Insulin - Genetically engineered Product

published by Pratik Umesh Parikh
Master of Pharmacy (Pharmaceutical Biotechnology)

1. Insulin – Genetically
Engineered Product
PRESENTED BY :
PRATIK UMESH PARIKH
MASTER OF PHARMACY
(PHARMACEUTICAL BIOTECHNOLOGY)
PRESENTED BY : PRATIK UMESH PARIKH

2. Contents :
Introduction of Insulin
Structure of Insulin
Pharmacological Action
Importance of Insulin
Production of Recombinant Insulin
Host cell used for Insulin production
Upstream process
Downstream process
Reference
PRESENTED BY : PRATIK UMESH PARIKH

3. Insulin :
oInsulin is a hormone central regulating carbohydrate and fat metabolism in the
body.
oInsulin is secreted by the Islets of Langerhans of pancreas which catabolizes
glucose in blood.
oInsulin causes liver cells, muscle cells and fat tissue to take up glucose from the
blood and store it as glycogen in the liver and muscle.
PRESENTED BY : PRATIK UMESH PARIKH

4. Structure of Insulin:
•Insulin is composed of two peptide chains referred to as the α chain and β chain.
•α and βchains are linked together by two disulfide
bonds, and an additional disulfide is formed within
the α chain.
•α chain consists of 21 amino acids and the β chain
of 30 amino acids.
•Molecular weight of Insulin about 6000.
PRESENTED BY : PRATIK UMESH PARIKH

5. Pharmacological Action of Insulin:
PRESENTED BY : PRATIK UMESH PARIKH

6. Importance of Insulin:
oWithout insulin, the blood glucose builds up in the blood and the cells are
starved of their energy source.
oSome of the symptoms that may occur include fatigue, constant infections,
blurred eye sight, numbness, tingling in the hands or legs, increased thirst, and
slowed healing of bruises or cuts.
oThe cells will begin to use fat, the energy source stored for emergencies.
oWhen this happens for too long a time the body produces ketones, chemicals
produced by the liver.
oKetones can poison and kill cells if they build up in the body over an extended
period of time. This can lead to serious illness and coma.
PRESENTED BY : PRATIK UMESH PARIKH

7. Production of Recombinant Insulin:
Recombinant human insulin :
a form of insulin (trade name Humulin) made from recombinant DNA which is
human insulin.

Recombinant Insulin approaches :
Eli Lilly ( bacterial host)
Novo nordisk (Yeast host)

PRESENTED BY : PRATIK UMESH PARIKH

8. Host cell used for Insulin production:
•Bacterial Host Cell :
Mainly E. Coli used as Bacterial Host cell in Insulin production.

•Yeast Host Cell :
Mainly Sacchromyces cerevisiae used as yeast host cell in Insulin production.
PRESENTED BY : PRATIK UMESH PARIKH

9. E. Coli as a System :
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
PRESENTED BY : PRATIK UMESH PARIKH

10. Loss of plasmid and antibiotic property
Unsolicited inducers for gene expression
Intracellular accumulation of heterologous proteins as inclusion bodies
Improper protein refolding
Lack of post- translational modifications (including unable to form disulphide bonds)
Protein-mediated metabolic burden and stress
Endotoxin contamination
Poor secretion
Proteolytic digestion complexity in downstream processing
DISADVANTAGES OF USING E.COLI AS A SYSTEM
PRESENTED BY : PRATIK UMESH PARIKH

11. ADVANTAGES OF USING SACCHAROMYCES CEREVISIAE
Nonpathogenic
Rapid growth (generation time ca. 80 min)
Dispersed cells
Ease of replica plating and mutant isolation
Can be grown on defined media giving the investigator complete control over
environmental parameters
Well-defined genetic system
Highly versatile DNA transformation system
PRESENTED BY : PRATIK UMESH PARIKH

12. Upstream Process :
PRESENTED BY : PRATIK UMESH PARIKH

13. Downstream Process :
•Removal of insoluble 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.
•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.
•Product polishing describes the final processing steps which end with packaging of the product
in a form that is stable, easily transportable and convenient.

PRESENTED BY : PRATIK UMESH PARIKH

14. Reference :
1.Kjeldsen, T. Yeast secretory expression of insulin precursors. Appl Microbiol
Biotechnol 54, 277–286 (2000).
2.Baeshen, N.A., Baeshen, M.N., Sheikh, A. et al. Cell factories for insulin
production. Microb Cell Fact 13, 141 (2014).
3.Ahmad B: Pharmacology of insulin. Br J Diabetes Vasc Dis. 2004, 4: 10-14.
4.https://www.slideshare.net/anandasaha773/insulin-production-profaksaha
5.https://www.slideshare.net/MohamedElasalyPTCKTP/insulin-production-and-
synthesis
PRESENTED BY : PRATIK UMESH PARIKH

15. PRESENTED BY : PRATIK UMESH PARIKH