Plant-Made Pharmaceuticals and
Biomaterials:
Novel Industrial Processes & Products
Relevant to Developing Countries
Yuri Gleba
Nomad Bioscience GmbH, Munich/Halle
IPBO Event, Gent, May 17, 2017
The Future of Plant Biotechnology
“Ultimately, the world will obtain
most of its food, fuel, fiber,
chemical feedstocks, and some of
its pharmaceuticals from
genetically altered vegetation and
trees.”
P.H. Abelson, “A Third Technological Revolution”,
Science, 279:2019 (1998)
New Green Revolution!
• 2015 global area: 180 M Ha (>12% of total 1.5 B Ha),
grew at double-digit rate for 19 consecutive years
• 2015 global market value of GM crops: over $15.7 B
• 2015 – 18 million farmers directly benefiting
• 28 countries grow, 90% in just five: USA, Argentina,
Brazil, Canada and India, (but also 5 EU countries)
• Americans have transgenics in their diet for 20 years
• No ill effects on health or environment found
• Just four crops: soybean, corn, cotton, rape seed
• Just two traits: herbicide tolerance, insect resistance
• Just two companies making serious money on it
ISAAA , 2014
New Green Revolution?
B$10.0
B$ 6.0
new
products
pharmaceuticals
food
processing
B$ 3.0
B$ 2.0
specialty
chemicals
agronomic
traits
1995
2000
2005
2010
Source: R. Fraley (1994)
Plant-Made Pharmaceuticals:
Entered Market Phase
most recent
trials rely on
transient
production
technology!
Several products advanced:
• Glucocerebrosidase/Protalix, approved
• Anti-caries Mab/Planet, approved
• Fabry Disease Therapy/Protalix, Phase I-II
• Anti-TNF Therapy/Protalix, Phase I
• Lactoferrin/Ventria, Phase I-II
• Anti-HIV Mab PharmaPlanta, Phase I
• NHL Vaccine/Icon-Bayer, Phase I
• Influenza Vaccine/Medicago, Phase I-II
• Influenza Vaccine/iBio, Phase I
• Anti-Ebola Mab, Mapp, Phase I-II
Financial support:
• Protalix, IPO, US$117 M cap, deal with Pfizer
• Icon/Nomad, over US$175 M invested by Bayer
• Icon/Nomad, US$85 M deal with Denka
• Medicago, deal with Philip Morris, US$357 M deal with Mitsubishi
Tanabe Pharma
• DARPA program for PMP, over US$250 M
SWOT Analysis
Animals
Yeasts CHO cells
Plants
E. coli
Plant cells
Technology Principle
• Van Larebeke N, Engler G, Holsters M, Van der Elsaker
S, Zaenen I, Schilperoort RA, Schell J, Large plasmid in
Agrobacterium tumefaciens essential for crown-gallinducing ability. Nature 252, 169 (1974)
• Van Larebeke N, Genetello C, Schell J, Schilperoort RA,
Hermans AK, Van Montagu M, Harnalsteens JP,
Acquisition of tumour-inducing ability by non-oncogenic
agrobacteria as a result of plasmid transfer. Nature, 255,
742 (1975)
Technology: Deconstructed Virus
Delivered by Agrobacterium
viral vector:
POL
MP
CP
UV
magnICON®:
agrodelivery:
POL
magnICON®:
the Ultimate Yield, Speed
GFP
4-7 g protein per /kg of leaf biomass
or up to 80% TSP
4-7 days
200
fluorescence units
pICH18711 leaf a
180
pICH18711 leaf b
160
pICH16707 leaf a
pICH16707 leaf b
140
120
100
80
60
40
20
0
0
2
4
6
days post infiltration
state of the art magnifection
8
10
12
magnICON®: Extreme Expression
Levels (up to 80% TSP or 7g/kg)
TMV-Protein A nanoparticles
Werner et al., 2006
NHL patient IgG1
Bendandi et al., 2010
Bayer’s first Batch of a PMP:
100mg of purified Aprotinin from Tobacco
Aprotinin
Yersinia antigens
Santi et al., 2006
cGMP Facility in Halle,
Germany
Plant-based
GMP facilities:
Icon/Denka
KBP/R.J. Reynolds
Protalix
Medicago/M-Tanabe
Greenovation
Fraunhofer Aachen
Fraunhofer USA
Caliber/iBio
magnICON®: Large-Scale
Vacuum Infiltration
The ICON process is
fully scalable, requires
growing plants in trays
UV
KBP/Bayer Deal:
Pilot plant,
1.2 ton biomass/day
NOMADIC® Technology:
Choice of Production Platforms
magnICON®
NOMADIC®
transfection using vacuum infiltration
ideal for high-cost products,
e.g. biopharmaceuticals, vaccines
magnICON®
transgenic
ethanol-inducible amplification
ideal for high-volume products,
e.g. biomaterials, antimicrobials
transfection using spraying
ideal for low-cost products,
e.g. industrial enzymes,
agronomic traits
NOMADIC®
seed biopriming
ideal for agronomic traits
Cost of Enzyme, Capital
and Operating Expenses
Daniel et al., 2014
SuperPro Designer® v.8.5 (Intelligen)
modeling software:
- process stimulation
- flowsheet development
- mass energy balances
- equipment sizes
- batch scheduling, etc.
Green Biomass Versus Seed:
15-100X Better Acre Utilization
Corn seed
Seed promoter
3-6 t/ha seed
max. 15-30kg API/Ha
1st gram: 2 years
Tobacco leaf
CP promoter
100.-300. t/ha leaves
max. 500-1500kg API/Ha
1st gram: 7 days
But: 10X more biomass
to process
Plant-Made Medicines & Foods
• Novel antivirals for rapid response during
outbreaks (e.g. Ebola, Zica)
• Inexpensive plant-made vaccines
• Inexpensive ‘biobetter’ therapeutic
antibodies
• Non-caloric natural sweeteners to replace
new product concepts, low cost and
sugar
manufacturing speed essential, greener
• Natural non-antibiotic antibacterials
Mapp Biopharmaceutical:
Ebola Immunotherapy
Mapp therapeutics pipeline:
Ebola virus
Marburg virus
Junin virus
same concept:
antibacterials
Ann Depicker’s group
new product concept
manufacturing speed essential
Phase I-II studies completed
17
Biosimilars/Biobetters From Plants
“It ain’t bragging, if you’ve done it!”
attributed to Walt Whitman
Icon
Originator
Infliximab
(8,0 B $)
Icon
Originator
Icon
Originator
existing product concept
essential improvements
Trastuzumab
Rituximab
lower cost of goods
(5,5 B $)
(6,7 B $)
Xyl-/Fuc- Rituximab: Removal
of B-cells in Transgenic Mice
%
days
anti-fucose
wt
RNAi
7xKO
mice model – C57 BL6 containing human CD20 transgene;
mAb doze – 100mkg/day 0;
Flow cytometry measurement of circulating B-cells was
done at day 0; 1; 2 and 7 after treatment with mAb
anti-xylose
Thaumatin
One third of people are overweight,
0.5 Billion are obese,
primarely due to sugar consumption
• 22 kDa protein; 207 amino acids; 8 disulphide bonds;
not glycosylated; water soluble; resistant to heating;
stable under acidic pH; easily purified and crystallized
• Natural source: fruit arils of Thaumatococcus daniellii
• 100,000 times as sweet as sucrose on molar basis;
2000-3000 times sweeter than sucrose on w/w basis
• Introduced in early 70ies by Tate & Lyle
• Approved in USA, EU, Japan, etc., as sweetener,
flavour modifier
• Limited supply of natural plant substance (160 t/year
in 2016), price of bulk substance $250/kg
• Microbial production not competitive
• NOMAD’s proposition:
unlimited supply of recombinant Thaumatin;
significantly lower COGs; GRAS approval in USA in 2018
Thaumatin-2: Expression Yield
and Purification by Heat
leaf tissue (7dpi) extracted in
-
1.5 g thaumatin
per kg fresh leaf
30-50% tsp
correct taste
1.) 3vol 50mM Acetat, 150mM NaCL, pH 4
incubated at 60°C for 0, 5, 10, 20, 40 and 60min
in water bath, vortex before taking sample
2.) 3vol water pH 4
incubated at 60°C for 0, 10, 20, 40 and 60min
in water bath, centrifuge before taking sample
- crude extract mixed 1:2 with 2x Lämmli
- 10µl (corresponding to 1.7 mg leaf tissue )
loaded on 15% SDS gel
M
NC
0‘
10‘
20‘
40‘
60‘
S
M
V
C
Per capita Sugar 21 kg/y (34 kg in USA)
lane 10 and 11: per capita 7-10 g/y or >0.05%
Thaumatin
sample from Acetat extraction after 60min at 60°C
(from 16.10.2013, stored at 4°C)
2016
global Sugar production: 171.000.000 ton;
V = vortex before taking sample
C = centrifuged before taking sample
Global
Thaumatin:
80.000
ton or >0.05%
S: BSA-Standard
(1mg/ml) in Lämmli
+ ME, 3µl
= 3µg
2016sugar
production
area: 12.000.000 Ha
Calculated
yield:
extracted in water pH 4
1mg THM / gram leaf tissue (partially purified)
Global Thaumatin area needed: 570.000 Ha or 5%
THM
2
Acetat
pH4
CGE analysis
Foodborne Diseases
Bacterial pathogens:
Campylobacter spp.*
Escherichia coli *
Listeria monocytogenes
Salmonella spp. *
Bacillus cereus
Clostridium spp.
Shigella spp.*
Vibrio spp.*
Staphylcoccus aureas
Enterococcus spp.
Yersinia spp.*
* Gram-negative bacteria
Newell et al 2010
Viral pathogens:
Hepatitis A
SARS
Rotaviruses
Norovirus
newly emerging
viruses
Parasitic pathogens:
Nematodes
-Ascaris
-Trichinella
Platyhelmints
Protozoa
-Cryptosporidia
Rise and Fall of Antibiotics
• 1999 US study: the introduction of antibiotics in 1936 caused deaths in
the US to fall by 220 per 100,000 people within 15 years. All other
medical technologies combined over the next 45 years reduced deaths by
only 20 per 100,000 people
• The golden age of antibiotics took place in the 1930s to 1970s, with at
least 11 new classes discovered; since then, only two new classes of
antibiotics
• Antibiotic resistance potentially puts everyone else at higher risk
• Dame Sally Davies: "Antimicrobial resistance poses a catastrophic threat.
Any one of us could go into hospital in 20 years for minor surgery and die
because of an ordinary infection that can’t be treated byantibiotics.“
• About 25,000 patients a year die in the European Union from an
infection caused by an MDR bacterium – and on current trends this is
predicted to grow to 390,000 a year by 2050.
Foodborne Diseases Worldwide, 2010
‘The
United States
spends $500Deaths
million
Pathogen
Illnesses
per
victim of terrorism,
Norovirus
677 millionand piddling
214 thousand
$10,000
death.’ 73 thousand
E. coli per cancer
233 million
ETEC
‘Food
menace. Every
year,
E. coliis a mortal
81 million
121 thousand
one
in six Americans gets sick, and 3,000
EPEC
die
from food-borne
illness. Your
burger
Shigella
188 million
64 thousand
is Total
a bigger threat
radical
Islam.’
1800than
million
600
thousand
T. Egan, Intern. N. Y. Times, June 6-7, 2015
Pires et al., 2015
CDC USA: E. Coli Outbreaks
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
2016
2016
2015
2014
2014
2013
2013
2012
2012
2011
2011
2011
2011
2010
2010
2010
2009
2009
2009
2008
2007
2007
2006
0121
O157
0157:H7
0121
0157:H7
0157:H7
0121
0157:H7
O26
O157:H7
0104
O157:H7
O157:H7
0157:H7
O145
O157:H7
O157:H7
O157:H7
O157:H7
O157:H7
O157:H7
O157:H7
O157:H7
flour
alfalfa sprouts
chicken salad
raw clover sprouts
ground beef
ready-to-eat-salads
frozen food products
organic spinach
raw clover sprouts
romaine lettuce
organic bean sprouts
bologna
in-shell hazelnuts
cheeses
romaine lettuce
beef
beef
beef
cookie dough
beef
pizza
beef
fresh spinach
26
source: www.cdc.gov/ecoli/outbreaks.html
Antibacterial Colicin Family
T
R
•
•
•
•
•
Colicins are a class of bacteriocins –
antibiotic proteins- produced by and toxic
to some strains of Escherichia coli.
Colicins are released to reduce
competition from other bacterial strains.
Pore-forming colicins are transmembrane proteins that depolarize the
cytoplasmic membrane, leading to
dissipation of cellular energy.
Colicins may also act as nuclease to
hydrolyze DNA or RNA of the target cell,
or they inhibit cell wall synthesis.
Colicins have a 3 domain structural
design:
–
–
–
N terminus Translocation domain (T)
Receptor binding domain is at the center of
the peptide (R)
C terminus Cytotoxic domain (C)
C
27
Plant Expression of All Known Colicins
in planta colicin
expression –I
(estimated)
Yield
%
TSP
wt
wt
95
95
72
55
*
*
*
*
*
*
*
72
*
55
*
43
34
26
*
43
*
*
*
*
*
*
*
*
*
(mg colicin/
g FW)
in planta colicin
expression –II
(estimated)
Yield
% TSP
(mg colicin/
g FW)
ColE2/ImmE2
20
2.3
17
2.6
ColE3
10
0.95
10
1.25
ColE5/ImmE5
-
-
-
-
ColE6/ImmE6
38
4.37
25
4.0
ColE7/ImmE7
13
1.5
14
2.0
ColE8/ImmE8
13
1.6
10
1.6
ColE9/ImmE9
13
1.7
17
2.8
ColD/ImmD
7
1.0
10
1.5
DF13/ImmDF13
25
3.25
20
3.3
ColA
15
1.2
15
2.0
ColN
10
0.85
7
0.98
ColS4
20
2.8
19
2.9
ColK
20
2.9
50
7.8
Col5
25
2.75
50
8.3
Col10
20
2.8
38
6.7
ColU
25
2.9
25
3.5
ColR
25
2.37
25
3.3
Col28b
25
2.75
17
2.1
ColY
20
1.7
15
2.0
ColB
10
0.7
25
0.7
ColIa
25
2.13
20
3.9
ColIb
33
3.3
20
3.0
ColM
38
3.99
30
4.7
*
34
26
*
12% SDS-PAGE and Coomassie staining, 7.5 µl of TSP extracts corr. to 1.5 mg FW
 all colicins (except colE5) well expressed
 Expression range 1.5-8.3 g/kg or 13-50% TSP
Colicins: E.coli Growth
Reduction
E. coli O157:H7
Colicin M
causes a
drastic reduction of living
cells in broth culture for
all
analyzed
protein
concentration
at
all
analyzed timpoints!
3-5 log reduction
in CFU counts!
Effect of ColM on E. coli O157:H7
on Fresh Steak Meat
log cfu/g of meat
4
3
2
1
0
Reduction of E. coli O157:H7 cells (Δlog)
1h, 10°C 1d,
10°C
3d, 10°C
Comparison no/carrier treatment
-0.073
-0.0248
-0,7
Comparison no/colicin treatment
2.3
2.7
2.6
Comparison carrier/colicin treatment:
2.3
3.0
3.26
30
First GRAS Regulatory Approval
for NOMAD’s Colicins in USA
• "GRAS" is an acronym for the phrase Generally
Recognized As Safe under sections 201(s) and 409 of
the U.S. Federal Food, Drug, and Cosmetic Act.
NOMAD’s R&D Pipeline, 2017
(Antimicrobials)
Antibacterial/antiviral
Pathogen
Product candidates
Colicins
Escherichia coli EHEC
Food additives/proc. aids
Salmocins
Salmonella enteridis
Food additives/proc. aids
Endolysins
Listeria monocytogenes
Food additives/proc. aids
Endolysins
Clostridium perfringens
Food additives/proc. aids
Endolysins
Campylobacter jejunii
Food additives/proc. aids
Pyocins
Pseudomonas
aeruginosa
Pharmaceuticals
Endolysins
Clostridium difficile
Pharmaceuticals
Bacteriocins
To be defined
Oral Pharmaceuticals
Bacteriocins
To be defined
Oral Pharmaceuticals
Mabs, anti-Norovirus
Norovirus
Oral Pharmaceuticals
Transgenic Versus Transient Process
2017, Flash drive, 512 GB memory
2017, 8.4 GHz CPU, 10 TB memory
3 KB
1977, CPU 1 Mhz, 4 KB memory
GM Agrobacterium
Transiently modified plant
GM plant
Where Do We Go From Here?
“I offer a prediction: the early twenty-first century
is going to see a struggle between
information technology and biotechnology on the one hand
and environmental degradation on the other.
Biotechnology is going to be our most powerful tool.
It will let us miniaturize things, avoid waste, and produce
more value without producing and processing more stuff.
The substitution of information for stuff
is essential to sustainability.”
R. Shapiro, Monsanto, CEO,
1997 interview to ‘Harvard Business Reviews’