Download - ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals
-
8/6/2019 ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals.
1/16
O R I G I N A L P A P E R
Optimisation of contained Nicotiana tabacum cultivation
for the production of recombinant protein pharmaceuticals
Richard Colgan Christopher J. Atkinson Matthew Paul Sally Hassan Pascal M. W. Drake Amy L. Sexton Simon Santa-Cruz David James Keith Hamp Colin Gutteridge Julian K-C. Ma
Received: 5 May 2009 / Accepted: 21 June 2009 / Published online: 9 July 2009
Springer Science+Business Media B.V. 2009
Abstract Nicotiana tabacum is emerging as a crop of
choice for production of recombinant protein pharma-
ceuticals. Although there is significant commercial
expertise in tobacco farming, different cultivation
practices are likely to be needed when the objective is
to optimise protein expression, yield and extraction,
rather than the traditional focus on biomass and
alkaloid production. Moreover, pharmaceutical trans-
genic tobacco plants are likely to be grown initially
within a controlled environment, the parameters for
which have yet to be established. Here, the growthcharacteristics and functional recombinant protein
yields for two separate transgenic tobacco plant lines
were investigated. The impacts of temperature, day-
length, compost nitrogen content, radiation and plant
density were examined. Temperature was the only
environmental variable to affect IgG concentration in
the plants, with higher yields observed in plants grown
at lower temperature. In contrast, temperature, supple-
mentary radiation and plant density all affected the
total soluble protein yield in the same plants. Trans-
genic plants expressing a second recombinant protein
(cyanovirin-N) responded differentlyto IgG transgenicplants to elevated temperature, with an increase in
cyanovirin-N concentration, although the effect of the
environmental variables on total soluble protein yields
was the same as the IgG plants. Planting density and
radiation levels were important factors affecting
variability of the two recombinant protein yields
in transgenic plants. Phenotypic differences were
observed between the two transgenic plant lines and
non-transformedN. tabacum, but the effect of different
growing conditions was consistent between the three
lines. Temperature, day length, radiation intensity andplanting density all had a significant impact on biomass
production. Taken together, the data suggest that
recombinant protein yield is not affected substantially
by environmental factors other than growth tempera-
ture. Overall productivity is therefore correlated to
biomass production, although other factors such as
purification burden, extractability protein stability and
quality also need to be considered in the optimal design
of cultivation conditions.
R. Colgan C. J. Atkinson C. Gutteridge
East Malling Research, New Road, East Malling, Kent
ME19 6BJ, UK
M. Paul S. Hassan P. M. W. Drake
A. L. Sexton
J. K-C. Ma (&
)CMM, 2nd Floor Jenner Wing, St. Georges Hospital
Medical School, Cranmer Terrace, London SW17 0RE,
UK
e-mail: [email protected]
S. Santa-Cruz D. James
Empharm Ltd., New Road, East Malling, Kent ME19 6BJ,
UK
K. Hamp
Unigro Ltd., Gay Dawn Offices, Valley Road, Fawkham,
Kent DA3 8LY, UK
123
Transgenic Res (2010) 19:241256
DOI 10.1007/s11248-009-9303-y
-
8/6/2019 ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals.
2/16
Keywords Contained cultivation
Nicotiana tabacum Molecular farming
Recombinant antibody
Introduction
As the first recombinant pharmaceuticals for humans
from transgenic plants approach clinical trials (Ma
et al. 2005), the regulatory framework for manufac-
turing processes of such products is becoming
established (Spok et al. 2008). Of the various stages
in the production process, the early steps involving
plant cultivation, harvest and initial processing rep-
resent the procedures that are specific to plants, and
require the most innovation and development. The
later downstream processing and product purificationsteps are more familiar as they would be essen-
tially similar to those already established for other
recombinant protein expression systems, such as
CHO cells and E. coli.
Tobacco is a major agricultural non-food crop that
is grown worldwide. It is also emerging as a
transgenic production crop of choice for plant-made
recombinant pharmaceuticals (PMPs) (Sparrow et al.
2007) and a number of recombinant proteins, includ-
ing monoclonal antibodies, have been successfully
produced in tobacco (Valdes et al. 2003). Conven-tionally, tobacco is grown in open fields, and the aim
of agricultural practices is to maximise leaf produc-
tion and that of the alkaloid compounds (such as
nicotine). Optimisation for protein expression and its
accumulation has not been considered, and limited
information is currently available. Moreover, molec-
ular farming is most likely to progress, at least
initially, under greenhouse containment. This segre-
gation measure minimises the potential for gene flow,
accidental exposure to animals and humans, contam-
ination of the pharmaceutical crop and also allowsmuch tighter control of environmental conditions for
plant cultivation. However, tobacco is not normally
grown under greenhouse containment; so again, little
horticultural information is available for optimised
growth, particularly with respect to environmental
control.
The primary goal in horticultural management of
plants for PMP production is to maximise recombi-
nant protein production. However, a number of other
factors need to be considered. The stability of the
recombinant protein is important (Stevens et al.
2000). Another key issue is the purification burden
that is determined by the level of indigenous, plant
compounds that are extracted alongside the target
recombinant protein, as well as undesirable degrada-
tion or aggregation products of the target proteinitself. A further important issue is uniformity of
production. An underlying principle of current Good
Manufacturing Practice (cGMP) regulatory oversight
is the achievement of uniformity of product through
consistency of production process. Thus cultivation
procedures for transgenic plants that predispose to
high levels of variability in protein production
between plants are undesirable.
The aim of this study was to identify optimal
environmental conditions for the cultivation of
transgenic Nicotiana tabacum (var. xanthii) for theproduction of recombinant proteins in greenhouse
containment. Two transgenic lines were studied,
expressing either a monoclonal antibody or a cyano-
bacterial protein. The effect of environmental vari-
ables such as temperature, day length, compost
nitrogen availability, radiation intensity (photosyn-
thetically active radiation PAR), and growing density
have been investigated in terms of impact not only on
plant growth and biomass production, but specifically
with regard to the yield of the two recombinant
pharmaceutical proteins. In addition, the plant-to-plant variability of such protein production has been
assessed for each environmental variable.
Materials and methods
Transgenic plant lines
Two well established homozygous transgenic plant
lines were used (Ma et al. 1994, Sexton et al. 2006).
The Guys 13 line expresses a murine IgG1 monoclo-nal antibody (MAb) that binds specifically to the SA I/
II protein ofStreptococcus mutans (Smith et al. 1984).
The CV-N plant line expresses cyanovirin-N (CV-N),
an 11 kDa protein from Nostoc ellipsosporum, which
has potent neutralising activity against HIV (Boyd
et al. 1997).
The Guys 13 line seeds are from the T4/F3
generation, that is they result from the sexual cross
between T0 transgenic plants individually expressing
242 Transgenic Res (2010) 19:241256
123
-
8/6/2019 ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals.
3/16
the immunoglobulin light and heavy chains and are
the third generation seed containing both transgenes.
The CV-N line seeds are from the T2 generation
that is the third generation seed containing the single
transgene.
Controlled environment cultivation
The GroDome facility is a secure, contained, double-
skinned, polycarbonate building, divided into six
independent walk-in growth rooms. Each room has a
controlled environment, that allows accurate control
of carbon dioxide concentration with controllable
pressure and air movement, temperature control
(within a 1C tolerance in a preset range of
530C) and ambient or supplementary lighting, with
integrated control software monitors for conditions in
each room. Supplementary radiation was supplied viasodium lamps (Osram Vialox Navcson_T) to deliver
*3,400 W per chamber over the growing area. The
facilities are designed to meet level II containment
requirements and are controlled and monitored by a
building management system which provides full
data storage of internal conditions.
Five environmental variables were investigated
day-time temperature (32/21 vs. 23/21C), day length
(18/6 vs. 12/12 h), compost nitrogen levels (180 vs.
108 g/m3), radiation intensity (ambient vs. 660 lmol/
m2 /s) and planting density (69 vs. 6/m2). Radiationintensity was measured over the plant growing area
within the chamber and is an average. Two identical
growth chambers were used, under the same condi-
tions with the exception of the test variable. In
addition, CO2 levels were maintained at [twice
ambient, i.e. 800 ll CO2 l-1.
Plant cultivation
Seeds of wild type, Guys 13 and CV-N tobacco lines
were sown on the top of Levington ProfessionalMedia (seed and modular compost F2 ? S (sand) pH
5.56) and germinated, with bottom heat, under
containment glass at ambient conditions. After
emergence, supplementary lighting (*150 lmol/m/
s) was provided and seedlings were pricked out into
12 cm square plastic pots in compost mixture (Rich-
moor compost, Whitemoss, Kirby, UK enriched with
osmacote (N:P:K 18:10:11) 4.4 g/l, ScottsMiracle-
Gro, The Netherlands). Seedlings were placed in a
controlled growth chamber at set temperature of 20C
with ambient radiation intensities and day length and
allowed to grow until plants had produced two true
expanded leaves, then transferred into another
GroDome chamber(s) set for experimental parame-
ters. Each individual experiment depending on con-
ditions utilised a GroDome chamber and at least 50replicate plants of each type were grown per exper-
iment. Plants were cultivated, unless density was
variable, at around nine plants per m2 to avoid any
self-shading. The plants were watered twice a day.
There was no regulation of humidity.
Plant sampling
Duplicate leaf discs were sampled either side of the
mid vein from the tip region of leaves avoiding any
secondary veins, using a sharp 7 mm cork-borer. Discswere taken from the top most fully expanded leaf and
from the lowest, visually non-senescent leaf. Pairs of
leaf discs were placed immediately into 1.5 ml
Eppendorf tubes and flash frozen in liquid nitrogen
prior to storage at -80C. For each experimental
growing condition, at least 10 replicate plants were
sampled from both bottom and topleaves of each plant.
After sampling at the end of each experiment,
stems were cut off at soil level. Leaf and stem
material was separated and placed into a drying oven
at 80C for 48 h (until dry) and dry leaf and stemsweights were measured individually.
For specific protein determination and total soluble
protein measurement, frozen leaf discs were homog-
enised in 300 ll ice cold phosphate buffered saline
(PBS) using a mill (Retsch, UK; MM301). The
samples were centrifuged at 20,000g for 3 min and
the supernatants analysed.
Recombinant protein assays
Plant extract supernatants were transferred to micro-titre plates (Immulon, Nunc, UK) pre-coated with a
specific ligand to determine the extractable levels of
functional recombinant protein.
For detection of fully assembled and functional
(antigen-binding) IgG, the microtitre plates were
coated with a recombinant fragment of streptococcal
antigen I/II at 1:5,000 dilution (van Dolleweerd et al.
2003) and blocked with 5% (w/v) non-fat dry milk in
PBS. For detection of functional cyanovirin-N, the
Transgenic Res (2010) 19:241256 243
123
-
8/6/2019 ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals.
4/16
microtitre plates were coated with recombinant HIV
gp120 at 1 lg/ml (EVA 607, NIBSC CFAR) and
blocked with 2.5% bovine serum albumin (Sigma
UK). Plant samples were assayed in duplicate and in
serial dilutions. Incubation was for 2 h at 37C after
which the plates were washed with 0.1% Tween 20
(dH2O).Bound recombinant immunoglobulin was detected
by incubation with a horseradish peroxidase-labelled
goat anti-mouse IgG-gamma chain antiserum
(1:1,000, The Binding Site), for 2 h at 37C, followed
by addition of TMB (3,30,5,50-tetramethylbenzidine,
Sigma, UK) as the substrate. The colour reaction was
stopped with 2 M sulphuric acid, and the absorbance
was determined at 450 nm (Sunrise, Tecan, UK).
Antibody concentration was calculated by compari-
son of binding curves with a pre-existing standard
(Guys 13 hybridoma supernatant).Bound recombinant cyanovirin was detected by
incubation with a CV-N specific polyclonal rabbit
antiserum (courtesy of Dr. Barry OKeefe) at 1:1,000
dilution, followed by a horseradish peroxidase
labelled anti-rabbit IgG antiserum (Sigma, UK) at
1:1,000 dilution. These incubations were for 2 h at
37C or overnight at 4C. The addition and detection
of substrate was as described above.
Total soluble protein assay
Total protein concentrations in soluble plant extracts
were determined by the bicinchonic acid assay (BCA
Protein Assay, Pierce Chemical Co., Rockford, IL).
Statistical analyses
Data were subject to analysis of variance (ANOVA)
using Genstat version 10, least significance difference
(LSD) values for treatment interactions were calcu-
lated at P\ 0.05 level. For testing environmental
effects on inter-plant variability, data was subject toBartletts test of homogeneity.
Results
Sampling variability
An initial requirement was to assess the variability in
sampling from individual plants as a basis for the
ensuing studies. IgG transgenic plants were grown
under a 28C/21C day/night cycle with 18 h day
length, 660 lmol supplementary radiation and 800 ll
CO2 l-1, until they developed five leaves and were
approximately 15 cm high. Ten samples (2 leaf discs
each) were taken from a bottom leaf and a further ten
samples were taken from a top leaf from each of fourplants. The concentrations of functional IgG and total
soluble protein were determined and are shown in
Fig. 1.
The most striking observation was the difference
in IgG concentrations seen between the samples taken
from bottom and top leaves of the same plants. It has
previously been observed that recombinant IgG
concentrations were higher in the uppermost leaves
of transgenic tobacco plants (Stevens et al. 2000) and
this was confirmed here (Fig. 1a). In all four plants,
the differences between the mean extracted IgG ofsamples taken from the top and the bottom leaves
were statistically significant (P\ 0.001). The same
trends between top and bottom leaves were observed
in total soluble protein (TSP) concentrations
(Fig. 1b), although this only reached statistical sig-
nificance for plants 2 and 3 (P\ 0.001).
The difference between top and bottom leaf
concentrations was a consistent finding throughout
this study and is correlatively linked with the stage of
maturation of the plant tissue. It is not due to
significant differences in the mass of the plantsamples, as the bottom leaf discs were invariably
thicker than those taken from top leaves.
The data also indicates the degree of plant to plant
variation in antibody yields (ie comparing the top
leaves from all four plants, or the bottom leaves).
This variation was generally less than the variability
between samples taken from visually similarly loca-
tions on different parts of a single leaf (Fig. 1a),
indicating that plants from this transgenic line
expressed a relatively uniform level of IgG. This
notion was supported by similar findings for totalsoluble protein concentrations between plants, and
between samples from the same leaf (Fig. 1b). The
variability within samples taken from the same leaf
could be due to a number of reasons, the most likely
being anatomical differences across the leaf with
varying amounts of palisade, spongy mesophyll, and
vascular tissues, within a relatively small tissue
sample. The variability would of course be eliminated
at scale-up when whole leaves are sampled. In all
244 Transgenic Res (2010) 19:241256
123
-
8/6/2019 ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals.
5/16
(a) Extracted IgG (ng/ml)
800.00
1000.00
1200.00
1400.00
1600.00
ng/ml
0.00
200.00
400.00
600.00
800.00
IgGn
Bottom TopBottom TopBottom TopBottom Top
Bottom Top Bottom TopBottom Top Bottom Top
(b) Extracted TSP (g/ml)
2000.00
2500.00
Plant 4
Plant 4
Plant 3Plant 2Plant 1
Plant 1
500.00
1000.00
1500.00
TSPug/ml
S l ( 10) M
0.00
Plant 3Plant 2
Samp e n= Mean sd
Plant 1 Bottom leaf 0.033 0.012
Plant 1 Top leaf 0.078 0.013
Plant 2 Bottom leaf 0.035 0.008
Plant 2 Top leaf 0.070 0.011
(c) Extracted IgG (%TSP)
Plant 3 Bottom leaf 0.058 0.029
Plant 3 Top leaf 0.085 0.017
Plant 4 Bottom leaf 0.032 0.017
Plant 4 Top leaf 0.067 0.015
Fig. 1 Variability of IgG
and total soluble protein
TSP yields due to sampling.
a IgG concentration in
processed samples from
each four individual plants.
Data is shown from bottom
andtop
leaves (Eachcolumn contains 10 data
points from one plant. The
mean of those points is
shown as a black square in
each case); b TSP
concentration in the same
processed samples from the
same plants (10 samples
each, and the mean is
shown as a black square);
and c IgG expressed as a
percentage of TSP. Data is
shown as mean and sd for
the top and bottom leafsamples from the same four
plants
Transgenic Res (2010) 19:241256 245
123
-
8/6/2019 ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals.
6/16
subsequent experiments an attempt was made to
minimise this variability by sampling in a reproduc-
ible way from a standard position on each leaf.
Expressing the recombinant IgG expression in
terms of a percentage of the TSP may mitigate the
effects of the sampling variability arising from
differences in sample mass. It also gives an indicationof the purification burden for later downstream
processing steps (Fig. 1c, Hassan et al. 2008). The
results confirmed that the yield of IgG was highest
from the top leaves, and this was statistically
significant for all four plants (P\ 0.001).
Environmental variables have little impact
on recombinant antibody expression
and accumulation
Five environmental variables have been assessed withregards their potential impacts on expression and
accumulation of recombinant IgG MAb. These were
air temperature, day length, compost nitrogen con-
tent, radiation intensity (PAR) and plant density (pot
number per m2) (Table 1). These studies were
possible due to the sensitivity and reproducibility of
environmental control achievable between individual
growth chambers in the GroDome.
For each experiment, all but one of the five
parameters was kept constant. In addition, the CO2
concentration was maintained at 800 ll CO2 l-1
throughout. The experimental duration was varied
between experiments only if the experimental vari-
able under investigation enhanced, or reduced the rate
of biomass accumulation and maturity dramatically.
Experimental duration did not however vary within
each separate experiment. Of necessity, these exper-
iments were performed at different times of the year
(Table 1) and so ambient light and day length varied
across experiments.
In all the experiments, as shown earlier, the yield
of MAb from top leaves was consistently greater thanthat from lower leaves. Yield in these studies is
represented by the concentration of IgG recovered
from each two leaf disc sample. However, with the
exception of the temperature experiment, no signif-
icant differences were observed in expression levels
between plants grown under the different conditions.
In the temperature experiment, plants grown in a day/
night temperature cycle of 23/21C accumulated
approximately double the amount of MAb, in both
bottom and top leaves compared to plants grown on a
32/21C cycle; these differences were significant
(P\0.05).
Environmental variables affect total soluble
protein accumulation differently to IgG
accumulation in transgenic plants
In contrast to the findings for IgG, the concentration
of total soluble protein (TSP) were not consistently
elevated in top leaves as compared with bottom
leaves (Table 2). Indeed, this was only observed
when plants were grown at high temperatures (32/
21C) or at high density (69 plants/m2) irrespective of
the lighting supplied.
In the temperature experiment, a lower TSP at
higher temperature was observed in the bottom
leaves, unlike the fall in IgG concentration at highertemperatures which was also observed in the top
leaves (Table 1).
In the density experiment, plants grown at low (6/
m2) density had consistently higher TSP levels than
those grown at high (69/m2). In this experiment,
supplementary radiation also caused a significant
increase in TSP, as compared with ambient lighting.
It is notable that this experiment was performed in the
summer, when ambient day light and length is
maximal.
This finding confirmed the result in the radiationexperiment in which 660 lmol/m2 /s PAR also
induced higher TSP concentrations than ambient
light.
Day length and compost nitrogen content had no
significant effect on TSP concentration.
The effect of environmental variables
on cyanovirin-N accumulation in transgenic
plants differs from IgG
In a second series of experiments, the effects oftemperature, compost nitrogen content and radiation
intensity on expression and accumulation of a second
recombinant protein, cyanovirin-N (CV-N) were
studied (Table 3).
A notable finding was that the extractable CV-N
concentration was not lower in the bottom leaves in any
of the three experiments, as observed for IgG. Indeed,
in all cases, the concentrations were either the same or
higher, and the differences reached significance after
246 Transgenic Res (2010) 19:241256
123
-
8/6/2019 ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals.
7/16
5 days in the temperature experiment and in the
nitrogen experiment.
In contrast to the IgG transgenic plants, higher
concentrations of CV-N were recovered after 5 days
from plants grown at the higher day temperature
(32C). This was observed in both bottom and top
leaf samples (0.89 vs. 0.63 and 0.82 vs. 0.49;
P\ 0.05). In this experiment, a second series of
Table 1 The effect of five environmental parameters on IgG yield from transgenic plants
IgG Temperature
(C)
Day/night
cycle
(hours)
Radiation
(lmol/
m2/s)
Density
(plants/
m2
)
Nitrogen
(g/m3
)
Time of year Expt.
duration
(days)
Bottom
(IgG
lg/ml)
Top
(IgG
lg/ml)
LSD0.05
Temperature 23/21 18/6 660 16 180 January
February
2008
5 0.13b 0.16a 0.04 on
72 df
*
32/23 18/6 660 16 180 January
February
2008
5 0.05c 0.07c
23/21 18/6 660 16 180 January
February
2008
14 0.09d 0.15ab
32/23 18/6 660 16 180 January
February
2008
14 0.02ce 0.01e
Day-length 28/21 18/6 660 16 180 October
December
2006
35 0.64 0.94 0.45 on
36 df
*
28/21 12/12 660 16 180 October
December
2006
35 0.63 1.092
N2 28/21 18/6 660 16 180 December
March 2007
83 1.62a
4.07b
1.56 on
33 df
*
28/21 18/6 660 16 108 December
March 2007
83 1.48a
2.91ab
Radiation 28/21 18/6 660 16 180 AprilMay
2007
19 1.92a
6.61b
2.46 on
36 df
*
28/21 18/6 Ambient 16 180 AprilMay
2007
19 2.27a
7.20b
Density 28/21 18/6 660 69 180 AugustSeptember
2007
23 0.20a
0.29c
0.05 on72 df
28/21 18/6 660 6 180 August
September
2007
23 0.22ab
0.21a
*
28/21 18/6 Ambient 69 180 August
September
2007
23 0.17a
0.25bc
28/21 18/6 Ambient 6 180 August
September
2007
23 0.17a
0.20a
The cultivation conditions are described for each experiment, and the single modified parameter in each case is identified. The time ofyear the experiment was performed is shown along with the duration of the experiment (time that the plants were subjected to the
specific environmental conditions). The mean IgG yield results are shown individually for bottom and top leaves. Data that are
statistically significant from other data in that experiment are in italics and underlined and marked with a letter (ae), with different
letters denoting statistical significance (P\0.05). The least significant difference (LSD) is shown. The asterisks in the final column
denote groups that were cultivated under the same conditions, except for the experimental duration and time of year
Transgenic Res (2010) 19:241256 247
123
-
8/6/2019 ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals.
8/16
plants was also sampled at maturity on day 14. There
were higher concentrations in the more mature plant
samples, although this only reached significance in
the plants grown at the lower (23C) day-time
temperature (0.63 vs. 1.02 and 0.49 vs. 0.99;
P\ 0.05).
However like IgG, no significant differences were
observed in CV-N concentrations between transgenic
plants grown under different compost nitrogen levels,
or under different radiation intensities.
Environmental conditions affect TSP
accumulation in CV-N transgenic plants
in the same way as IgG transgenic plants
The results for TSP accumulation were consistent
with those found for the IgG plants (Table 4). Thus,
at a higher temperature, the TSP was reduced in the
bottom leaves of plants after 5 days. Surprisingly,
this was not seen after 14 days. There was also a
statistically higher TSP concentration in top leaves
from plants grown under more intense lighting in the
Radiation experiment, but in the bottom leaves there
was no significant reduction in TSP levels.
The effect of environmental variables
on transgenic plant biomass and leaf area
Data for total leaf area and plant biomass in each
experiment are shown for IgG transgenic, CV-N
transgenic and wild-type plants in Table 5. In gen-eral, IgG transgenic plants and wild-type non-trans-
genic plants performed in the same way, in terms of
biomass production. At high temperature (32/21C),
long day length (18/6 h) and ambient radiation, there
was no significant difference in final dry tissue
weights between the two plant lines. However, at low
temperature, short day length and with supplementary
lighting there were statistically significant lower
biomass yields in the IgG plants.
Table 2 The effect of five environmental parameters on TSP yield from IgG transgenic plants
IgG-TSP Time of year Expt. duration
(days)
Bottom
(TSP mg/ml)
Top
(TSP mg/ml)
LSD0.05
Temperature
23/21C February 2008 5 2.12a 2.37a 0.32 on 72 df
32/21C February 2008 5 1.55a
2.15a
23/21C 14 1.90a 2.07a
32/21C 14 1.65ab 1.84a
Day-length
18/6 h OctoberDecember 2006 35 3.11 2.93 1.00 on 36 df
12/12 h OctoberDecember 2006 35 2.41 3.51
N2
180 g/m3
DecemberMarch 2007 83 1.97 2 0.47 on 36 df
108 g/m3
DecemberMarch 2007 83 2.07 1.94
Radiation
660 lmol/m2 /s AprilMay 2007 19 2.75
a 3.67c 0.51 on 36 df
Ambient AprilMay 2007 19 1.95b 2.35ab
Density
660 lmol/m2 /s 69 plants/m
2AugustSeptember 2007 23 0.22
ad0.8
b0.14 on 72 df
660 lmol/m2 /s 6 plants/m
2AugustSeptember 2007 23 1.14
c 1.16c
Ambient 69 plants/m2
AugustSeptember 2007 23 0.10d 0.26a
Ambient 6 plants/m2
AugustSeptember 2007 23 0.36ae 0.43e
The cultivation conditions are the same as shown in Table 1, here only the modified parameter in each case is shown. The time of
year the experiment was performed is shown along with the duration of the experiment (time that the plants were subjected to the
specific environmental conditions). The mean TSP yield results are shown individually for bottom and top leaves. Data that are
statistically significant from other data in that experiment are in italics and underlined and marked with a letter (ae), with different
letters denoting statistical significance (P\0.05). The least significant difference (LSD) is shown
248 Transgenic Res (2010) 19:241256
123
-
8/6/2019 ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals.
9/16
The CV-N plants had a very different growing
habit that was obvious visually. Overall, they took
considerably longer to germinate and establish as
seedlings (usually 4 weeks, compared with 2 weeks
for IgG and WT plants). The established plants were
also more compact and slower growing, which is
reflected in the biomass data. In all experiments, the
CV-N plant biomass at harvest were significantly
lower than WT and IgG plants cultivated under the
same conditions (P\ 0.05).
Table 3 The effect of three environmental parameters on CV-N yield from transgenic plants
CV-N Temperature
(C)
Day/night
cycle
(hours)
Radiation
(lmol/
m2/s)
Density
(plants/
m2
)
Nitrogen
(g/m3
)
Time of year Expt.
duration
(days)
Bottom
(CV-
N lg/ml)
Top
(CV-
N lg/ml)
LSD0.05
Temperature 23/21 18/6 660 16 180 February 2008 5 0.63a 0.49b 0.14 on
72 df
32/21 18/6 660 16 180 February 2008 5 0.89c 0.82c
23/21 18/6 660 16 180 February 2008 14 1.02c 0.99c
32/21 18/6 660 16 180 February 2008 14 0.92c 0.87c
N2 28/21 18/6 660 16 180 December
March 2007
83 0.80a
0.69b
0.08 on
38 df
28/21 18/6 660 16 108 December
March 2007
83 0.81a 0.64b
Radiation 28/21 18/6 660 16 180 AprilMay
2007
19 2.1 2.03 0.35 on
36 df
28/21 18/6 Natural 16 180 AprilMay
2007
19 1.87 1.96
The cultivation conditions are described for each experiment, and the single modified parameter in each case is identified. The time of
year the experiment was performed is shown along with the duration of the experiment (time that the plants were subjected to the
specific environmental conditions). The mean CV-N yield results are shown individually for bottom and top leaves. Data that are
statistically significant from other data in that experiment are in italics and underlined and marked with a letter (ac), with different
letters denoting statistical significance (P\0.05). The least significant difference (LSD) is shown
Table 4 The effect of three environmental parameters on TSP yield from CV-N transgenic plants
CV-N-TSP Time of year Expt. duration
(days)
Bottom
(TSP mg/ml)
Top
(TSP mg/ml)
LSD0.05
Temperature
23/21C February 2008 5 1.39a
1.45a
0.34 on 72 df
32/21C February 2008 5 0.72b
1.17a
23/21C February 2008 14 1.65ac
1.79c
32/21C February 2008 14 1.38a
1.06ab
N2
180 g/m3 DecemberMarch 2007 83 0.55 0.54 0.26 on 36 df
108 g/m3
DecemberMarch 2007 83 0.78 0.8
Radiation
660 lmol/m2 /s AprilMay 2007 19 0.94
a0.80
a0.33 on 36 df
Ambient AprilMay 2007 19 0.91a
0.48b
The cultivation conditions are the same as shown in Table 3, here only the modified parameter in each case is shown. The time of
year the experiment was performed is shown along with the duration of the experiment (time that the plants were subjected to the
specific environmental conditions). The mean TSP yield results are shown individually for bottom and top leaves. Data that are
statistically significant from other data in that experiment are in italics and underlined and marked with a letter (ac), with different
letters denoting statistical significance (P\0.05). The least significant difference (LSD) is shown
Transgenic Res (2010) 19:241256 249
123
-
8/6/2019 ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals.
10/16
With regards the effect of the different environ-
mental variables on each plant line, day time temper-
ature had a variable effect on leaf area in the juvenile
phase over the first 5 days in IgG and WT plants, but
the observed differences were not significant. In the
CV-N plants, a significantly higher leaf area was
achieved in plants grown at the 32/21C regime bothover 5 and 14 days. These differences did not
however, translate to significant differences in leaf
dry weight. In all cases, the higher temperature regime
led to a significant increase in stem dry weight.
Indeed, it was visually evident that the plants grown at
higher temperature reached maturity earlier, which
can be explained by a re-partitioning of carbohy-
drate from leaves into stem extension growth and
flower initiation. Thus growing plant lines at lower
temperatures may slow development and allow pro-
duction of plants with higher leaf:stem ratios, thereby
increasing the proportion of leaf biomass available for
extraction of recombinant protein and reducing the
amount of waste, particularly stem biomass.
Day length had a variable impact on leaf area, but
the differences did not reach significance. However,there was a consistent and significantly higher leaf
and stem dry weight for all the plant lines under the
18/6 h day length schedule.
Leaf area data was not collected from the compost
nitrogen experiment. However, no significant differ-
ences were observed in leaf or stem biomass
measurements, except for the wild-type line which
unexpectedly did not respond to additional compost
nitrogen.
Table 5 The effect of environmental parameters on dry weight and leaf area in IgG and CV-N transgenic and WT plants
Variable Leaf area per plant (cm2
) Dry leaf (g) Dry stem (g)
IgG CV-N WT IgG CV-N WT IgG CV-N WT
Temperature 32/21 (C) 890 979 1,124 7.54 4.70 7.97 0.96 0.77 1.09
5-day sample 23/21 (C) 983 746 998 6.98 4.96 8.76 0.43 0.31 0.68
LSD0.05 54 df 142.3 1.01 0.17
Temperature 32/21 (C) 1,755 1,799 1,815 11.30 9.70 12.62 6.51 4.97 7.71
14-day sample 23/21 (C) 1,903 1,313 1,708 16.10 9.06 15.49 6.49 2.00 6.50
LSD0.05 54 df 276 1.45 0.88
Day-length 18/6 h 2,433 1,467 2,234 16.20 10.70 16.00 11.50 7.24 11.52
12/12 h 2,289 1,657 2,394 11.40 8.60 12.80 7.00 5.72 8.43
LSD0.05 54 df 289.0 1.56 1.22
Nitrogen 180 (g/m3
) 7.20 2.50 4.00 4.80 2.30 3.10
108 (g/m3
) 7.10 2.60 9.30 4.90 2.50 6.50
LSD0.05 54 df 2.10 1.43
Radiation 660 (lmol/m
2
/s) 1,644 1,636 1,941 12.10 9.40 14.30 4.20 2.41 4.80Ambient 1,842 1,552 1,999 5.30 3.40 5.60 2.10 1.06 2.80
LSD0.05 54 df 257.5 1.28 0.72
Density 660 (lmol/m2/s)
69 plants/m2
3.00 3.30 0.90 1.30
660 (lmol/m2/s)
6 plants/m2
7.20 7.90 1.20 1.70
Ambient light
69 plants/m2
0.80 1.40 0.20 0.40
Ambient light
6 plants/m2
1.70 2.60 0.30 0.50
LSD0.05 42 df 0.97 0.25
The cultivation conditions are shown. Dry weights are shown for leaves and stems and represent the total mass for the ten plants
studied. Leaf area data also represents total area for the ten plants combined. The least significant difference (LSD) is shown for each
data set, and those results which are significantly different (P\0.05) are in italics and underlined
250 Transgenic Res (2010) 19:241256
123
-
8/6/2019 ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals.
11/16
In the radiation experiment, supplementary light-
ing at *660 lmol/m2 /s is sufficient to saturate
the photosynthetic assimilation of tobacco plants
(Henkes et al. 2001) and the effect was to more than
double total dry biomass of IgG, WT and CV-N
plants through a large increase in leaf biomass and a
smaller increase in stem dry weight, leading to higherthan average leaf to shoot ratios. No significant
differences however, were seen in mean total plant
leaf areas and the differences in biomass were
attributable to differences in leaf thickness.
A related issue was apparent in the density
experiment with supplementary radiation, where
plants grown at a density of 6 plants/m2 had
consistently higher leaf and stem biomass than their
counterparts grown at 69 plants/m2, with the increase
in leaf biomass being more prominent than that of the
stems. A similar tendency was observed in the plantsgrown under ambient radiation, but this did not reach
significance. At both densities, the plants grown with
supplementary radiation had significantly higher
biomass compared with those grown under ambient
lighting. The addition of supplementary lighting
reduced the impact of planting at higher densities.
Although the highest biomass per plant was achieved
with supplementary lighting and low density plant-
ing, on a batch basis, the overall increase in yield per
area attained with increased planting densities would
compensate for the reduction of yield per plant.
Temperature, day length and nitrogen do not
influence the variability of expression within
transgenic plants, but plant density and radiation
are important
The impact of environmental conditions on the
variability of recombinant protein accumulation and
extraction in different transgenic plants was investi-
gated. In this case, data representing recombinant
protein as a percentage of total soluble protein wasused to minimise any impact of variation in sampling
and processing.
The results for IgG transgenic plants are shown in
Fig. 2. The shaded box contains data between 25 and
75% and the outer bars represent data at the 10 and
90% levels. The mean and median data are repre-
sented by a bold line and a line within the shaded box
respectively. In the temperature experiment (Fig. 2a),
there is a tendency for greater inter-plant variability
in the top leaves at 23/21C as compared with top
leaves grown at 32/21C. This difference did not
however, reach significance. No such difference was
observed in the bottom leaves.
In the day length experiment, a significant differ-
ence (P = 0.002) was observed with higher variabil-
ity observed in bottom or top leaves grown at 12/12 hday length regime (Fig. 2b). Under high nitrogen
levels (Fig. 2c), the variability in top leaves was
significantly greater than in the three other samples
(P = 0.04).
In the density/radiation experiment, more substan-
tial differences were observed (Fig. 2d). Under both
660 lmol supplementary radiation and ambient light-
ing conditions, the inter-plant variability was sub-
stantially decreased under less dense conditions in
both bottom and top leaves (compare columns 1 vs. 3,
2 vs. 4, 5 vs. 7 and 6 vs. 8 P\ 0.001). In the highdensity planting, there was little difference in the IgG
yield in bottom leaves under different radiation
regimes (compare columns 1 vs. 5), but in the top
leaves there was less variation in the plants grown
with supplementary lighting (compare columns 2 vs.
6; P\ 0.001). It was also noteworthy, that under
conditions that favoured limited inter-plant variation,
the mean yield was much lower than achieved under
other conditions.
The results from IgG transgenic plants was
supported by the CV-N transgenic plants (Fig. 3).In the temperature experiment, at the 5-day time
point (columns 14) no differences in variability were
observed. At the 14-day sample however, (columns
58), there was increasing variability in the plants
grown under the higher temperature regime (compare
columns 5 vs. 7 and 6 vs. 8 P\ 0.05). These data
also demonstrate an increase in variation in top leaves
at the higher temperature with time (compare
columns 4 vs. 8; P\0.05).
In the CV-N radiation experiment, supplementary
lighting reduced variability in the CV-N yield in topleaves (P\ 0.05), but no difference was observed in
the bottom leaves.
Discussion
The instinctive approach to growing plants is to
optimise conditions for plant health and vigour (bio-
mass accumulation) based on visual phenotype.
Transgenic Res (2010) 19:241256 251
123
-
8/6/2019 ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals.
12/16
However, although this may lead to larger healthylooking plants, the conditions may not be optimal for
recombinant protein production where other parame-
ters may be important. These include the levels of
protein expression, variability of the protein, the
stability and accumulation of the protein and finally
the extractability of the protein. In this study, we have
focused on recombinant IgG, as this is a major
commercial target for molecular farming (Ma et al.
2005). However in order to determine whether our
observations are specific for IgG or generic forrecombinant proteins, we have also investigated a
second non-immunoglobulin protein that is also
targeted to the plant secretory pathway. Both trans-
genes were expressed from the same vector, using the
same controlling elements including the Cauliflower
mosaic virus 35S promoter, and a murine leader
peptide which directs the transgene product to the
endomembrane system. However, unlike IgG,
which is a large (150 kDa) multimeric glycosylated
1.0
1.2
1.4
(b) Day length(a) Temperature
0.014
0.016
0.018
LSD0.05
IgG(%
TSP)
0.2
0.4
0.6
0.8 LSD0.05Plot 1
IgG%
TSP
0.004
0.006
0.008
0.010
0.012
18hours
: Bott
omleaf
18hours
: Top
leaf
12hours
: Bott
omleaf
12hours
: Top
leaf
0.0
23/21
CBotto
mleaf
23/21
CTop l
eaf
32/21
CBotto
mleaf
32/21
CTop l
eaf
0.000
0.002
(d) Density / Radiation(c) Nitrogen
0.5
0.6
0.7
LSD0.05
0.3
0.4
0.5
LSD0.05
IgG(%TSP)
0.1
0.2
0.3
0.4
Plot 1
IgG(%TSP)
0.1
0.2
High N
2,Bottom lea
f
High
N2, Top lea
f
Low
N2, Bott
om leaf
Low
N2, Top lea
f
0.0
H.D.
660
mol,
Bot le
af
H.D.
660
mol,
Top
leaf
L.D. 660
mol,
Bot le
af
L.D.
660
mol,
Top
leaf
H.D.
ambie
nt, Bo
t leaf
H.D.
ambie
nt,Top
leaf
L.D.
ambie
nt, Bo
t leaf
L.D.
ambie
nt,Top
leaf
0.0
Fig. 2 Effect of cultivation conditions on functional IgG
expression in transgenic tobacco plants grown: a under two
temperature regimes; b under two day length regimes; c with
two different compost nitrogen contents; and d under two
different planting density and two different radiations intensi-
ties. In each case, data is shown for bottom and top leaf
samples (n = 10 in each case). The shaded box represents the
25th and 75th interquartile range, whiskers delineate the 10th
and 90th percentile. The dark line within the box marks the
mean and the fainter line the median. Outlying data points are
represented by dots. LSD0.05 for the interaction between
temperature regime and leaf position is 0.0014 on 36 df.
LSD0.05 for the interaction between day length and leaf
position is 0.097 on 36 df. LSD0.05 for the interaction between
leaf position and nitrogen regime is 0.099 on 36 df. LSD0.05 for
density and lighting regime and leaf position is 0.0621 at 71 df
252 Transgenic Res (2010) 19:241256
123
-
8/6/2019 ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals.
13/16
mammalian protein, CV-N is a small (11 kDa) single
non-glycosylated bacterial polypeptide. Reliable
assays exists in both cases to confirm antigenicity as
well as molecular functionality, and it is important
here that we have always measured functional pro-
tein activity rather than total recombinant protein
concentration.
The post-translational events that affect the con-
centration of protein expression include efficiency of
protein folding, assembly and sub-cellular localisa-
tion. Perhaps surprisingly, environmental parameters
associated with plant stress such as temperature and
day-light hours, which might up-regulate the indig-
enous stress response did not have a consistent and
significant impact on protein concentraton, neither for
IgG nor CV-N.
The stability and accumulation of recombinant
proteins in planta is another important factor. Growing
plants at higher day time temperatures reduced the
amount of IgG that could be recovered by a significant
amount. This is in agreement with earlier findings,
where transgenic tobacco plants grown at 25C
yielded less recombinant IgG than plants grown at
15C (Stevens et al. 2000). This is counter-intuitive, as
tobacco thrives in hot climates, but this is an example
where conditions that are optimal for biomass pro-
duction and alkaloid levels are clearly not suited for
protein production. A likely explanation is that at
higher temperatures, the turnover or degradation of
accumulated IgG increases. Similarly, the reason for
greater IgG levels in top leaves as compared with
bottom leaves is probably due to this mechanism, with
degradative processes increased in bottom leaves,
which are likely to have entered a senescence process.
Indeed, Stevens et al. have demonstrated higher in
vitro degradation of IgG using crude plant extracts
from bottom leaves as compared with higher leaves in
the same plant (Stevens et al. 2000).
This hypothesis is also supported by the findings
from the TSP analysis. No significant differences
were observed between bottom and top leaves, nor
did temperature appear to make an important differ-
ence. TSP represents a mixture of proteins throughout
the plant cell, but at least 50% of the TSP comprises
RUBISCO (Geada et al. 2007) which is compart-
mentalised in the relatively stable environment of the
chloroplast and not exposed to the same array of
proteases encountered by secreted IgG.
1.80.30
(a) Temperature 5/ noitaidaR(b)syad41
1.2
1.4
1.6
LSD0.05
0.20
0.25 LSD0.05
CV-N(%
TSP)
0.4
0.6
0.8
1.0
CV-N(%TSP)
0.05
0.10
0.15
0 mo
l, bott
omleaf
660
mol, t
opleaf
mbien
t, bott
omleaf
Ambie
nt,top
leaf
0.0
0.2
botto
mleaf
,S1
1C
tople
af,S1
botto
mleaf
,S1
1Cto
ple
af,S
1
botto
mleaf
,S2
1C
tople
af,S
2
botto
mle
af,S2
1C
tople
af,S
20.00
1 2 3 4 5 6 7 8
660 6 Am
b
23/21C
bo
23/21C
32/21C
bo
32/2
23/21Cbo
23/21C
32/21Cbo
32/21C
Fig. 3 Effect on cultivation conditions on functional CV-N
expression in transgenic tobacco plants grown: a under two
temperature regimes sampled after 5 (S1) and 14 (S2) days;
and b under supplementary (660 lmol, m2
, s-1
) or ambient
light. In each case, data is shown for bottom and top leaf
samples (n = 10 in each case). The shaded box represents the
25th and 75th interquartile range, whiskers delineate the 10th
and 90th percentile. The dark line within the box marks the
mean and the fainter line the median. Outlying data points are
represented by dots. In a, numbers 18 are provided for ease of
reference to the text; LSD0.05 for the interaction between
temperature regime and leaf position is 0.0295 on 72 df.
LSD0.05 for the interaction between nitrogen regime and leaf
position is 0.2486 on 36 df
Transgenic Res (2010) 19:241256 253
123
-
8/6/2019 ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals.
14/16
It is interesting that CV-N yields were affected
differently by alterations in temperature, but this may
also be explained on the basis of protein stability.
Unlike IgG which is known to be readily degraded in
tobacco plants (Ma et al. 1995), CV-N is extremely
stable and remarkably resistant to proteolytic degra-
dation under a variety of conditions (Colleluori et al.2005). Thus we suggest that the higher CV-N levels
found in plant samples grown at higher temperature
result from the combination of higher recombinant
protein synthesis and lower degradation of accumu-
lated product. Unlike IgG, no significant difference
was observed between the CV-N expression levels in
bottom and top leaves. If the hypothesis for IgG
degradation in ageing leaves is correct, this too could
be explained by the greater stability of CV-N.
As the rate of degradation of accumulated recombi-
nant protein determines the extractable yield, theselection of optimal harvest time is an important
consideration. Here, we have not yet formally assessed
the optimal time of harvest. These are complicated and
lengthy experiments requiring very large amounts of
greenhouse space and will be the subject of future
studies. However, a preliminary indication can be
gained by comparing the results from different exper-
imental groups (marked with an asterisk in Table 1)
that were subject to near identical conditions. For both
IgG and CV-N, the data suggest maximal expression
after 19 days in the environmental chamber. Thisfinding needs to be interpreted with caution and
supported by further studies however, as of practical
necessity, the experiments were performed during
different parts of the year (as indicated). This had an
impact on the germination and development time for
the plantlets before they were introduced into the
controlled environment chamber. In addition, although
the radiation was theoretically saturating at 660 lmol/
m2 /s, there would have been variability in ambient
radiation wavelengths between experiments (although
the variation would have been very small during asingle experiment).
The evaluation of extracted total soluble protein
concentration provided a useful reference against
which to compare IgG and CV-N yield. Most notably,
plants grown under supplementary radiation consis-
tently yielded higher TSP, however this was not
linked to a parallel increase in recombinant protein
yield. Similarly, low density planting increased TSP
yield, but had no effect on IgG yield. Stevens et al.,
reported a parallel effect of climatic conditions on
IgG as compared with TSP (Stevens et al. 2000), but
this was not the case in our studies.
Although day length, radiation and planting den-
sity had little effect on recombinant protein yield,
they did, as expected, have significant impacts on
plant biomass production. Our data indicate thatincreasing day length would favour protein manu-
facture by improving biomass, whilst having little
effect on IgG or TSP yield. In the case of supple-
mentary radiation and low density planting, which
also increase biomass production per plant, more
careful consideration needs to be given, as the
biomass gains must be balanced by the recombinant
protein yield as well as the increased purification
burden (extractability) resulting from a higher rela-
tive burden of total soluble protein.
In addition to studying the effect of differentenvironmental factors on recombinant protein levels,
we were also interested to study the possibility that
different environmental factors might either increase
or decrease the inter-plant variation in these levels.
This is an important factor when considering the
design of a facility for establishment of a uniform and
highly reproducible cGMP compliant production
process. It is also an important consideration for the
definition of batches during manufacture. The effect
of senescence on the glycosylation profile of endog-
enous tobacco proteins has been reported, but it wasalso shown that different growth conditions had no
impact (Elbers et al. 2001). Both the IgG and CV-N
seed lines were homozygous for each transgene locus
and should have been genetically homogeneous. The
uniformity of conditions achievable in the GroDome
allowed us to investigate inter-plant variability under
different cultivation conditions. The results suggest
that inter-plant variation is limited, which contrasts
with the findings of Hassan et al., (Hassan et al. 2008),
but is probably explained by the level consistency of
environmental conditions that is achieved in theGroDome, as compared with a standard laboratory
growth facility. This variation was largely unaffected
by temperature and compost nitrogen content. How-
ever, short day lengths and high plant density (69/m2)
significantly increase variability. The effect of high
plant density could be mitigated to some extent in top
leaves by supplementary radiation, but not in bottom
leaves. Together these results point to light availabil-
ity being the most important factor for ensuring
254 Transgenic Res (2010) 19:241256
123
-
8/6/2019 ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals.
15/16
uniformity of recombinant protein yield in transgenic
plants.
Overall, the selection of optimal cultivation con-
ditions for protein production in transgenic plants is
complex. The only significant effect on IgG and CV-N
yield was observed under different day-time temper-
ature conditions. In the case of IgG, although higheryields were observed at a lower growing temperature,
using higher temperatures may reduce the amount of
time required for plants to attain sufficient maturity
and allow increased numbers of crops to be grown
each year. Extractability and purification burden are
important considerations, as the ratio of recombinant
protein to total protein needs to be compatible with
purification processes. Thus, the use of supplementary
lighting should be balanced in view of the finding that
it resulted in a significant increase in TSP, but not of
IgG or CV-N. Increasing the photo-period from 12/12to 18/6 h had an important effect on increasing leaf
biomass per plant without affecting purification
burden and in decreasing inter-plant variability.
However, although increasing planting density also
increased biomass per unit area, and the addition of
supplementary lighting mitigated the impact of higher
density planting on the yield per plant, these condi-
tions also increased inter-plant variability in terms of
recombinant protein yield.
An important challenge to be faced in using
tobacco for PMP production will be to control stemelongation and flower development. It is envisaged
that only leaves will be harvested for PMP extraction,
therefore without control of stem growth and flower
formation a diversion of resources into the growth of
non-usable plant product will impact on leaf biomass
production and lead to an increase in the amount of
GM plant waste requiring disposal through autoclav-
ing or other approved channels.
Finally, although the IgG transgenic plants
behaved broadly similarly to the non-transgenic wild-
type plants, a clear difference was observed with CV-N transgenic plants. It is likely that subtle phenotypic
alterations would result from the demands of
constitutive protein expression, particularly under
sub-optimal cultivation conditions. The additional
differences seen in CV-N transgenic plants may be
due to transgene positional effects, or due to direct
effects of CV-N in planta. Given its mannose-binding
properties, it is quite possible that the expression of
CV-N in the plant endomembrane system could affect
the synthesis and/or targeting of other plant
glycoproteins.
Acknowledgments The authors would like to acknowledge
DTI (TP/3/BIO/6/I/17346), Plant Vaccines Ltd., the EU Pharma-
Planta Integrated Project and the Hotung Foundation for
supporting the project. Also, Dr. Barry OKeefe for providing
CV-N specific antiserum, and Dr. Gillian Arnold for statisticalsupport. We also thank Mick Buss for supervising the
horticultural operations and Mike Davies for technical support
at EMR.
References
Boyd MR, Gustafson KR, McMahon JB, Shoemaker RH,
OKeefe BR, Mori T, Gulakowski RJ, Wu L, Rivera MI,
Laurencot CM, Currens MJ, Cardellina JH, Buckheit RW
Jr, Nara PL, Pannell LK, Sowder RC, Henderson LE
(1997) Discovery of cyanovirin-N, a novel human
immunodeficiency virus-inactivating protein that bindsviral surface envelope glycoprotein gp120: potential
applications to microbicide development. Antimicrob
Agents Chemother 41:15211530
Colleluori DM, Tien D, Kang F, Pagliei T, Kuss R, McCor-
mick T, Watson K, McFadden K, Chaiken I, Buckheit RW
Jr, Romano JW (2005) Expression, purification, and
characterization of recombinant cyanovirin-N for vaginal
anti-HIV microbicide development. Protein Expr Purif
39:229236
Elbers IJ, Stoopen GM, Bakker H, Stevens LH, Bardor M,
Molthoff JW, Jordi WJ, Bosch D, Lommen A (2001)
Influence of growth conditions and developmental stage
on N-glycan heterogeneity of transgenic immunoglobulin
G and endogenous proteins in tobacco leaves. PlantPhysiol 126:13141322
Geada D, Valdes R, Escobar A, Ares DM, Torres E, Blanco R,
Ferro W, Dorta D, Gonzalez M, Aleman MR, Padilla S,
Gomez L, Del CN, Mendoza O, Urquiza D, Soria Y, Brito
J, Leyva A, Borroto C, Gavilondo JV (2007) Detection of
Rubisco and mycotoxins as potential contaminants of a
plantibody against the hepatitis B surface antigen purified
from tobacco. Biologicals 35:309315
Hassan S, van Dolleweerd CJ, Ioakeimidis F, Keshavarz-
Moore E, Ma JK (2008) Considerations for extraction of
monoclonal antibodies targeted to different subcellular
compartments in transgenic tobacco plants. Plant Bio-
technol J 6:733748
Henkes S, Sonnewald U, Badur R, Flachmann R, Stitt M
(2001) A small decrease of plastid transketolase activity
in antisense tobacco transformants has dramatic effects on
photosynthesis and phenylpropanoid metabolism. Plant
Cell 13:535551
Ma JK, Lehner T, Stabila P, Fux CI, Hiatt A (1994) Assembly
of monoclonal antibodies with IgG1 and IgA heavy chain
domains in transgenic tobacco plants. Eur J Immunol
24:131138
Ma JK, Hiatt A, Hein M, Vine ND, Wang F, Stabila P, van DC,
Mostov K, Lehner T (1995) Generation and assembly of
secretory antibodies in plants. Science 268:716719
Transgenic Res (2010) 19:241256 255
123
-
8/6/2019 ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals.
16/16
Ma JK, Barros E, Bock R, Christou P, Dale PJ, Dix PJ, Fischer
R, Irwin J, Mahoney R, Pezzotti M, Schillberg S, Sparrow
P, Stoger E, Twyman RM (2005) Molecular farming for
new drugs and vaccines. Current perspectives on the
production of pharmaceuticals in transgenic plants.
EMBO Rep 6:593599
Sexton A, Drake PM, Mahmood N, Harman SJ, Shattock RJ,
Ma JK (2006) Transgenic plant production of Cyanovirin-
N, an HIV microbicide. FASEB J 20:356358
Smith R, Lehner T, Beverley PC (1984) Characterization of
monoclonal antibodies to Streptococcus mutans antigenic
determinants I/II, I, II, and III and their serotype speci-
ficities. Infect Immun 46:168175
Sparrow PA, Irwin JA, Dale PJ, Twyman RM, Ma JK (2007)
Pharma-planta: road testing the developing regulatory
guidelines for plant-made pharmaceuticals. Transgenic
Res 16:147161
Spok A, Twyman RM, Fischer R, Ma JK, Sparrow PA (2008)
Evolution of a regulatory framework for pharmaceuticals
derived from genetically modified plants. Trends Bio-
technol 26:506517
Stevens LH, Stoopen GM, Elbers IJ, Molthoff JW, Bakker HA,
Lommen A, Bosch D, Jordi W (2000) Effect of climate
conditions and plant developmental stage on the stability
of antibodies expressed in transgenic tobacco. Plant
Physiol 124:173182
Valdes R, Gomez L, Padilla S, Brito J, Reyes B, Alvarez T,
Mendoza O, Herrera O, Ferro W, Pujol M, Leal V, Lin-
ares M, Hevia Y, Garcia C, Mila L, Garcia O, Sanchez R,
Acosta A, Geada D, Paez R, Luis VJ, Borroto C (2003)
Large-scale purification of an antibody directed against
hepatitis B surface antigen from transgenic tobacco plants.
Biochem Biophys Res Commun 308:94100
van Dolleweerd CJ, Chargelegue D, Ma JK (2003) Charac-
terization of the conformational epitope of Guys 13, a
monoclonal antibody that prevents Streptococcus mutans
colonization in humans. Infect Immun 71:754765
256 Transgenic Res (2010) 19:241256
123