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
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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 2016sugar 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’
