Growth and Development of Biotechnology - the website of ...

mutebabiesBiotechnology

Dec 6, 2012 (4 years and 10 months ago)

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1

Dr. P.K. Ghosh

President Biotechnology

Cadila Pharmaceuticals Limited, Ahmedabad

Inaugural Session Lecture of GIBioN

18 Dec 2005

Growth and Development of Biotechnology:

Past, Present and Future


2

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
;

bio
-
fertilisers
;

bio
-
pesticides
;

plant

growth

stimulants
;

extraction

and

isolation

of

active

principles

from

plants

or

animals

or

parts

thereof

etc
.


3

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!

4

Beginning of Understanding

Science of Biotechnology

* 1590 Janssen invented the microscope.

* 1802 : The terms biology first appeared in literature.

* 1830 : Proteins were discovered.

* 1865 : Mendel discovered the existence of heredity.

* 1860 : Louis Pasteur’s experiments abolished the theory


of spontaneous generation of living organisms.

* 1897 : Buchner’s discovered the biochemical basis of


life process.

* 1919 : A Hungarian agricultural engineer coined


the term Biotechnology.

5

Domain of Biotechnologists (Schematic)
Basic Biotechnologists:
Use molecular and biological keys and tools
Cellular tools / keys
Genomics &
Proteomics
Molecular tools/keys
Libraries of small Molecules :
natural, combinatorial &
computational
Libraries of large molecules:
proteins, carbohydrates,
nucleotides etc
.
Study of molecular
interactions: large
-
large,
large
-
small molecules etc
Molecular evolutions:
large and small molecules
Structural
Functional
Pharmacological
Informatics: Internet,
sequence data, data
searching, predictive
methods etc.
Engineering Biotechnologists
provide inputs for easing production
Products to Market
Feed back loop
Biotic Diversities &
Interactions
Evolutionary
Relationships
Metabolic Studies
Biological Models
Creating relation
-
ships
among these disciplines
Main applications emerged
in Healthcare Products,
Agriculture, Environment
Management & Others
6

The Hershey
-
Chase Blender Experiment, 1952


No labeled sulphur detected in cells

Lal
Labeled sulphur detected

in supernatant

Labeled Phosphorus detected in cells

No labeled phosphorus

Detected in supernatant

7

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
.

8

DNA Double Helical Structure


9



Restriction Enzymes

10

Sanger’s Dideoxy Chain Termination Method for
DNA Sequencing


11

The Genetic Code


12

The Basic Constituents of lac Operon

13

The Central Dogma of Life

14

From Gene to Protein

15

Principle Of PCR


16

Gene Cloning (Recombinant DNA Technology)

17

Expression of Recombinant Proteins

Recombinant proteins are expressed as:


1) Intracellular Substances



Insoluble protein


Soluble protein


Cytoplasmic


Periplasmic

2) Extracellular Substances

18

Recombinant Protein

Purification : ISSUES



Folding for activity



Purity

Primary Techniques for purification



Ion exchange chromatography



Hydrophobic interaction chromatography



Gel filtration Chromatography



Affinity Chromatography


Precipitation
-

pH and salt addition

19

Intracellular Insoluble Proteins: as Inclusion Bodies

Dense aggregates of mainly the desired protein

Concentration based

Unable to fold correctly in the environment

Simple purification steps using centrifugation are used

Cell lysis: Enzyme treatment


Sonication


French press


Dynomill


Freeze
-
thaw

20

1 2 3 4 5 6 7 8 9 10


14.3


68.0


43.0


29.0


20.0

1.
Mol. Wt marker

2.
Total cell no.1

3.
Inclusion Bodies no.1

4.
Total cell no. 2

5.
Inclusion Bodies no.2

6.
Total cell no.3

7.
Inclusion Bodes no.3

8.
Total cell no.4

9.
Inclusion Bodies no.4


Expression and Purity of Intracellularly Expressed
and Isolated Insoluble Proteins

Product obtained
-

insoluble and non
-
native

In
-
vitro refolding is essential

21

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


22

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

23

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

24

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.

25


Microarray Technology


26

DOLLY
-

The Cloning Glory


27

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


Overlap

of

the

Segments

of

the

rest

of

the

world











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


Estimated Global Consumption of Modern Biotech Products


28

Strategies for Technology Development

In Developing Country Industries

Alliances through

Research

Grants

Alliances through

Institutions & Companies

:In
-
Country & Abroad

Sponsored

Research

Technology

Sourcing &


Acquisitions

A

Developing Country

Company

Technologies

In
-
House R&D

Production Infrastructure

Products and Services

Commercialization

29



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

Indian Strength in Biotechnology Development

30

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.

31


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


Concluding Remarks