Plant Biotechnology Journal (2010) 8, pp. 638–654
doi: 10.1111/j.1467-7652.2009.00495.x
Review article
Production of pharmaceutical-grade recombinant
aprotinin and a monoclonal antibody product using
plant-based transient expression systems
Gregory P. Pogue1,2, Fakhrieh Vojdani3, Kenneth E. Palmer4, Ernie Hiatt1, Steve Hume1, Jim Phelps1,
Lori Long1, Natasha Bohorova5, Do Kim5, Michael Pauly5, Jesus Velasco5, Kevin Whaley5, Larry Zeitlin5,
Stephen J. Garger6, Earl White7, Yun Bai8, Hugh Haydon1 and Barry Bratcher1,*
1
Kentucky BioProcessing, LLC, Owensboro, KY, USA
2
Emergent Technologies, Inc., Austin, TX, USA
3
Novici Biotech, LLC, Vacaville, CA, USA
4
Department of Pharmacology and Toxicology and James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY, USA
5
Mapp Biopharmaceutical, Inc., San Diego, CA, USA
6
Bayer HealthCare, Inc., Berkeley, CA, USA
7
MDx BioAnalytical Laboratory, Inc., Tucson, AZ, USA
8
Bioprocessing Consultants, San Diego, CA, USA
Received 1 September 2009;
revised 9 December 2009;
accepted 14 December 2009.
*Correspondence (fax +1 270 689 2571;
e-mail gppogue@yahoo.com)
Summary
Plants have been proposed as an attractive alternative for pharmaceutical protein
production to current mammalian or microbial cell-based systems. Eukaryotic protein
processing coupled with reduced production costs and low risk for mammalian pathogen contamination and other impurities have led many to predict that agricultural
systems may offer the next wave for pharmaceutical product production. However,
for this to become a reality, the quality of products produced at a relevant scale
must equal or exceed the predetermined release criteria of identity, purity, potency
and safety as required by pharmaceutical regulatory agencies. In this article, the ability of transient plant virus expression systems to produce a wide range of products
at high purity and activity is reviewed. The production of different recombinant proteins is described along with comparisons with established standards, including high
purity, specific activity and promising preclinical outcomes. Adaptation of transient
plant virus systems to large-scale manufacturing formats required development of
virus particle and Agrobacterium inoculation methods. One transient plant system
case study illustrates the properties of greenhouse and field-produced recombinant
aprotinin compared with an US Food and Drug Administration-approved pharmaceutical product and found them to be highly comparable in all properties evaluated. A
second transient plant system case study demonstrates a fully functional monoclonal
antibody conforming to release specifications. In conclusion, the production capacity
Keywords: virus vector, monoclonal
of large quantities of recombinant protein offered by transient plant expression sys-
antibody, aprotinin, plant expression,
tems, coupled with robust downstream purification approaches, offers a promising
therapeutic proteins, biologics, manu-
solution to recombinant protein production that compares favourably to cell-based
facturing.
systems in scale, cost and quality.
638
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd
Transient production of pharmaceutical proteins 639
Introduction
Cancer, infectious and chronic diseases continue to exact
a high toll on human life. More than 560 000 people die
each year from cancer and associated complications in the
United States alone, and the emergence of a new and
deadly swine flu strain in 2009 provides ample illustration
of this reality (Jemal et al., 2008; Sym et al., 2009). While
medical science has made great strides, traditional small
molecule drugs do not adequately address many disease
conditions requiring treatment. The advent of recombinant, biologically derived products (biologics) has revolutionized the practice of medicine through the use of
monoclonal antibodies (mAbs), vaccines and other therapeutic proteins. Biologics offer new patient therapies and
often show increased efficacy and less off-target, offmechanism effects in comparison with small molecule
therapies or chemotherapies (Szymkowski, 2004; Platis
and Labrou, 2008). The desirability of these therapies is
best characterized by biologic sales that have experienced
annual double digit increases for most years since their
introduction in the early 1980s and are predicted to
increase from 26% of the pharmaceutical market to 40%
by 2013 (Goodman, 2009). Although the biologics market
continues to represent the fastest growing segment in the
biopharmaceutical industry because of their new and
broad clinical applications, the financial challenges facing
the industry necessitate alternative cost-effective solutions.
Plants have been gaining market acceptance as an
attractive alternative production system for biologics by
overcoming several challenges facing the biopharmaceutical industry. In this article, we review two major market
challenges and, utilizing two case studies, demonstrate
the ability of transient plant expression systems to meet
the stringent demands for high quality biologics at competitive scale and competitive cost of current manufacturing systems while overcoming some complications with
current systems.
One imminent challenge to existing biologic products is
competition from follow-on biologics. Currently, the market is partially insulated from follow-on biologic threat
because of the ‘process is product’ stance of the US Food
and Drug Administration (FDA) and the willingness of governments to provide extended product protection periods
to biologics (Datamonitor, 2008). However, change is on
the horizon. The European Agency for the Evaluation of
Medicinal Products has issued formal guidance for follow-on
biologics and has approved 13 follow-on products—67%
of the applications filed since 2004 (Greb, 2009). In the
United States, discussions have begun regarding follow-on
biologic approval pathways by healthcare reform groups
who desire significant cost reduction in pharmaceutical
products (Greb, 2009). As pressure mounts to reduce the
cost of biologics, biopharmaceutical companies will seek
ways to recapture the high costs of research and development, along with the reduced profit margins associated
with follow-on biologics.
Production costs represent a second challenge to successful biologic products which are manufactured primarily
using microbial or mammalian cell-based expression systems. Cell-based systems are inherently more complex and
expensive than the production of most small molecule
drugs (Yina et al., 2007). Cell-based manufacturing
requires considerable capital and time to construct the
requisite facilities, including both upstream, cell-based fermentation and downstream production, purification and
formulation, capabilities. The typical costs associated with
these facilities are $300–$500 million and require from 4
to 5 years to complete construction, validation, and to
gain regulatory approval (Thiel, 2004). Each facility has a
basic production capacity that must be continually
deployed for necessary amortization of construction costs;
however, this capacity cannot be readily expanded without
construction of replicate facilities. Such costs and capital
commitment inevitably affect the costs of resulting products necessitating manufacturing systems that offer less
capital-intensive scaling to accommodate product requirements (Garber, 2001).
Because of their eukaryotic protein processing and
established success surrounding agricultural products,
plants are viewed as an attractive alternative production
system for many biologics (Ma et al., 2003; Floss et al.,
2007; Lico et al., 2008; Plasson et al., 2009). Agriculture
allows upstream manufacturing capacity to be scaled in a
capital-efficient manner, offering both flexibility and cost
savings that cannot be easily matched by fermentation
technologies. Such efficiencies make plants particularly
attractive with the threat of follow-on biologics and rising
capital costs. Downstream handing of plant biomass
requires unique biomanufacturing solutions (Plesha et al.,
2009). In spite of different methods employed, the purified product must show the same quality as produced by
traditional cell-based systems (Pogue et al., 2002; Sharma
and Sharma, 2009). To this end, many competing technologies have been developed to produce recombinant proteins in plants using stable transformation methods that
modify the genetic complement of the production plant
species, such that prodigy inherits the foreign gene
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 638–654
640 Gregory P. Pogue et al.
sequence and expression capacity (Floss et al., 2007;
Sharma and Sharma, 2009). Although this method has
shown robust expression of several pharmaceutically relevant proteins such as growth hormone, a-1 antitrypsin,
various mAbs and recombinant vaccines (Ma et al., 2003),
2009). The flexibility of the Agro-infiltration system allows
for the efficient expression of the biopharmaceutical protein of interest and provides the required cofactors to
improve pharmaceutical protein yield and processing. The
co-expression of silencing suppressor proteins has been
it does have notable drawbacks. The process of genetic
transformation is slow, requiring months to years to derive
sufficient seed for significant plantings. Horizontal transmission of the recombinant gene is a concern and has led
shown to be a key factor for optimized yields (Mallory
et al., 2002; Hellens et al., 2005; Azhakanandam et al.,
2007). Such methods have been used to produce a range
of biopharmaceutical proteins (Joh and VanderGheynst,
to complex regulatory oversight to prevent pollen transfer
or regrowth of transgenic crops through tissue or seed
dispersal. Finally, many food or feed crops are often
employed as production hosts leaving significant human
2006; Benchabane et al., 2009; Sourrouille et al., 2009)
and offer strategies to modify the plant enzymatic machinery, producing more stable and ‘human’-like recombinant
proteins, including glycan structures (Benchabane et al.,
food supply or livestock contamination concerns (Belson,
2000; Pogue et al., 2002) as described both in the media
and in official documentation from agricultural regulatory
authorities in Europe and the United States (European
2008; Vézina et al., 2009).
Virus-based replicating systems offer advantages over
standard integrative plant expression systems by exploiting
the cytoplasmic replication cycle of the virus vector (Pogue
Food Safety Authority, 2009). The risks associated with
non-food, non-feed crops are significantly less than consumable plants and are strongly favoured by regulatory
authorities (Belson, 2000).
In the face of these challenges, transient protein expres-
et al., 2002; Gleba et al., 2008; Lico et al., 2008). The
ability of virus-based systems to sequester host and virally
encode enzymes facilitates the amplification of messenger
RNA leading to increased pharmaceutical protein accumulation (Gleba et al., 2008; Lico et al., 2008). These systems
sion strategies bring the significant advantages of plantbased bioreactors at considerably reduced costs to current
cell-based manufacturing systems while avoiding the less
desirable properties of stable plant transformation. Tran-
also offer rapid and efficient expression characteristics,
new genes can be tested for expression and test quantities
of the recombinant protein can be obtained in as little as
4–8 weeks, while leaving no heritable changes to the pro-
sient systems have been demonstrated as safe and environmentally friendly in both indoor and outdoor tests
since 1991, and 16 products produced by transient systems were shown safe in early-stage human clinical trials
duction plant (no foreign gene is transmitted in the pollen
or by insects and is therefore contained within its boundaries; Pogue et al., 2002). Virus-based replicating systems
generally fall into two categories: ‘independent-virus’ or
as personalized vaccines administered to non-Hodgkin’s
lymphoma patients (Pogue et al., 2002; McCormick et al.,
2008). Additional advantages compared with traditional
cell-based fermentation approaches include: (i) speed and
‘minimal-virus’. Independent-virus vectors are inoculated
as virus particles or viral RNA and exploit virus-encoded
cell-to-cell and systemic movement activities to infect host
plants. Replicating independent-viruses spread systemically
low cost of genetic manipulation; (ii) rapid manufacturing
cycles; (iii) no mammalian pathogen contamination; (iv)
minimal endotoxin concentrations and (v) economical production (Pogue et al., 2002; Ma et al., 2003; Gleba et al.,
2008; Lico et al., 2008; Vézina et al., 2009).
from a small number of initially infected cells to infect the
majority of the phloem sink tissue of a host. Expression of
messenger RNAs encoding recombinant proteins is mediated by either the activity of virus subgenomic promoter
or polyprotein translational expression mechanisms. Inde-
Two approaches dominate transient expression: standard integrative plant expression vectors and virus-based
replicating systems (Ma et al., 2003; Floss et al., 2007;
Lico et al., 2008; Sharma and Sharma, 2009). Standard
pendent-virus systems have been derived from the
genomes of potexviruses (including potato virus X; PVX),
tobamoviruses (including tobacco mosaic virus; TMV),
comoviruses (including cowpea mosaic virus), potyviruses,
integrative plant expression vectors are introduced into
intact plants using an Agrobacterium tumefaciensmediated transfer-DNA delivery system (Agro-infiltration;
vacuum infiltration of aerial parts of the plant to introduce
tobraviruses, closteroviruses and several others (Pogue
et al., 2002; Lico et al., 2008).
In contrast, minimal-virus systems are capable of functions supporting RNA replication. This approach increases
Agrobacterium cells containing expression vectors into the
plant cells; Joh and VanderGheynst, 2006; Vézina et al.,
the genetic load carried by the minimal-virus systems
allowing efficient expression of larger recombinant
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 638–654
Transient production of pharmaceutical proteins 641
proteins (Giritch et al., 2006). Because these systems are
incapable of movement in inoculated plants, they must be
delivered to the majority of plant cell to produce meaningful amounts of recombinant proteins. This is usually
accomplished through Agro-infiltration of host plants to
(MP) and CP, respectively (Figure 1). GENEWARE also
exploits the strength and duration of the viral subgenomic
promoter’s activity to reprogram the translational priorities
of the plant host cells so that virus-encoded proteins are
synthesized at similar high levels as the TMV CP (Shivpra-
launch the infection process (Gleba et al., 2005, 2007,
2008). Minimal-virus systems do not require the delays
associated with systemic plant movement and have the
ability to replicate to high levels, often yielding greater
sad et al., 1999). A foreign gene encoding the protein for
overexpression is added in place of the virus CP, so it will
be expressed from the endogenous virus CP promoter
[illustrated by green fluorescent protein (GFP) in Figure 1;
amounts of recombinant proteins in a shorter period of
time than independent-virus systems (Gleba et al., 2007,
2008). Minimal-virus systems primarily exploit the subgenomic promoter activities and genomes of potexviruses
Shivprasad et al., 1999]. A second CP promoter of lower
transcriptional strength, divergent in sequence from the
endogenous (TMV U1) CP promoter, is placed downstream of the heterologous coding region, and a virus CP
and tobamoviruses (Gleba et al., 2008).
This review will focus on two case studies of transient,
viral-based plant expression technologies: Kentucky BioProcessing, LLC’s (KBP) GENEWARE, a independent-virus sys-
gene is then added. This encodes a third subgenomic RNA
allowing the virus vector to express all requisite genes for
virus replication and systemic movement in addition to the
tem and Icon Genetics (Halle, Germany), GmbH’s
magnICON, a minimal-virus system. We will consider the
advantages offered by each and explore two specific biologic examples, recombinant aprotinin and a mAb binding
the chemokine (C–C motif) receptor-5 (CCR5), each produced at multi-gram scale under current good manufacturing practices. These case studies will illustrate the
flexibility and power of transient plant expression systems
to provide recombinant protein products of the quality
(a)
T7 Promoter
Replicase
(b)
T7 Promoter
Replicase
MP
MP
CP
Product
(GFP) CP
(c)
2 dpi
6 dpi
and quantity required for clinical development.
White
Light
Case study I: GENEWARE system
GENEWARE is a hybrid replicon derived from TMV, principally strains U1 and U5. Tobamoviruses have a plus sense
single-stranded RNA genome of 6400 nucleotides helically encapsidated in rigid rod-shaped particles composed
of 2100 copies of the 17.5 kDa coat protein (CP). The
viral proteins involved in RNA replication are directly transcribed from the genomic RNA, whereas expression of
internal genes is through the production of subgenomic
RNAs (Dawson and Lehto, 1990). The production of subgenomic RNAs is controlled by sequences in the tobamovirus genome, which function as subgenomic promoters.
The CP is translated from a subgenomic RNA and is the
most abundant protein, and RNA produced in the infected
cell (Turpen, 1999). In a tobamovirus-infected plant, there
are several milligrams of CP produced per gram of
infected tissue.
GENEWARE expression system takes advantage of
independent-virus functions, including cell-to-cell and systemic movement activities mediated by movement protein
UV
Light
Figure 1 Genomic structure of tobacco mosaic virus (TMV) and illustration of construction and utility of GENEWARE expression system. (a)
Shows the genomic organization of TMV and the positions of two subgenomic promoters (bent arrows) driving expression of subgenomic
messenger RNAs encoding movement protein and coat protein,
respectively. Replicase proteins are translated from the genomic RNA.
GENEWARE vectors are constructed by insertion of an additional subgenomic RNA promoter and multiple cloning site for insertion of foreign
genes (b) such as the green fluorescent protein (GFP) shown. Infectious
cDNA clones of the recombinant TMV genome are transcribed from the
T7 bacteriophage RNA promoter, followed by infection of plants, such
as Nicotiana benthamiana, with infectious RNA transcripts, shown in (c).
The plants in (c) are shown 2 and 6 days post inoculation under white
light (top) and ultraviolet light illumination (bottom). Expression of GFP
and systemic spread of GFP carrying GENEWARE are clearly visible
under UV light.
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 638–654
642 Gregory P. Pogue et al.
heterologous gene intended for overexpression (Figure 1).
GENEWARE vectors infect various tobacco-related species
(genus Nicotiana), including tabacum, benthamiana and a
KBP-proprietary Nicotiana hybrid species, Nicotiana excelsiana (Fitzmaurice, 2002). The infectious vector RNA enters
system. Selected examples of highly purified proteins that
have been subjected to potency testing are shown in
Table 1. The results obtained from these proteins
expressed from the GENEWARE system in Nicotiana hosts
were extracted using either tissue homogenization and
plant cells via wounds induced by an abrasive. The virus
replicates in the initial cell, moves to adjacent cells to produce round infection foci and then enters the plants’ vascular system for transport to aerial leaves. There, it
clarification methods or leaf infiltration and isolation of
interstitial fluids (Pogue et al., 1998; Turpen, 1999) and
purified through differential separation and standard chromatographic separations (see references in Table 1). Purity
systematically infects the majority of cells in each infected
leaf (as illustrated using GFP in Figure 1). The foreign gene
is expressed in all cells that express other virus protein
products, including the replicase, MP and CP. The foreign
of the recombinant proteins was determined by densitometric analysis of overloading of Coomassie brilliant
blue-stained sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) gels and high pressure liquid
protein is deposited in the site dictated by its protein
sequence, either naturally or purposely engineered
(Turpen, 1999; Pogue et al., 2002).
A range of human enzymes, antimicrobials, cytokines,
chromatography (HPLC), when appropriate. Potency determinations were made testing the specific activities of each
product with appropriate enzymatic or cytokine controls.
In each case, highly purified proteins with specific activities
subunit vaccine components and immunoglobulin fragments have been produced using the GENEWARE
matching established controls were observed demonstrating the broad classes of proteins that can be effectively
Table 1 Qualities and bioequivalence of GENEWARE produced pharmaceutical proteins and peptides
Product
Size (kDa)
a Galactosidase A (human)*†
48.5
Results
>98% purity, comparable enzymatic activity with CHO cell-derived controls
and preclinical efficacy demonstrated
Aprotinin (bovine)à
6.5
>99% purity, comparable specific activity with pharmaceutical product
Trasylol
§
Granulocyte colony-stimulating factor (human)
18.8
>95% purity, bioequivalence to Neupogen using specific cell proliferation
Griffithsin (Griffithsia)–
12.7
>99% purity, bioequivalence with natural product and potent neutralization of
Hepatitis B core antigen (Hepatitis B virus)§
31
>95% purity, conservation of virus-like particle structure and immunoreactivity
Interferon a 2a (human)§
19.3
>99% purity, bioequivalence with WHO standard in antiviral and
Interferon a 2b (human)§
19.3
>99% purity, bioequivalence with WHO standard in antiviral and
Interleukin-2 (human)§
15.4
>97% purity, bioequivalence in cell proliferation assays with interleukin-2
Lysosomal acid lipase (human)**
50.6
>99% purity, bioequivalence with standards and preclinical efficacy
Lysozyme (bovine)§
14
>85% purity, comparable enzymatic activity with natural and yeast derived
Papillomavirus capsid fusion (Human Papillomavirus)††
19
>99% purity, preclinical efficacy demonstrated in two different models
activity assay
12 different human immunodeficiency virus strains
antiproliferative activity assays
antiproliferative activity assays
standards
demonstrated
protein standards
Single chain antibody fragments (human)àà
30
>95% purity for 16 different human idiotypic proteins, preclinical efficacy and
clinical safety and immunogenicity
*Protein name and species of origin is indicated.
†
Gelderman et al. (2004).
à
This report.
§
Kentucky BioProcessing, LLC, unpubl. data.
–
O’Keefe et al. (2009).
**Du et al. (2008).
††
Grill et al. (2005); Palmer et al. (2006).
àà
McCormick et al. (1999, 2003, 2008).
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 638–654
Transient production of pharmaceutical proteins 643
expressed and purified from plants treated with transient
expression systems. In each of the examples presented
above, the control proteins used were either obtained
from established research vendors or were proteins manufactured under good laboratory practices.
plant media or 3.7% of secreted protein present in Spirodela (duckweed) growth media (Rival et al., 2008) and
0.5% total soluble protein in selected leaves in transplastomic tobacco (Tissot et al., 2008). These measurements
of yield are in general difficult to compare because of the
Biologic example: aprotinin
vastly different levels of protein present in the various targeted tissues or the efficiencies in extraction from these
tissues. This is most clearly seen with r-aprotinin expressed
in transgenic corn seed. Total soluble protein concentra-
Background
Aprotinin is a 58 amino acid active serine protease inhibitor of bovine origin that is processed from a preproprotein
precursor (Laskowski and Kato, 1980). The active protein
tion is much lower when the entire seed is extracted compared with the germ (Zhong et al., 2007). Optimized
extraction methods and selective extraction from the germ
resulted in a >10-fold increase in recovered r-aprotinin
conformation requires three disulphide bridges and appropriate processing from both N-terminal and C-terminal
prepropeptides. Aprotinin has been explored for clinical
applications for four decades for a variety of clinical indi-
activity from corn seed (Zhong et al., 2007). These results
illustrate the critical nature of downstream processing efficiencies to ensure yield and quality of purified protein
product (Zhong et al., 2007; Plesha et al., 2009). Further,
cations (Beierlein et al., 2005). Bayer HealthCare Pharmaceuticals’ Trasylol, natural aprotinin, was a FDA-approved
product indicated for prophylactic use to reduce perioperative blood loss and the need for blood transfusion in
patients undergoing cardiopulmonary bypass in the course
purified proteins showed comparable protein size and
trypsin inhibitory activity (Zhong et al., 2007; Rival et al.,
2008; Tissot et al., 2008). However, extensive analysis of
the identity, purity and potency of the product was not
presented. In each cited study, the limited product accu-
of coronary artery bypass graft surgery (CABG; Munoz
et al., 1999; Sedrakyan et al., 2004). The drug, manufactured from residual bovine lung materials, was approved
in the United States in 1993. However, recent interna-
mulation required detection by immunoassay or activity
assays—visualization of product in crude plant lysates by
protein gel analysis was not provided. In contrast to transgenic expression strategies, transient plant expression vec-
tional studies have indicated increased risk of in-hospital
death and 5-year mortality rates among aprotinin recipients when compared with non-recipients (Mangano et al.,
2006, 2007). In late 2008, Bayer HealthCare announced
tors generally offer higher yield potential enabling product
analysis in direct plant lysates and development of appropriate product release tests. Aprotinin serves as a promising product candidate well suited for transient plant
that marketing of the product was temporarily suspended
pending review of additional clinical studies (Stamou et al.,
2009). In spite of the adverse events associated with the
drug in CABG patients, clinical studies continue to explore
expression.
the application of aprotinin in other indications, both prophylactic and therapeutic, where the control of pathophysiological inflammatory cascades is desirable. These
ongoing studies suggest that the market for aprotinin
could expand once again provided an alternative active
A synthetic cDNA of the mature bovine aprotinin gene
was constructed as an in-frame fusion with the Nicotiana
benthamiana (N. benthamiana) extensin signal peptide
(Figure 2). This genetic arrangement was chosen to simplify the post-translational processing of r-aprotinin with
pharmaceutical ingredient (API) to bovine tissue could be
more reliably produced without raising concerns over animal-associated adventitious agents, such as bovine spongiform encephalopathy prions (Maffulli et al., 2008; Orchard
regard to its preproprotein structure. The expression cassette was sub-cloned into the TMV-based GENEWARE
vector under the control of the T7 RNA polymerase promoter to produce expression plasmid construct pKBP2602.
et al., 2008; Rademakers et al., 2009).
To date, several groups have expressed and purified
recombinant bovine aprotinin (r-aprotinin) from transgenic
plant materials. The crude yields of r-aprotinin varied per
RNA transcripts were prepared and inoculated on
N. benthamiana. Characteristic viral symptoms, vein clearing and leaf curling, were noted 6–12 days post-inoculation (dpi). Representative plants were extracted at 14 dpi
system, examples include 0.17% total protein in the corn
seed (Azzoni et al., 2002; Zhong et al., 2007), 0.65 mg ⁄ L
by total leaf and stem homogenization and analysed for
the presence of r-aprotinin and activity. The pH of this
GENEWARE r-aprotinin production
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 638–654
644 Gregory P. Pogue et al.
(a)
(b)
1 2 3 4
(a)
ATGGGAAAAATGGCTTCTCTATTTGCCACATTTTTAG
TGGTTTTAGTGTCACTTAGCTTAGCTAGCGAAAGCT
CCGCCCGGCCTGACTTCTGCCTAGAGCCTCCATAT
ACGGGTCCCTGCAAGGCCAGAATTATCAGATACTTC
TACAACGCCAAGGCTGGGCTCTGCCAGACCTTTGT
ATATGGCGGCTGCAGAGCTAAAAGAAACAATTTCAA
GAGCGCAGAGGACTGCATGAGGACCTGTGGTGGTG
CTTAG
MGKMASLFATFLVVLVSLSLASESSARPDFCLEPPYTG
PCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAE
DCMRTCGGA
22 kD
TMV CP
14 kD
Figure 2 The recombinant bovine aprotinin expression construct is
described in both nucleic acid coding strand (a) and deduced amino
acid sequence (b). The modified aprotinin gene sequence is shown
with Nicotiana extensin signal peptide underlined (nucleic acid and
deduced amino acid) and mature aprotinin sequence (nucleic acid and
deduced amino acid) not underlined. The DNA sequence of the synthetic aprotinin gene was constructed using codon biases based on
the tobacco mosaic virus coat protein sequence.
6.5 kD
Aprotinin
(b)
1
2
3
4
5
6
7
kDa
homogenate was acidified and clarified by centrifugation.
The expression of r-aprotinin was evaluated by reducing
SDS-PAGE and showed the accumulation of the TMV CP
and the r-aprotinin that co-migrates with the Trasylol
97.4
66.3
55.4
36.5
control (Figure 3a). The molecular mass of r-aprotinin in
the clarified homogenate was the expected molecular
mass of 6512 Da as determined by matrix-assisted laser
desorption–ionization time-of-flight mass spectrometry.
Further, significant inhibition of serum protease activity
31.0
21.5
14.4
was also determined in the extract using trypsin inhibition
assays and conversion of activity into trypsin inhibitory unit
(TIU). Inhibition activity showed 7100 TIU ⁄ mg of extract
protein, comparable to that observed for native bovine
aprotinin (Table 2; Fritz and Wunderer, 1983).
Based on these promising results, large-scale manufacturing of aprotinin was conducted using plants grown
under greenhouse conditions (N. benthamiana) or in open
field cultivation (N. excelsiana). TMV virions were isolated
from plants infected with transcripts derived from
pKBP2602 plasmid DNA. The virion was confirmed for its
ability to produce aprotinin in inoculated plants, and
reverse transcriptase polymerase chain reaction was used
to confirm the presence of the aprotinin expression cassette in the recombinant virus genome (data not shown).
Virions were mixed with an abrasive and spray inoculated
on either greenhouse-grown or field-grown plants. Plants
were monitored for virus symptoms and were harvested in
bulk 14 days post infection. R-aprotinin accumulated in
the leaf and recovered by extraction of the interstitial fluid
of leaves or through total leaf homogenization. Maximum
yields (40% enhanced protein recovery; data not shown)
6.5
3.5
Trasylol®
1.5 µg/lane
r-aprotinin
1.5 µg/lane
MW
Marker
Figure 3 Expression and extraction of recombinant bovine aprotinin
(r-aprotinin) in Nicotiana plants. Virion preparations containing
tobacco mosaic virus expression vector encoded by plasmid pKBP2602
were inoculated on Nicotiana benthamiana plants. Plants were harvested 14 days post inoculation. (a) Initial plant homogenate is represented in lane 2, while the supernatant derived from clarified
homogenate is shown in lane 3. Trasylol, bovine-purified aprotinin
(2 lg), is provided in lane 4 for control. Molecular weight marker is
shown in lane 1 and relevant markers with known molecular weight
provided at left. (b) Trasylol (1.5 lg ⁄ lane) was loaded in triplicate in
lanes 1–3. Purified r-aprotinin (1.5 lg ⁄ lane) was loaded in triplicate in
lanes 4–6. Molecular weight markers containing known molecular
weight proteins are loaded at right. Proteins were analysed using
4–12% Bis–Tris sodium dodecyl sulphate polyacrylamide gel electrophoresis gels and subjected to Coomassie Brilliant blue-staining.
were noted by total homogenization extraction; therefore,
this approach was adopted for large-scale manufacturing.
The leaves were homogenized and clarified followed by
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 638–654
Transient production of pharmaceutical proteins 645
Table 2 Test methods and results of aprotinin comparisons
Assay
Comparative attribute
R-aprotinin
Trasylol
Identity by tryptic digest
Conforms with bovine lung aprotinin
Conforms
Conforms
MALDI-TOF MS mass mapping
predicted tryptic fragments and fragment
derivatives (84% amino acid coverage)
Identity by MALDI-TOF MS
6512 Da ± 0.05%
6512 Da
6512 Da
Identity by amino acid analysis
Conforms with bovine lung aprotinin
Conforms
Conforms
Purity by SDS-PAGE
Purity
>99%
>99%
Purity by RP-HPLC
Purity
87.6% + 12.4(Ox)%
86.3% + 5.7(Ox)%
Purity by GC ⁄ MS small molecular
Purity
Comparable levels of target
Comparable levels of target
Clear, colorless, free of visible particles
Clear, colorless, particle free
Potency by specific activity
>6500 KIU ⁄ mg protein or >5.0 TIU ⁄ mg protein
7175 KIU or >5.7 TIU
6859 KIU or 5.4 TIU
Endotoxin
<1 EU ⁄ 28 mg
<1 EU ⁄ 28 mg
<1 EU ⁄ 28 mg
amino acid composition
weight host toxicants
Purity by appearance
compounds
compounds
Clear, colorless, particle free
RP-HPLC method separates non-oxidized and oxidized forms of r-aprotinin.
MALDI-TOF MS, matrix-assisted laser desorption–ionization time-of-flight mass spectrometry; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel
electrophoresis; RP-HPLC, reverse phase high pressure liquid chromatography; GC, gas chromatography; MS, mass spectrometry; KIU, Kallikrein inactivation
unit; TIU, trypsin inhibitory unit; EU, endotoxin units; r-aprotinin, recombinant bovine aprotinin.
concentration of the r-aprotinin using ultrafiltration. The
r-aprotinin was purified using cation exchange chromatography followed by reverse phase chromatography. The
final product was concentrated across a 1 kDa molecular
weight cut-off membrane (MWCO), pH adjusted, sterile
scale more
systems.
competitive
than
traditional
production
filtered and vialed.
Because of differences in soluble protein content in various plant extracts, KBP and its collaborators report protein accumulation as milligrams per kilograms of fresh
The purified r-aprotinin was subjected to rigorous analytical testing. Table 2 shows the types of release tests performed on the plant-produced r-aprotinin lots and a
comparison of results from greenhouse-produced product
weight of extracted tissues. This approach takes into
account variables in the extraction and the efficiency of
the method used and provides a basic and relevant crude
production level from which to base predictable econom-
with that of Trasylol. The identity of the proteins was virtually identical as determined by tryptic peptide analysis,
amino acid analysis and reactivity with anti-aprotinin mAb
(Table 2; data not shown). Further, the molecular mass of
ics. GENEWARE production of r-aprotinin in greenhousegrown Nicotiana plants showed crude and purified yields
of 750 and 400 mg ⁄ kg, respectively. Field-produced
plants showed crude and purified yields of 300 and
both proteins was found to be identical at 6512 Da
(Table 2). The potency of the r-aprotinin was consistently
higher than Trasylol, as measured by Kallikrein inactivation units per milligram of purified protein (Table 2). Purity
150 mg ⁄ kg, respectively. However, costs of plant agronomic practice were approximately fivefold less in open
fields compared with greenhouse production plants modulating the reduction in absolute protein expression. In
spite of the differences in methods of reporting protein
analyses showed no detectable protein impurities by overloaded SDS-PAGE, exact migration pattern on gels, reverse
phase HPLC (RP-HPLC; Figure 3b; Table 2) and immunoassays (data not shown). Neither Trasylol nor GENEWARE
r-aprotinin product showed any immunoreactivity with a
yield, these results suggest transient expression offers
superior yields than transgenic approaches (Azzoni et al.,
2002; Zhong et al., 2007; Tissot et al., 2008). The exploitation of agriculture scale allows production of 1 kg of
polyclonal antibody generated against crude Nicotiana protein extracts demonstrating an absence of host-derived proteineous impurities in the final product (data not shown).
Detailed RP-HPLC analysis revealed minor aprotinin variants.
purified r-aprotinin from 2500 square feet of greenhouse
space or 1.5 acres of field transfected Nicotiana plants.
These results demonstrate that transient plant production
systems can provide product quantity and economies of
Truncated aprotinin species, including desAla58 and desAla58Gly57, and various oxidized aprotinin species were
quantitated in the Trasylol product as 8% and 5.7%,
respectively. R-aprotinin showed no detectable truncated
GENEWARE r-aprotinin characterization
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 638–654
646 Gregory P. Pogue et al.
Table 3 Test methods and results of r-aprotinin comparisons (greenhouse versus field grown)
Assay
Comparative attribute
R-aprotinin (Greenhouse)*
R-aprotinin (field)†
Identity by ESI-TOF MS
Average molecular mass between
6511.4 Da
6511.8 Da
>99%
>99%
‡5.0 mg ⁄ mL
21.3 mg ⁄ mL
18.3 mg ⁄ mL
Clear, colorless to amber, free of
Clear, light yellow, particle free
Clear, light yellow, particle free
6508.2–6514.8 Da
Purity by SDS-PAGE (reduced)
‡95% of r-aprotinin as determined
by densitometry (% band)
Protein concentration by UV
absorbance
Purity by appearance
visible particles
Potency by TIU
>5.0 (B) TIU ⁄ mg protein
5.7 TIU ⁄ mg
5.6 TIU ⁄ mg
Endotoxin
<1 EU ⁄ 28 mg
<1 EU ⁄ 28 mg
<1 EU ⁄ 28 mg
*Lot 07A0009.
†
Lot O8A0025.
ESI-TOF MS, electrospray ionization time-of-flight mass spectrometry; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; TIU, trypsin
inhibitory unit; EU, endotoxin units; r-aprotinin, recombinant bovine aprotinin.
species, although it contained oxidized forms at 12.4% of
the final product. It is well known that oxidation is common
on methionine 52 (Concetti et al., 1989). Indeed, oxidation
of the methionine residue was noted in chloroplast-produced aprotinin in specific plant lines (Tissot et al., 2008).
pharmaceutical products, such as Trasylol, demonstrating
the ability of the GENEWARE transient plant-expression
system to produce product matching those of FDAapproved biologics.
The oxidated species did not exhibit reduced inhibition activity (data not shown; Concetti et al., 1989). Exploitation of
changes in physicobiochemical behaviour of the oxidized
protein allowed efficient removal of the oxidized forms
Case study II: magnICON system
Independent-virus systems, such as GENEWARE, must
maintain all activities of the virus to successfully colonize
using a second, subsequent reverse phase chromatography
method (data not shown).
Comparison of field-produced r-aprotinin with greenhouse produced revealed virtually identical products
an infected host plant, in addition to subgenomic promoter and RNA replication functions responsible for production of recombinant protein products. The necessity of
MP and CP coding regions reduces the genomic capacity
(Table 3). Purity and identity analyses revealed identical
results (examples provided electrospray ionization timeof-flight mass spectroscopy, appearance and SDS-PAGE).
Protein concentrations of the final bulk drug differed
of the virus and reduces the size of proteins efficiently
before vialing because of degree of concentration, yet
both were under predetermined bulk drug release specifications. The potency of the field product was comparable
with that of the greenhouse-produced API (Table 3). The
stability of the greenhouse-produced r-aprotinin, in liquid
Assay
Table 4 Stability testing of r-aprotinin product*
Protein concentrationà
Potency§
Purity† (%)
(mg ⁄ mL)
(TIU ⁄ mg)
0
100
21.6
6.1
3
Not determined
21.5
5.7
6
Not determined
21.3
5.4
form, was monitored over a 31-month period with realtime storage at 4 C (Table 4). No significant changes in
the purity, protein concentration and specific activity were
observed at any point in the 31-month test period or
12
Not determined
21.0
6.4
16
Not determined
21.0
5.1
24
Not determined
21.3
5.5
31
>99
21.6
6.1
when the initiation point was compared with terminal
time point (Table 4). These results demonstrate the consistency and quality of the GENEWARE r-aprotinin produced from transiently transfected Nicotiana plants of
r-aprotinin, recombinant bovine aprotinin.
different species and production conditions. Further, the
data presented show comparable results with that of
(# months)
*Lot 07A0009.
†
Purity—sodium dodecyl sulphate polyacrylamide gel electrophoresis and
densitometry; release specification ‡95%.
à
Concentration—OD280 and bicinchoninic acid method; release
specification ‡5.0 mg ⁄ mL.
§
Trypsin inhibitory unit (TIU); release specification ‡5.0 TIU ⁄ mg.
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 638–654
Transient production of pharmaceutical proteins 647
produced by such systems to <70 kDa. The magnICON
system represents a distinct minimal-virus approach for
using tobamovirus-based vectors where systemic movement functions are eliminated to transiently express heterologous proteins in permissive hosts, such as
production, two magnICON virus expression vectors are
delivered by Agro-infiltration into the same plant. Each
vector replicates independently and expresses heavy and
light chains (HC and LC) in the same cells. The two chains
self-assemble into authentic and functional mAbs and are
N. benthamiana (Marillonnet et al., 2004; Gleba et al.,
2005, 2007, 2008). In magnICON vectors, the MP and
CP genes may be eliminated through genetic deletion,
and the gene encoding a pharmaceutical protein is placed
secreted to the apoplastic space at yields up to 1 g ⁄ kg
fresh weight (Giritch et al., 2006; Gleba et al., 2007,
2008).
To date, most published studies concerning transient
under the control of the endogenous CP subgenomic promoter. This minimal-virus strategy provides increased
genomic capacity to express larger proteins than typically
compatible with independent-virus systems. The magnI-
expression systems have detailed expression under laboratory conditions yielding milligram to gram levels of product
(see references in this article and Floss et al., 2007;
Sharma and Sharma, 2009). The ability to scale manufac-
CON system utilizes the Agro-infiltration system to introduce the plant viral vector expression system, as intact
virus vectors or in distinct modules, including a module
containing the gene(s) of interest (Marillonnet et al., 2004;
turing to multi-kilogram quantities of plant material is a
critical step to validate the use of plant systems for therapeutic protein production. To accomplish this task with
transient expression systems, plant inoculation as well as
Gleba et al., 2008). If the distinct module strategy is used,
the components are assembled in planta and the resulting
DNA is transcribed, spliced and translated, resulting in
high yields of the expressed protein (Marillonnet et al.,
2004). Numerous heterologous proteins have been pro-
protein extraction and purification methodologies must be
adapted. Although plant processing and purification methods can be modelled from food processing and standard
biomanufacturing systems (Doran, 2000; Pogue et al.,
2002), inoculation methods for magnICON vectors
duced using this system, including cytokines, interferon,
bacterial and viral antigens, growth hormone, single chain
antibodies and mAbs at levels of 1–10 g ⁄ kg (Giritch et al.,
2006; Gleba et al., 2007, 2008).
require adaptation of the traditional laboratory-based
Agro-infiltration method to a robust, large-scale process.
Working in cooperation with Bayer Innovation, GmbH
and Icon Genetics, KBP adapted the Agro-infiltration pro-
Nicotiana benthamiana plants are ideally suited for the
magnICON expression system because it relies on Agrobacterium infection to mediate initial entry and introduction of the viral expression vectors. Nicotiana benthamiana
cess to accommodate the infiltration of kilograms of
plants per hour, allowing 25–75 g of antibody to be produced per greenhouse lot using the magnICON vectors
(KBP Agro-infiltration system shown in Figure 4). The pro-
is known to be nearly universally susceptible to plant
viruses, partially based on a defective form of RNA-dependent RNA polymerase found in its genome (Yang et al.,
2004). This viral susceptibility allows external viral repli-
cess begins with seeding plants in a tray system that, as
plants grow through a hole in the tray lid, the aerial portion of the growing plant is physically separated from the
soil and root components. The trays are grown in a con-
cases, delivered as part of the magnICON expression system, to successfully replicate the delivered genes. The
combination of ease of infection with bacterial and viral
components and a long history of experimental use have
made N. benthamiana a common host for the expression
trolled growth environment until reaching appropriate size
and then manually loaded onto a conveyor, inverted 180
and moved through a vacuum rated autoclave with reservoirs containing the Agrobacterium solution. Sufficient
vacuum is applied and then released to allow entrance of
of many recombinant proteins. The flexibility of Agro-infiltration of N. benthamiana also offers the ability to introduce more than one expression vector into a host plant in
a given treatment. The magnICON system exploits this
the Agrobacterium solution into the interstitial spaces of
the submerged plant tissues. Upon completion of vacuum
cycle, trays are placed into an upright position and transported back to a controlled growth environment
advantage to be an efficient system for the production of
heteromeric recombinant proteins, such as mAbs. For the
production of mAbs, the magnICON system employs two
non-competitive virus vectors: one based on turnip vein-
(Figure 4). The system has been designed to operate with
450–750 kg of green biomass in an 8-h production cycle,
depending on plant growth conditions and protein product design. Following Agro-infiltration, plants are grown in
clearing tobamovirus (TVCV) and the other based on PVX
(Giritch et al., 2006; Hiatt and Pauly, 2006). In mAb
greenhouses for 7–14 days depending on product-specific
optimization of plant biomass and yield (Figure 4).
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 638–654
648 Gregory P. Pogue et al.
(c)
(a)
(d)
(b)
Figure 4 ‘At scale’ Agrobacterium tumefaciens-mediated transfer-DNA delivery (Agro-infiltration) system. (a) Plants are seeded in trays with specially designed lid to permit growth, yet provide a barrier for soil and root components. Following plant growth to appropriate size, ten trays are
loaded on each of four conveyors to enter the vacuum-rated chamber, shown in (b) with both for and aft doors open and empty. Conveyors
rotate 180 and enter the chamber (c), plants are submerged in Agrobacterium-containing solution and vacuum is applied and released. Plants are
removed from chamber and rotated to upright position using conveyors and subsequently transferred to greenhouses for growth and product
accumulation (d).
Following growth period, plants are harvested and subjected to standard protein extraction methods. To demonstrate the adaptability of the magnICON plant virus
transient expression system for large-scale, multi-gram,
biomanufacturing, the production of a neutralizing mAb
the prevention of sexual transmission of HIV-1 (Gaertner
et al., 2008). Further, CCR5 appears to be non-essential
for human health because individuals with CCR5-D32
alleles (essentially a CCR5 knockout) are healthy (Dean
et al., 1996).
binding the CCR5 co-receptor follows.
Anti-(a) CCR5 mAbs are currently in clinical development as HIV therapeutics (Jacobson et al., 2008) due to
their potent blockage of CCR5-mediated HIV-1 cell entry
in vitro (Trkola et al., 2001; Murga et al., 2006; Shearer
Biologic example: mAb
mAbs represent the fastest growing sector in the biopharmaceutical market ($35 billion in 2008 revenue; La Merie
et al., 2006). Despite the fact that small molecule CCR5specific drugs are potent chemokine antagonists, neutralizing antiviral concentrations of CCR5 mAbs [inhibitory
concentration (IC)50 0.1–1 lg ⁄ mL] did not block the natu-
Business Intelligence, 2009) and are used therapeutically in
many different clinical areas, including infectious disease,
oncology, inflammation, allergy and cardiovascular (Hoentjen and van Bodegraven, 2009; Weiner et al., 2009).
ral activity of CCR5 in vitro, although CCR5 antagonism
was observed at higher concentrations (IC50 of 45 lg ⁄ mL;
Olson et al., 1999). Similarly, at concentrations ranging to
100 lg ⁄ mL, the mAbs had no effect on lymphocyte prolif-
Many companies are exploring broader applications of
mAbs, including their use to block the entry of viruses into
cells to prevent infection (Trkola et al., 2001; Murga et al.,
2006; Shearer et al., 2006; Jacobson et al., 2008). For
example, CCR5 acts as a co-receptor for human immuno-
eration in response to mitogenic and allogeneic stimulation (Gardner et al., 2003) and did not mediate significant
levels of antibody-dependent cellular cytotoxicity or complement-dependent lysis of CCR5-expressing cells. A CCR5
mAb has been shown to neutralize escape mutants raised
deficiency virus type 1 (HIV-1) entry into cells (Moore
et al., 1997), and it has been suggested that a microbicide
acting to block CCR5 may serve as a possible strategy for
against small molecule CCR5 inhibitors (Pugach et al.,
2008). Systemic delivery (intravenous and subcutaneous)
of an aCCR5 mAb has shown strong antiviral activity in
Background
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 638–654
Transient production of pharmaceutical proteins 649
Phase 1b (Jacobson et al., 2008) and later stage clinical trials (Olson and Jacobson, 2009) as well as a good safety
profile. Although the potent anti-HIV activity of aCCR5
mAb suggests this molecule could be quite valuable in
prophylactic as well as therapeutic applications to control
mAb DNA
PROVECTOR DNA
(aCCR5-LC, aCCR5-HC)
(31660-LC, 26211-HC)
Amplify in E. coli
HIV infection, the costs of aCCR5 mAb mammalian production are prohibitive.
Plants were first shown to correctly fold and produce
antibodies in 1989 with continued demonstration of a vari-
Plasmid DNA Purification
ety of antibody candidates through the efforts of many
investigators (see Ma et al., 2003 and references therein).
However, production levels and characterization of these
products have been slow to emerge in the published litera-
Linearized, purified
provector DNA
ture. Reported expression levels of mAbs expressed via
transgenic plants are rather low (<30 mg ⁄ kg plant tissue;
Fischer et al., 2003; Floss et al., 2007; Gaertner et al.,
2008; Ma et al., 2003; Valdés et al., 2003). Indeed, a highly
efficient process showing protein purity of >90% was
demonstrated from transgenic tobacco plants with a yield
of recombinant antibody of 25 mg ⁄ kg fresh weight
tissues (Valdés et al., 2003). However, the time required to
construct, select and grow these lines for large-scale production is predicted to be >24 months. Transient plant
expression offers a solution to this challenge. Using the
magnICON system, the time and subsequent cost
efficiency of agricultural-scale production of mAbs offer a
viable manufacturing option for products, including mAbs
as microbicides for the prevention of HIV-1 infection and a
means to apply promising products to a broader range of
individuals.
magnICON mAb production
Mapp Biopharmaceutical, Inc. is developing a aCCR5 mAb
as an intravaginal topical antimicrobial agent to reduce
mucosal transmission of HIV-1. This humanized mAb
specifically binds the ligand-binding domain of the human
chemokine receptor and HIV co-receptor, CCR5. Using the
magnICON system, HC and LC of the aCCR5 mAb are
inserted in two different virus expression vectors, TVCV
and PVX (Figure 5). To express the aCCR5 mAb in plants,
Working Cell Banks (WCB) of Agrobacterium cell lines,
containing mAb LCs and HCs (HC; 31 160-LC, 26 211-
Ligate mAb and provector DNA
+ electrocompetent Agrobacterium tumefaciens
Electroporation
Transformed Agrobacterium (2 cell lines per mAb)
(31660 aCCR5-LC, 26211-aCCR5-HC)
Figure 5 Flow diagram of the anti-chemokine (C–C motif) receptor-5
(aCCR5) Master Cell Bank construction. Strain development process is
presented and used for infiltration inoculation of Nicotiana benthamiana plants for the production of aCCR5 monoclonal antibody.
two Agrobacterium cell banks (311 600-LC, 26 211-HC)
and allowed to grow for 10 dpi (Figure 4). At this time, all
aerial portions of the treated plants were harvested and
aCCR5 mAb extracted and purified. Briefly, plant materials
were homogenized (typically 40–60 kg of plant material ⁄ extraction). The homogenized materials were then
subjected to a horizontal screw press to separate plant
fibre and ‘green juice’ extract. The pH of the extract was
adjusted and clarified using a plate and frame filter press.
The clarified extract was then loaded on a protein A
column, and bound antibody was further treated by filtration and multi-ion exchange resin column. The column
eluant was pooled and diafiltered using a 30-kDa MWCO
HC, respectively), were derived from Master Cell Banks
(MCB; see Figure 5 for flow diagram of aCCR5 MCB construction). WCBs were amplified, and overnight cultures
were mixed and diluted in infiltration buffer. Nicotiana
membrane, sterile filtered using a 0.2-lm filter and vialed.
benthamiana plants were subjected to the KBP Agro-infiltration process using an infiltration buffer containing the
During the manufacturing process, the purity and protein
concentration of the mAb product were monitored via
magnICON mAb characterization
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 638–654
650 Gregory P. Pogue et al.
SDS-PAGE gels and OD280 measurements. Presence of
endotoxin was also monitored to ensure appropriate
recovery and freedom from contaminating materials. Bulk
drug substance for the mAb was vialed immediately and
stored as drug product. The final aCCR5 mAb product
tion levels reported for transgenic systems (Valdés et al.,
2003). The quality of the vialed product supports its use in
early-stage clinical investigations as a novel, biologic
microbicide to stem the tide of HIV-1 infection within
at-risk populations and provides further proof-of-concept
was subjected to rigorous release testing in accordance
with predefined acceptance criteria specifications
(Table 5). Example results showed high purity obtained
through this purification process, >99% by SDS-PAGE and
for transient plant expressions systems.
Conclusions
97% monomer by size exclusion HPLC. The potency was
also measured using a CCR5-specific enzyme-linked immunosorbent assay and revealed a highly active mAb product
with expected specific activity. Further, non-protein impu-
Plants have been touted as an attractive alternative for
pharmaceutical protein production to the current mammalian or microbial cell-based systems. The potential for
reduced production costs coupled with the low risk for
rities, such as nicotine, were reduced to parts per billion
levels (Table 5). The product showed no significant levels
of endotoxin, and no detectable bioburden per millilitre
(Table 5). These are important findings because the Agro-
contamination with human-tropic adventitious agents and
other impurities have led many to hypothesize that agricultural systems may offer the next wave for pharmaceutical product production (Ma et al., 2003; Floss et al., 2007;
infiltration process involves infiltration of all aerial portions
of the plants with a solution containing Agrobacterium
strains encoding the production viruses. The copious quantities of bacteria would provide opportunity for the retention of these contaminants and impurities. Nevertheless,
Lico et al., 2008; Plasson et al., 2009). However, for this
to be a reality, the quality of products produced at a relevant scale must match the common release criteria in the
pharmaceutical industry. A review of the literature demonstrates the variety of recombinant proteins that can be
the purification strategy and aseptic environment led to
efficient removal.
The aCCR5 mAb was produced in a scalable manner by
the magnICON system. Processing greenhouse-propa-
produced in transgenic and transient plant virus expression
systems, as well as the quality of the resulting purified
products (Ma et al., 2003; Floss et al., 2007; Lico et al.,
2008; Plasson et al., 2009; Sharma and Sharma, 2009).
gated plants provides for 25–75 g purified product lots at
an expected yield of 250 mg ⁄ kg fresh weight of plant
materials. These levels are 10-fold greater than produc-
Detailed review of the GENEWARE and magnICON systems provides further demonstration that quality biologics,
such as r-aprotinin and aCCR5 mAb, respectively, can be
Table 5 Release test specifications and results for anti-chemokine (C–C motif) receptor-5 monoclonal antibody
Parameter
Test method
Release specification
Production batch results
Appearance
Visual
Clear, colorless to amber, liquid
Clear, colorless, liquid
Protein concentration
OD280
0.7–1.3 mg ⁄ mL
1.1 mg ⁄ mL
Identity*
Isoelectric focusing
4–5 bands pI range 8.4–9.7
5 bands pI 8.4–9.7
Purity
SDS-PAGE
‡95% (sum of heavy and light chain)
>99%
Purity
Size exclusion HPLC
‡90% monomer
97% monomer
£10% aggregation
0.44% aggregation
£10% LMW
2.63% LMW
0.08 lg ⁄ mL
Potency
Viral neutralization
IC50 < 1 lg ⁄ mL
Physical ⁄ chemical properties
pH
5.5–6.5 pH units
6.2 pH units
Physical ⁄ chemical properties
Conductivity
9.15 mS ⁄ cm ± 0.5
9.15 mS ⁄ cm
Safety
Endotoxin
<10 EU ⁄ mL
0.5 EU ⁄ mL
Safety
Bioburden
<10 CFU ⁄ mL
<1 CFU ⁄ mL
Impurities
1-methyl-2-[3-pyridyl]-pyrrolidine
For information only
<50 ppb
For information only
<0.2%
(nicotine) concentration
Impurities
Residual host cell protein
OD, optical density; PAGE, Isoelectric focusing polyacrylamide gel electrophoresis gels; pI, isoelectric point; SDS-PAGE, sodium dodecyl sulphate PAGE; HPLC,
high pressure liquid chromatography; LMW, low molecular weight; IC, inhibitory concentration; EU, endotoxin units; CFU, colony forming units.
*Isoelectric focusing PAGE.
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 638–654
Transient production of pharmaceutical proteins 651
produced using non-food ⁄ feed, non-genetically modified
plants.
In addition to the quality of proteins produced from
transient plant expression systems, speed to develop the
virus inoculum and MCB for the expression of a given protein provides significant advantages. As described by Hiatt
and Pauly (2006), the ability of transient systems to produce milligrams of product can be as little as 2 weeks and
production of grams may take only a few weeks more.
These timeframes are much shorter than the requirements
to transfect, select, establish and characterize mammalian
cells, transgenic animal or traditional plant-based systems.
Both the GENEWARE and magnICON systems, through
the adaptation of both virus inoculation and Agro-infiltration methods to large-scale biomanufacturing systems, can
yield productivity of recombinant proteins at levels of
200–1000 mg ⁄ kg fresh weight tissue in as little as
3 months. Further, the yields that can be expected from
these systems can be quite high, ranging from 0.25 to
0.75 g ⁄ kg when extracting >100 kg of crude plant material. These values are 10-fold greater than production levels of the same proteins in transgenic plant systems.
Lastly, these transient systems yield correctly folded monomeric and multimeric proteins that show release properties
comparable with standard pharmaceutical products attesting to the robustness of plant expression capabilities. The
data reviewed here strongly support the contention that
transient plant expression systems have moved beyond the
proof-of-concept stage in development and offer a legitimate cost-competitive alternative for recombinant protein
production.
Acknowledgements
We appreciate the efforts of Terri Cameron, Mark Smith,
Sarah Doucette, Steve Reinl, Long Nguyen, Amanda
Lasnik, Lee Hamm, Hal Padgett, Wayne Fitzmaurice and
Peter Roberts for contributing to GENEWARE expression
results. We also acknowledge the contributions of Jennifer
Bleckmann, Cara Working, Josh Morton and Jennifer
Poole in the production and characterization of the r-aprotinin product. We thank Dr David Montefiori (Duke University) for performing the HIV neutralization assays. This
project was supported in part by Award Number U19 AI
62150 from the National Institute of Allergy and Infectious
Diseases. The content is solely the responsibility of the
authors and does not necessarily represent the official
views of the National Institute of Allergy and Infectious
Diseases or the National Institutes of Health.
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