Post on 06-Jun-2020
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
“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)
The Future of Plant Biotechnology
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?
Source: R. Fraley (1994)
agronomic
traits
food
processing
pharmaceuticals
1995 2000 2005 2010
B$10.0
B$ 6.0
B$ 3.0
B$ 2.0
new
products
specialty
chemicals
Plant-Made Pharmaceuticals:Entered Market Phase
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
most recent
trials rely on
transient
production
technology!
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-gall-
inducing 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:
magnICON®:
CPPOL MP
POL
agrodelivery:
UV
magnICON®: the Ultimate Yield, Speed
GFP
0
20
40
60
80
100
120
140
160
180
200
0 2 4 6 8 10 12
days post infiltration
flu
orescen
ce u
nits
pICH18711 leaf a
pICH18711 leaf b
pICH16707 leaf a
pICH16707 leaf b
state of the art magnifection
4-7 g protein per /kg of leaf biomass
or up to 80% TSP
4-7 days
TMV-Protein A nanoparticles
Werner et al., 2006
Yersinia antigens
Santi et al., 2006
Aprotinin
Bayer’s first Batch of a PMP:
100mg of purified Aprotinin from Tobacco
magnICON®: Extreme Expression Levels (up to 80% TSP or 7g/kg)
NHL patient IgG1
Bendandi et al., 2010
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
UV
KBP/Bayer Deal:
Pilot plant,
1.2 ton biomass/day
The ICON process is
fully scalable, requires
growing plants in trays
ideal for high-cost products,
e.g. biopharmaceuticals, vaccinesideal for low-cost products,
e.g. industrial enzymes,
agronomic traits
ideal for high-volume products,
e.g. biomaterials, antimicrobials
ethanol-inducible amplification
transfection using vacuum infiltration transfection using spraying
magnICON®
NOMADIC®
NOMADIC® Technology:
Choice of Production Platforms
magnICON®
transgenic
seed biopriming
ideal for agronomic traits
NOMADIC®
Cost of Enzyme, Capital
and Operating Expenses
SuperPro Designer® v.8.5 (Intelligen)
modeling software:
- process stimulation
- flowsheet development
- mass energy balances
- equipment sizes
- batch scheduling, etc.
Daniel et al., 2014
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
sugar
• Natural non-antibiotic antibacterials
new product concepts, low cost and
manufacturing speed essential, greener
Mapp Biopharmaceutical:
Ebola Immunotherapy
Phase I-II studies completed
Mapp therapeutics pipeline:
Ebola virus
Marburg virus
Junin virus
17
new product concept
manufacturing speed essentialsame concept:
antibacterials
Ann Depicker’s group
Rituximab
(6,7 B $)
Trastuzumab
(5,5 B $)
Infliximab
(8,0 B $)
Icon Icon IconOriginator Originator Originator
“It ain’t bragging, if you’ve done it!” attributed to Walt Whitman
Biosimilars/Biobetters From Plants
existing product concept
essential improvements
lower cost of goods
Xyl-/Fuc- Rituximab: Removal of B-cells in Transgenic Mice
%
RNAi 7xKOwt
days
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-fucose
anti-xylose
• 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
One third of people are overweight,
0.5 Billion are obese,
primarely due to sugar consumption
leaf tissue (7dpi) extracted in
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
S: BSA-Standard (1mg/ml) in Lämmli + ME, 3µl
= 3µg
M NC 0‘ 10‘ 20‘ 40‘ 60‘ S M V
C
THM
2
extracted in water pH 4 Acetat
pH4
lane 10 and 11:
sample from Acetat extraction after 60min at 60°C
(from 16.10.2013, stored at 4°C)
V = vortex before taking sample
C = centrifuged before taking sample
CGE analysis
Calculated yield:
1mg THM / gram leaf tissue (partially purified)
Thaumatin-2: Expression Yield
and Purification by Heat
- 1.5 g thaumatin
per kg fresh leaf
- 30-50% tsp
- correct taste
Per capita Sugar 21 kg/y (34 kg in USA)
Thaumatin per capita 7-10 g/y or >0.05%
2016 global Sugar production: 171.000.000 ton;
Global Thaumatin: 80.000 ton or >0.05%
2016 sugar production area: 12.000.000 Ha
Global Thaumatin area needed: 570.000 Ha or 5%
Bacterial pathogens:
Campylobacter spp.*
Escherichia coli *
Listeria monocytogenes
Salmonella spp. *
Bacillus cereus
Clostridium spp.
Shigella spp.*
Vibrio spp.*
Staphylcoccus aureas
Enterococcus spp.
Yersinia spp.*
Viral pathogens:
Hepatitis A
SARS
Rotaviruses
Norovirus
newly emerging
viruses
Parasitic pathogens:
Nematodes
-Ascaris
-Trichinella
Platyhelmints
Protozoa
-CryptosporidiaNewell et al 2010
Foodborne Diseases
* Gram-negative bacteria
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
Pathogen Illnesses Deaths
Norovirus 677 million 214 thousand
E. coli
ETEC
233 million 73 thousand
E. coli
EPEC
81 million 121 thousand
Shigella 188 million 64 thousand
Total 1800 million 600 thousand
Pires et al., 2015
‘The United States spends $500 million
per victim of terrorism, and piddling
$10,000 per cancer death.’
‘Food is a mortal menace. Every year,
one in six Americans gets sick, and 3,000
die from food-borne illness. Your burger
is a bigger threat than radical Islam.’
T. Egan, Intern. N. Y. Times, June 6-7, 2015
CDC USA: E. Coli Outbreaks
• 2016 0121 flour
• 2016 O157 alfalfa sprouts
• 2015 0157:H7 chicken salad
• 2014 0121 raw clover sprouts
• 2014 0157:H7 ground beef
• 2013 0157:H7 ready-to-eat-salads
• 2013 0121 frozen food products
• 2012 0157:H7 organic spinach
• 2012 O26 raw clover sprouts
• 2011 O157:H7 romaine lettuce
• 2011 0104 organic bean sprouts
• 2011 O157:H7 bologna
• 2011 O157:H7 in-shell hazelnuts
• 2010 0157:H7 cheeses
• 2010 O145 romaine lettuce
• 2010 O157:H7 beef
• 2009 O157:H7 beef
• 2009 O157:H7 beef
• 2009 O157:H7 cookie dough
• 2008 O157:H7 beef
• 2007 O157:H7 pizza
• 2007 O157:H7 beef
• 2006 O157:H7 fresh spinach
source: www.cdc.gov/ecoli/outbreaks.html26
Antibacterial Colicin Family
• 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 trans-
membrane 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)
T
C
R
27
Plant Expression of All Known Colicins
12% SDS-PAGE and Coomassie staining, 7.5 µl of TSP extracts corr. to 1.5 mg FW
wt wt
95
72
55
43
34
26
95
72
55
43
34
26
* * * * * *
*
*
*
*
** *
* *
*
*
*
* *
*
all colicins (except colE5) well expressed
Expression range 1.5-8.3 g/kg or 13-50% TSP
in planta colicin
expression –I
(estimated)
in planta colicin
expression –II
(estimated)
%
TSP
Yield(mg colicin/
g FW)
% TSPYield(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
Colicin M causes a
drastic reduction of living
cells in broth culture for
all analyzed protein
concentration at all
analyzed timpoints!
E. coli O157:H7
3-5 log reduction
in CFU counts!
Colicins: E.coli Growth
Reduction
0
1
2
3
4
Effect of ColM on E. coli O157:H7 on Fresh Steak Meat
log
cfu
/g o
f m
ea
t
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
Reduction of E. coli O157:H7 cells (Δlog)
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
1977, CPU 1 Mhz, 4 KB memory
GM plant
Transgenic Versus Transient Process
Transiently modified plant
2017, 8.4 GHz CPU, 10 TB memory
2017, Flash drive, 512 GB memory
GM Agrobacterium
3 KB
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’