dr Marco Cacciuttolo
and Dr John Chon for helpful discussions and review
The effectiveness of plant-derived therapeutics may be
compromised, however, by plant-specific glycosylation.
All of the plant species used to date for therapeutic
protein production have the capacity to associate
bisecting β(1,2)-xylose and core α(1,3)-fucose residues
onto complex N-glycans; these residues are constituents
of the glyco-epitopes of some plant allergens and show
IgE binding and cause mediator release by human
basophils (10).
of the manuscript.
References
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Cell-Free Extract
Protein expression systems using cell extracts have been
available for decades but, until recently, have not been
shown to achieve production at a commercial scale.
Extracts derived from an optimised E. coli host have been
used to produce recombinant human GM-CSF
(rhGM-CSF), antibodies and cytokines using an open,
cell-free synthesis (OCFS) technology, as developed
by Sutro Biopharma. The OCFS system has been
designed to generate clinically validated protein-based
therapeutics in controlled biochemical reactions for use
in research and optimised for scale-up to 100 litres. An
added feature of OCFS is the independence from mass
transport, cell growth and viability, and incorporation of
the addition of non-natural amino acids to the protein
with charged tRNAs that can be directed to a specific
codon to deliver the non-natural amino acid to a
specific location on the protein – making the protein
amenable to specific modification or imparting a new
desired property (11).
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CONCLUSION
Campbell RL, In: Langer ES, Ed, Vol 1, Emerging
and novel technologies in biopharmaceutical
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Gasdaska JR et al, The Power of One: GlycoOptimised Therapeutic Antibodies in Lemna,
In Vitro Cell Dev Biol-Animal 44: S18-S29, 2008
Adopting new technologies continues to be fundamental
to improving product quality and cost. However, the
rapid adoption of expression technologies for
biomanufacturing has a steep hill to climb – in part due
to the regulatory hurdles that new technologies have to
overcome, and also a conservative preference for the
predictability of established systems. However, the
innovations associated with alternative expression
systems are attractive as they add value through
improvements in therapeutic properties and safety.
These new systems are providing higher yields,
often with disposable equipment allowing for further
cost savings and convenience. Alternative expression
systems enable much faster cell line development,
scale-up and processing and this will have
a direct bearing on speed to market – providing a
significant advantage for biosimilar products.
www.iptonline.com
10. van Ree R, Cabanes-Macheteau M, Akkerdaas
J et al, β(1,2) xylose and α(1,3) fucose residues
have a strong contribution in IgE binding to
plant glycoallergens, J Biol Chem 275:
pp11,451-11,458, 2000
11. Goerke AR and Swartz JR, Development of cell-free
protein synthesis platforms for disulfide bonded
proteins, Biotechnol Bioeng 99: pp351-367, 2008
Felipe Monteclaro is currently Director, Cell Line
Development at Percivia LLC. He was formerly at Bayer
Healthcare, California, US, and at Schering AG, Berlin, Germany.
He received his BSc in Microbiology and Immunology from the
University of California, Berkeley, US and his PhD in Molecular
and Cell Biology from the University of Southern California, US.
Email: [email protected]
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