This document discusses the production of human insulin using recombinant DNA technology. It begins by providing background on insulin and diabetes. Insulin is normally produced in the pancreas but some people do not produce enough. Recombinant DNA technology allows for the insertion of the human insulin gene into E. coli bacteria. The bacteria then produce human insulin which can be purified from cell cultures. This was the first pharmaceutical produced using recombinant DNA and was approved for diabetes treatment in 1982 under the name Humulin. The document details the cloning of the insulin gene, production in E. coli, purification downstream processing, and advantages of using this system.

1. INSULIN PRODCTION
USING RECOMBINANT
DNA TECHNOLOGY

2. Introduction.
• The Human Insulin is a peptide hormone secreted by the
β cells of the pancreatic islets of Langerhans
• It was discovered by sir Edward Sharpey Schafer (1916) while studying
Islets of Langerhans.
• maintains normal blood glucose levels by facilitating
cellular glucose uptake.

3. • People who do not produce the necessary amount of insulin
have diabetes.
• Type 1 diabetes develops when the cells of the pancreas stop
producing insulin. Without insulin, glucose cannot enter the
cells of the muscles for energy.
• Type 2 diabetes develops when the pancreas does not make
enough insulin and the insulin that is made does not work as
well as it should (also known as insulin resistance).
• As a result, the glucose begins to rise above normal levels in
the blood. Half the people with type 2 diabetes do not know
they have the condition because they have no symptoms.

4. • . It is formed of 51 aminoacids which are arranged in two
polypeptide chains, A and B.
• The polypeptide chain A has 21 amino acids while the
polypeptide chain B has 30 amino acids.
• Both A and B chains are attached together by disulphide
bonds.
Structure

5. • Insulin gene is found on chromosome (11).
• It is found on the short arm of chromosome 11 ( p arm).
• It is located region 1, band 5, sub- band 5.
Insulin gene

6. In the early years, insulin isolated and purified from the pancreas of pigs
and cows was used to treat diabetic patients.
Due to minor differences in the structure of the animal insulin as compared
to human insulin, it resulted in the occurrence of allergic reactions in some
diabetic patients.
Production of insulin by recombinant DNA technology started in the late
1970s. This technique involved the insertion of human insulin gene on the
plasmids of E.coli.
Insulin was the first ever pharmaceutical product of recombinant DNA
technology administered to humans.
The approval to use recombinant insulin for diabetes mellitus was given in
1982.
In 1986 human insulin was marketed under the trade name Humulin.
history

8. • Sal I and BamH I are restriction enzymes used to
facilitate the cloning of insulin gene

11. • 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.
Production

13. • Simple, well-understood genetics
• Ease of genetic manipulation
• Minimal culturing cost
• Fast expression (doubling time is only 20 - 30 mins).
• Well established labeling protocols for stability studies.
• Established regulatory track record.
• Fermentation: ease of scaling up.
• Ease of Inclusion bodies purification.
ADVANTAGES OF USING E. COLI
AS A SYSTEM

14. • 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.
DOWNSTREAM PROCESS

15. • Product purification is done to separate those contaminants that resemble
the product very closely in physical and chemical properties. operations
include affinity, size exclusion, reversed phase chromatography, ion-
exchange chromatography, crystallization and fractional precipitation.
• The polypeptide chains are synthesized as a precursor called
pre-pro insulin, which contains A and B segments linked by a
third chain (C) and preceded by a leader sequence.
• The leader sequence is removed after translation and the C
chain is excised, leaving the A and B polypeptide chains
• Product polishing describes the final processing steps which end with
packaging of the product in a form that is stable, easily transportable and
convenient.
DOWNSTREAM PROCESS

17. Questions & answers
Invite questions from the audience

18. THANK YOU