1. 1
Welcome

2. Production of
antibodies from
Cost per gram (USD)
Mammalian cell 1000.00
Transgenic plants 200.00
According to Monsanto’s Integrated
Protein Technologies
2

3. Plant biopharming: applications,
importance and its usage
Kalilu S. Donzo
2013-11-199
CPBMB
COH, Vellanikkara
KAU
3

4. Outline
 Concept
 General strategy in biopharming
 Different production systems
 Downstream processing
 Applications
 Case study
 Biosafety issues
 Conclusion
 Future lines
4

5. Concept
 Biopharming
Large scale production of recombinant proteins,
including therapeutics and industrial proteins, in
transgenic plants
• Biopharming is also known as molecular pharming
5
(Humphreys et al., 2000)

6. …concept
 Biopharming started about 20 years ago with the
promise to produce therapeutic molecules like
vaccines, antibodies etc.
 Some therapeutic molecules are very expensive to
produce using conventional systems
 Falls under the category of green biotechnology
6

7. Biopharming vs. Biofarming
Biopharming
PRODUCTION of active
pharmaceutical substances in
genetically modified
organisms (GMOs)
Used exclusively for
Pharmaceuticals
Biofarming
USE of genetically
modified organisms
(GMOs) as a production
platform
Used for both
pharmaceuticals and
others metabolites
7

8. 8
1986
1989
1997
The first plant-derived recombinant
therapeutic HGH protein produced in tobacco
and sunflower
Full-size IgG produced in tobacco
Avidin produced in maize – the first
commercialized plant-derived protein
Barta et
al., 1986
Hiatt et
al., 1989
Hood et
al., 1997
Milestones
1992
Hepatitis B virus surface antigen produced in
tobacco – the first plant-derived vaccine
candidate
Mason et
al., 1992

9. 9
2000
2003
2009
Human growth hormone produced in tobacco
chloroplasts
Bovine trypsin – the first marketed plant-
derived protein, targeted towards a broad
market
Highest transient expression of full-sized IgG
antibody in plants
Staub et
al., 2000
Woodard et
al., 2003
Vézina et
al., 2009
2006
Antibody against Hepatitis B – the first
commercialized plant-derived antibody
(marketed in Cuba)
Vermin and
Waltz, 2006
…milestones

10. Why plants for biopharming?
10
 Simple, cost-effective and faster
 High yield
 Stability – storage
 Safety - free from animal virus
Disadvantages
 Environmental safety- gene flow and wildlife exposure
 Health safety concern- some plants produce allergenic
compounds like alkaloids

11. Expression
system
Yeast Bacteria Plant
viruses
Transgenic
plants
Transgenic
animal
Animal
cell
culture
Cost of
maintaining
less
expensi
ve
less
expensi
ve
less
expensi
ve
less
expensive
Expensive Expensi
ve
Type of
storage(Celsiu
s)
-2.0 -2.0 -2.0 RT Liquid N2 Liquid
N2
Gene(protein)
size
unlimite
d
unlimite
d
limited unlimited limited limited
Production
cost
medium medium low low high high
Protein yield high medium very
high
high medium high
11(Ma et al., 2003 )
…why plants for biopharming?

12. Plants often used
12
 Tobacco
 Most popular used
 High biomass yield
 Rapid scalability
lettuce & alfalfa
 Immediately process
 Rapid degeneration of proteins in leaves-Less stable
(Ma et al., 2003)

13. …plants often used
13
 Cereal grains- rice and maize
 To avoid the problem of short shelf-life
 Easy to transform and manipulate
 Potatoes
 First system to be developed for edible vaccine
 Edible
 Protein stable in storage tissue
(Ma et al., 2003)

14. General strategy in biopharming
• Clone a gene of interest
• Transform the host
species
• Grow the host species,
recover biomass
• Process biomass
• Purify product of interest
• Deliver product of interest
14

15. Different production systems
15
 Stable nuclear transformation
 Plastid transformation
 Transient transformation
 Stable transformation for hydroponics
( Nikolov and Hammes, 2002)

16. Stable nuclear transformation
16
 Most common method
 Foreign genes are transferred via Agrobacterium
tumefaciens or particle bombardment
 Large acres can be utilized with the lowest cost- grains
 Long-term non-refrigerated storage of the seed upto 2yrs
 Manual labor required
 Lower yield and outcrossing

17. Plastid transformation
 First described by Svab et al. in 1990
 No transgenic pollen is generated
 Very high expression levels can be achieved
 Protein – upto 70% on dry weight but relatively stable
 No outcrossing
17

18. Transient transformation
18
 Depend on recombinant plant viruses to infect tobacco
plants like TMV
 Small amounts target protein is obtained in weeks
 Infection process is rapid
 No long term storage
 Target protein is temporarily expressed in the plant
 No stable transgenic plants are generated

19. Stable transformation for hydroponics
19
 Transgenic plants are grown on hydroponic medium
 Desired products are released as part of root fluid into
a hydroponic medium
 Plants are contained in greenhouse
 Easier purification but expensive to operate
 Not suitable for large scale production

20. Downstream processing and recovery
20
 Concerned with the isolation and purification of the product
from the raw biomass
 Regardless of the production system, downstream
processing represents up to 80% of overall production costs
 Basically, there are 4 stages:
 Removal of insolubles
 Product isolation
 Product purification
 Product Polishing
(Fischer et al., 2004)

21. 21(Fischer et al., 2004)
…downstream processing and recovery
Isolation

22. Applications
Parental therapeutics
intermediates-collagen
Industrial enzymes Monoclonal antibodies
Antigens for edible
vaccines
22

23. Monoclonal antibody
23
 Antibody that is produced by genetically engineered
plant is referred to as plantibody
 All current therapeutic antibodies produced are of the
IgG class
 Hiatt et al. were the first to demonstrate the production
of antibodies in tobacco plants in 1989
 The plantibody is the trademark of Biolex (North
Carolina)

24. Two main approaches to produce mAb in
plants
24
 Cross-pollination - transformed plants expressing
light or heavy chains
 Co-transformation of the heavy and light chain
genes on two or more expression vectors to
produce full-size mAb

25. 25
Method of production
harvested and downstream processing

26. Antibodies from transgenic plants
26
Plant Antibody type Purpose References
Tobacco IgG Catalytic
antibodies
Hiatt et al., 1989
Tobacco IgG-nematode Plant pathogen
resistance
Baum et al., 1996
Tobacco
&maize
IgG-HIV gp120
(2G12)
Therapeutic Rademacher et al.,
2008
Soybean,
rice
IgG-herpes virus Therapeutic Zeitlin et al., 1998
Tobacco IgG-colon cancer Systemic
injection
Verch et al., 1998;
Ko et al., 2004
Alfalfa IgG-human Dianostic Khoudi et al., 1999
Tobacco IgG-rabies virus Therapeutic Ko et al., 2003
Tobacco IgG-hepatitis B
virus
Immunopurificati
-on of hepatitis B
surface antigen
Valdes et al., 2003

27. Edible vaccines
27
 The concept of edible vaccine got incentive after
hepatitis B antigen was expressed in tobacco by
Arntzen et al. in 1992
 Developed by engineering a gene for an antigenic
protein into a plant

28. …edible vaccines
28
 Expressed in the edible portion like tubers, fruits
etc
 Due to ingestion, it releases the protein and get
recognized by the immune system

29. Edible vaccine production methods
29
 Expression of foreign antigens in plant via
stable transformation
Delivery of vaccine epitopes via plant virus
(Mishra et al., 2008)

30. …edible vaccine production methods
30(Mishra et al., 2008)

31. Examples of plant edible subunit
vaccines
31
(Mason et al. 2008)

32. 32
Outline
32

33. Industrial enzymes
33
 Avidin and β- glucuronidase first commercialized
industrial proteins from transgenic maize
 Trypsin produced prodiGene company
(proteolytic enzyme) on large scale using maize
 Avidin was the first commercial transgenic protein
produced via transgenic maize

34. Industrial enzymes from transgenic
plants
Proteins Plants Reference
ά Amylase Tobacco Seon et al.,2002
Avidin Corn/maize Hood et al.,1997
β
glucuronidase
Brassica Seon et al.,2002
Xylanase Brassica Seon et al.,2002
Hirudin Brassica Seon et al., 2002
34

35. …industrial enzymes
35(Seon et al., 2002)

36. Plant-derived pharmaceuticals in clinical stages of
development
36
 Vaccines:
Product Disease Plants Clinical
trial status
Company
Hepatitis B
antigen (HBsAg)
Hepatitis B Potato Phase II Arizona State
University
Fusion proteins Rabies Spinach Phase II T.J.University
Cancer vaccine Non-Hodgkin’s
lymphoma
Tobacco Phase II Large Scale
Biology, USA
Vibrio Cholerae Cholera Potato Phase I Arizona State
University
DoxoRX Side-effects of
cancer therapy
Tobacco Phase I
completed
Planet
Biotechnology
IgG (ICAM1) Common cold Tobacco Phase I Planet
Biotechnology
Lactoferon™ (α-
interferon)
Hepatitis B & C Duckweed Phase III Biolex
(Obembe, 2010)

37. 37
Current biopharming companies
(Horn et al., 2008)

38. 38
Case study

39. Production of highly concentrated, heat
stable hepatitis B surface antigen in maize
39(Hayden et al., 2012)

40. 40
Construct design
Transformation into maize and
propagation of seeds
Oil extraction
Experimental procedures

41. 41
Construct design

42. 42
Immunoblotting of maize
material
Antigen detection by ELISA
Confocal microscopy
…experimental procedures

43. 43
Results
HBsAg accumulation in single seeds from the first generation
(Hayden et al., 2012)
0.12%
0.31%
0.41%
0.51%
0.15%

44. 44
…results
HBsAg concentration in second generation (T2) ears with highest antigen
accumulation, as determined by ELISA
(Hayden et al., 2012)
0.05%
0.17%
0.27%
0.26%

45. …results
Effect of oil extraction and temperature on maize-produced HBsAg, as
determined by immunoblot.
45

46. 46
…results
Confocal microscopy :presence of protein(fast
Green) and lipids(Nile Red)
A- full fat
B- hexane
C- SFE (Hayden et al., 2012)

47. …results
47
Total soluble protein and HBsAg protein content in HBsAg maize seed stored at
−20°C, 55°C, and 80°C for one week
Hayden et al., 2012

48. Biosafety issues on biopharming
48

49. …biosafety issues in biopharming
 Gene and protein pollutions
 Vertical gene transfer- most prevalent form via
pollen/seed dispersal among partially compatible plant
 Horizontal gene transfer- between very different
taxonomic groups; and common in bacteria
49

50. …biosafety issues in biopharming
50
 Product safety- public concern about the potential
health and environmental risks associated with the
transgenic plants
 Economic risks to farmers and food industry as
result of co-mingling and contamination of MP
plants with food/feed chain

51. Conclusion
51
 Provides safe, economical and large-scale production of
pharmaceuticals, industrial enzymes and technical
proteins
 PMPs have already achieved preclinical validation in a
range of disease models like hepatitis B, rabies etc.
 We must ensure that the potential benefits are not
outweighed by risks to human health

52. Future lines
52
 Maximization of expression level
 Improvement of downstream processing
 Evaluation of dosage requirement
 Improve and establish a more reliable biosafety
system

53. 53