Introduction

• the earliest uses of biotechnology in pharmaceutical

manufacturing is the use of recombinant DNA technology to
modify Escherichia coli bacteria to produce human insulin,
which was performed at Genentech in 1978

• Insulin is a hormone produced by β-cells of islets of

Langerhans of pancreas. It was discovered by sir Edward
Sharpey Schafer (1916) while studying Islets of Langerhans

• People who do not produce the necessary amount of insulin

have diabetes.
Introduction
 Structure of insulin
• Chemically, insulin is small and simple protein
consist of 51 amino acids, 30 construct
polypeptide chain B and 21 amino acids
construct polypeptide chain A and both chains
linked by disulfide bond
Genetic engineering for
insuli
n production
Microorganisms
Main process of insulin
production
Microorganisms
Process of Producing Insulin Using
Recombinant DNA Technology
 First Step (Preparing)
• The human gene is isolated. The mRNA is taken from
the cell of islet of Langerhans.
 Messenger RNA is a molecule of RNA that encodes a
chemical
"blueprint" for a protein product.
 The isolated gene contains the code of the human DNA
for the production of insulin.
•The plasmid DNA of the bacterial cell is taken out of the
cell. NOTE: Escherichia coli (E. Coli) bacteria is widely
used in producing insulin but yeast may also be used.
 Second Step (Cutting)
• The plasmid DNA of the bacteria is cut
out producing plasmid ring which is an
empty segment of the DNA.
 A Restriction Enzyme is an enzyme that cuts
DNA at specific recognition nucleotide
sequences known as restriction sites.
 A segment of DNA known as sticky ends.
 Third Step (Combining)
• With the plasmid ring open,
the gene obtained from
human cell that contains the
code of protein responsible
for the production of insulin
is inserted into the plasmid
ring and the ring is closed.
• The human insulin gene is
now combined with the
bacterial DNA plasmid. Mix the recombinant
plasmid with bacteria.
 Fourth Step (Inserting)

• Transformed: resulting DNA is inserted

back to the bacteria.
 Production

• The cells need nutrients in order to grow,

divide, and live. While they live, the
bacterial cell processes turn on the gene
for human insulin and the insulin is
produced in the cell. When the bacterial
cells reproduce by dividing, the human
insulin gene is also reproduced in the
newly created cells.
Finding
another
cloning
system !
 HOST CELL USED FOR
INSULIN
PRODUCTION
SACCHAROMYCES CEREVISIAE
Is an eukaryotic microbe
system that is widely used for
protien expression that is
required for post-translational
modification
micro
s be
wide
ly
prot
ein
 ADVANTAGES OF USING
SACCHAROMYCES
CEREVISIAE

• Nonpathogenic
• Rapid growth (generation time ca. 80 min)
• Dispersed cells
• Ease of replica grown on defined media giving
the investigator complete control over
environmental parameters
• Well-defined genetic system
• Highly versatile DNA transformation system
 DISADVANTAGES OF
USING
SACCHAROMYCES
CEREVISIAE

• S. cerevisiae is not
productive as the E. coli.
 PHASES INVOLVED IN
INSULIN
PRODUCTION

Upstream Process

Downstream Process
 UPSTREAM
PROCESS PHASE
Shakingbioreactor
Advantages:
Easy ,Visible ,Cheap, Depyrogenation
feasible

Disadvantage:
Poor aeration
Impeller jams
Requires cleaning siliconizing & sterilization lHigh
space requirements in incubator
 UPSTREAM
PROCESS PHASE
Stir tank bioreactor
• Continuous bioreactor
processes continually
feed nutrients and
medium into the
bioreactor while also
continually harvesting
material from the
bioreactor.
 UPSTREAM
PROCESS PHASE
Stir tank bioreactor
• Agitator is introduced to
disperse the reactants
thoroughly into the reaction
mixture immediately as they
enter the reactor.
• Product is continuously drawn
out and that’s why known for
perfect mixing.
• Compositions at outlet and
inside reactor are the same.
ADVANTAGES OF STIR TANK
BIOREACTOR

• Multi-gas and pH control

• Increased Capacity( 5 L
to 500 L)
• Versatility
DISADVANTAGES OF STIR TANK
BIOREACTOR

• Costly
• High power due the presence of
mechanical pumps
• Limitation of Weight Preparation
• Requires siliconizing
• Requires constant cleaning,
• Sterilization, depyrogenation
• Requires high Maintenance -Chiller,
parts
 BIOREACTOR
CONDITIONS

• 10L total working

volume
• 31 hour growth
phase
• pH 7
• 37 °C
 DOWNSTREAM PROCESS
• Removal of insolubles is the first step and
involves the capture of the product as a solute in
a particulate-free liquid. Typical operations to
achieve this are filtration, centrifugation.

• Product isolation is the removal of those

components whose properties vary considerably
from that of the desired product. Solvent
extraction, adsorption, ultrafiltration, and
precipitation are some of the unit operations
involved.