Transcript Document

Growth and Development of Biotechnology:
Past, Present and Future
Dr. P.K. Ghosh
President Biotechnology
Cadila Pharmaceuticals Limited, Ahmedabad
Inaugural Session Lecture of GIBioN
18 Dec 2005
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What is Biotechnology?
• Biotechnology : techniques applied to organisms or parts thereof
to produce, identify or design substances, or to modify organisms
for specific applications.
• Cell fusion techniques, hybridomas, recombinant DNA technology,
cloning through whole genome transfer, protein engineering,
structure based molecular design, genomics, proteomics, bio
informatics including biochips, cell therapy including cell, tissue or
organ transplant, xeno - transplants or creation of artificial cells
are modern biotechnology.
• Conventional biotechnology includes fermentation or conversion
of substrates into desired products by biological processes; use of
microbes or enzymes; sera, vaccines and diagnostics;
reproduction, artificial insemination and embryo transfer
technology for animal breeding; methods for fish spawning
induction; plant cell or tissue culture; plant breeding; biofertilisers; bio-pesticides; plant growth stimulants; extraction and
isolation of active principles from plants or animals or parts
thereof etc.
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Conventional Biotechnology
Existed Since Ages!
* 4000 BC : Egyptians used yeast for bread & wine making.
* 3000 BC : Peruvians cultivated potatoes.
* 2000 BC : Egyptians, Sumerians and Chinese developed
techniques of fermentation, brewing and cheese making.
* 500 BC : Chinese used moldy soybean curds to treat boils.
* Indian ancient literature (> 4000 BC) refers to Sura &
Madira which are fermented beverages!
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Beginning of Understanding
Science of Biotechnology
* 1590 Janssen invented the microscope.
* 1802 : The terms biology first appeared in literature.
* 1830 : Proteins were discovered.
* 1860 : Louis Pasteur’s experiments abolished the theory
of spontaneous generation of living organisms.
* 1865 : Mendel discovered the existence of heredity.
* 1897 : Buchner’s discovered the biochemical basis of
life process.
* 1919 : A Hungarian agricultural engineer coined
the term Biotechnology.
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Domain of Biotechnologists (Schematic)
Basic Biotechnologists:
Use molecular and biological keys and tools
Molecular tools/keys
Libraries of small Molecules :
natural, combinatorial &
computational
Libraries of large molecules:
proteins,
carbohydrates,
nucleotides etc.
Cellular tools / keys
Biotic
Diversities
Interactions
&
Evolutionary
Relationships
Metabolic Studies
Genomics
Proteomics
&
Structural
Functional
Pharmacological
Biological Models
Study
of
molecular
interactions: large-large,
large-small molecules etc
Molecular
evolutions:
large and small molecules
Creating relation-ships
among these disciplines
Main applications emerged
in Healthcare Products,
Agriculture, Environment
Management & Others
Informatics:
Internet,
sequence
data,
data
searching,
predictive
methods etc.
Feed back loop
Engineering
Biotechnologists
provide inputs for easing production
Products to Market
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The Hershey-Chase Blender Experiment, 1952
No labeled sulphur detected in cells
LalLabeled sulphur detected
in supernatant
Labeled Phosphorus detected in cells
No labeled phosphorus
Detected in supernatant
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Rosalind Franklin’s X-ray Diffraction Photo of
Structure B of DNA
Sodium deoxyribose nucleate
from calf thymus, Structure B,
Photo 51, taken by Rosalind
E. Franklin and R.G. Gosling.
Linus Pauling's holographic
annotations are to the right of
the photo., May 2, 1952.
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DNA Double Helical Structure
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Restriction Enzymes
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Sanger’s Dideoxy Chain Termination Method for
DNA Sequencing
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The Genetic Code
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The Basic Constituents of lac Operon
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The Central Dogma of Life
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From Gene to Protein
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Principle Of PCR
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Gene Cloning (Recombinant DNA Technology)
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Expression of Recombinant Proteins
Recombinant proteins are expressed as:
1) Intracellular Substances
•Insoluble protein
•Soluble protein
Cytoplasmic
Periplasmic
2) Extracellular Substances
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Recombinant Protein Purification : ISSUES
• Folding for activity
• Purity
Primary Techniques for purification
•Precipitation - pH and salt addition
• Ion exchange chromatography
• Hydrophobic interaction chromatography
• Gel filtration Chromatography
• Affinity Chromatography
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Intracellular Insoluble Proteins: as Inclusion Bodies
Dense aggregates of mainly the desired protein
Concentration based
Unable to fold correctly in the environment
Cell lysis: Enzyme treatment
Sonication
French press
Dynomill
Freeze-thaw
Simple purification steps using centrifugation are used
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Expression and Purity of Intracellularly Expressed
and Isolated Insoluble Proteins
1
2
3
4
5
6
7
8
9
10
1.
Mol. Wt marker
2.
Total cell no.1
3.
Inclusion Bodies no.1
4.
Total cell no. 2
20.0
5.
Inclusion Bodies no.2
14.3
6.
Total cell no.3
7.
Inclusion Bodes no.3
8.
Total cell no.4
9.
Inclusion Bodies no.4
68.0
43.0
29.0
Product obtained - insoluble and non-native
In-vitro refolding is essential
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Intracellular Soluble Protein
Expressed protein is soluble
In-vitro refolding may not be required as
protein may attain native structure
Protein is extracted by simple lysis methods
Protein has large number of cellular protein
contaminants, compared with partially
purified inclusion bodies
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Purification of a Soluble Cytoplasmic Protein:
rp24 of HIV-1
Expression of the protein in E. coli
Lysis of the cells
Centrifugation
Supernatant
Ion exchange Chromatography
Gel filtration Chromatography
Ready for use in a formulation
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Important Globally Approved
Recombinant Therapeutics
Product
Therapeutic Indication
Factor VIII
Hemophilia Type A
Factor IX
Hemophilia Type B
Tissue Plasminogen activator
Acute myocardial infarction
Insulin
Diabetes
Human growth hormone
hGH deficiency in children
Thyrotropin-a
Thyroid Cancer
Erythropoietin
Treatment of anemia
GM-CSF / G-CSF
Chemotherapy induced neutropenia
PDGF
Diabetic Neuropathic ulcers
Interferon alpha 2a
Hairy cell leukemia
Interferon alpha 2b
Hairy cell leukemia / Hepatitis B & C
Interleukin – IL-2
Renal cell carcinoma
Hepatitis B surface antigen
Vaccine against Hepatitis B
Alpha-Glucocerebrosidase
Treatment of Gaucher’s disease
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Important Globally Approved
Genetically Modified Plants
Product
Genetically Altered Traits
Tomato
Delayed ripening: Gene sequence for polygalaturonase
production in tomato rearranged and reversed to minimise
its expression by Antisense technology.
Cotton
Bt gene incorporated plants (ballworm & budworm resistant):
CRY 1A c gene from Bt Kurstaki.
Soybean
Reisitant to glyphosate for control of weeds:
Enolpyruvylashikimate-3-phosphate synthase gene from
Agrobacterium sp.CP4
Potato
Bt gene incorporated (Colorado potato beetle resistant) : Cry
III (A) gene from Bt. Tenebrionis.
Maize/Corn
Bt gene incorporated (resistant to comborer) : Cry 1A b gene
from Bt. Kurstaki
Rapeseed / Canola
Altered oil composition (high lauric acid content): 12:0 acyl
carrier protein thioesterase gene from Umbellularia
californica.
Resistant to glufosinate for Male sterility properties
Squash
Resistant to viruses: Coat protein genes of watermelon
mosaic virus 2 and Zucchini yellow mosaic virus.
Papaya
Resistant to Papaya ring spot virus: Coat protein gene of p
type of PRSV HA-5-1 from Hawai.
Chicory
Male starility resistant to glufosinate and fertility restores
genes from bacteria.
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Microarray Technology
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DOLLY- The Cloning Glory
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Estimated Global Consumption of Modern Biotech Products
Global Segment
Estimated consumption in billion US$
2001
2005
2010
USA & Canada
28
34
50
Europe and Japan
7.5
10
15
Rest of the World
2.5
5
12
Total
38
49
77
India
0.1
0.3
1.0
China
0.6
1.2
3.0
South Korea
0.3
0.5
1.3
Latin American countries
and the rest of the world
1.5
3.0
6.7
Subtotal
2.5
5.0
12.0
Overlap of the Segments of
the rest of the world
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Strategies for Technology Development
In Developing Country Industries
Alliances through
Research
Grants
Sponsored
Research
A
Developing Country
Company
In-House R&D
Alliances through
Institutions & Companies
:In-Country & Abroad
Technology
Sourcing &
Acquisitions
Technologies
Production Infrastructure
Products and Services
Commercialization
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Indian Strength in Biotechnology Development
 Capacity in handling sterile fermentation processes
 Skills in handling microbes and animal cells
 Skills in plant cell/tissue culture
 Competence in chemical synthesis, proficiency in immunology and
hands-on experience in microbiology
 Capacity in downstream processing and isolation methods
 Skills in cloning
 Skills in extraction and isolation of plant and animal products
 Competence in plant and animal breeding
 Infrastructure and skills in fabricating bio-reactors and processing
equipment of diverse kinds
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India’s Entry Will Reduce Costs
• Health care products : Hepatitis B Vaccine, Interferon Alpha,
Erythropoietin and Insulin doses cost less.
• Agriculture : Genetically modified planting materials will be
available at cheaper prices.
• Prices of Industrial products like Enzymes will also come
down.
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Concluding Remarks

Modern Biotechnology has made tremendous progress during the
last 5 decades.

The number of Biotech products are increasing at a galloping speed
in Health care area, Agriculture and Industrial applications.

Industries in Developed Countries shall hold Major Technologies.

Developing countries incl. India shall also make progress.

All Biotech Industries shall need strong support from Governments.

Indian development would contribute to providing Biotech products
at cheaper prices though total contribution in global context would
be small.

Skilled Biotechnologists would have plenty of opportunities
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