The document discusses the production of hormones such as insulin through recombinant DNA technology. It describes how insulin was traditionally derived from animals but this led to disadvantages like immunogenicity and availability issues. The development of recombinant DNA technology in the 1980s allowed for the large-scale production of human insulin in microorganisms like E. coli and yeast. This eliminated immunogenicity concerns and provided a more reliable method of production. The document also discusses how this technology has enabled the development of engineered insulin analogues with altered properties and the production of other important hormones like glucagon, thyroid stimulating hormone, parathyroid hormone, and calcitonin.

1. “PRODUTION OF HARMONES”
PRESENTED BY: SAIMA, 15
SADAF, 08
SUMBAL, 19

2. HARMONES:
• Chemical messages which are secreted by
collection of glands (endocrine system)

3. ENDOCRINE SYSTEM
o The endocrine system is made up of glands
that produce and secrete hormones.
o Hormones regulate:
The body's growth,
Metabolism
Sexual development and function.

4. ENDOCRINE SYSTEM

5. “General Mechanisms of
Hormone Release”
(a) Humoral: in response to changing levels of
ions or nutrients in the blood.
(b) Neural: stimulation by nerves.
(c) Hormonal: stimulation received from
other hormones.

6. “INSULIN”

7. “Structure of Insulin”
• Gene encoding : Chromosome 11.
• Two polypeptide chains:
– A CHAIN: 21 amino acids
– B CHAIN: 30 amino acids.
• Chains are linked via a disulfide bond.

8. Why is insulin needed?

9. “PRODUCTION OF INSULIN”
• TRADITIONALLY:
– Animal derived,
– Potential disadvantages
– 1) Immunogenicity: (3 a.a difference)
– 2) Availability: (5000kg+)
• BY rDNA TECHNOLOGY:
– 1st approved in 1982.
– engineered insulin products: 1990’s

10. Why synthesize human insulin?
• No immunogenicity.
• Decline in the production of animal-derived
insulin.
• Need for a more reliable and sustainable
method

11. What types of skills required?

12. Affinity Chromatography purification
– Specific interaction with a ligand
bound to column
– “general” ligand e.g. chemical group
– Immobilised metal -Histidine
containing proteins
- Highly specific ligand
e.g : antibody

13. “EXPRESSION SYSTEM”
• Nowadays, recombinant human insulin is mainly produces
either in E.coli or S.cerevisiae.
• Using E. coli expression system, the insulin precursors
(IP) are produced as inclusion bodies
• and fully functional polypeptides are obtained finally by
solubilization and refolding procedures.

14.  Yeast based expression system yield soluble IP which is
secreted into the culture supernatant.
 Saccharomyces cerevisiae is the most preferred and
predominant yeast for large scale commercial production of
insulin,
 Besides, E.coli and yeast, mammalian cells, transgenic
animals and plant expression systems are also employed as
a host for large-scale production of recombinant insulin.

15. Figure 1 Percentage of biopharmaceuticals
produced in different expression systems

16. “E.coli AS AN EXPRESSION SYSTEM”
• Why use E.coli ?
– high growth rate
– simple media requirement
– easy to handle
– high yield
– very cost effective.
• However, there are some disadvantages using E. coli expression
system, such as:
– loss of plasmid
– intracellular accumulation of heterologous proteins as inclusion
bodies
– improper protein refolding
– lack of post- translational modifications
– Endotoxin contamination
– complexity in downstream process

17. INITIAL APPROACH:
 Inserting sequence coding for the insulin chain A and
B into two different E.coli cells.
 Cels cultured separately in large scale fermentation
vessels,
 chromatographic purification of the insulin chains
produced.
 The A and B chain are incubated together under
appropriate oxidizing conditions to promote
disulphide bond formation, forming ‘human inulin

19. “ENGINEERED INSULIN”
 rDNA technology facilitates not only production of
human insulin in microbial cell, but also generation of
insulin of modified a.a sequences.
 Their major aim:
a) Identification of insulin with altered
pharmacokinetic properties(faster or lower acting)
b) Identification of super-potent insulin forms
(insulin with higher receptor affinities)
 Number of analogues manufactured, with modified a.a
sequences:
• Histidine Glutamate (B10 position)
For example, FIVEFOLD HIGHER ACTIVITY
INVITRO *

20. Contd..
1. Insulin lispro (trade name: ‘Humalog’)
 1st such engineered short-acting insulin to come to
market.
2.Insulin Aspart.
 2nd fast acting engineered human insulin analogue.
 Proline B28 replaced by aspartic acid.
3.Optisulin or Lantus (trade names)
 Gained marketing approval in 2000.
4. Levemir (trade name)
 Long acting insulin product.
 Gained approval for medical use in 2004.

21. Additional recombinant hormones
now approved:
• which recently gained marketing
approval:
a) Glucagon
b)Thyroid stimulating hormone
c) Parathyroid hormone
d) calcitonin

22. a) Glucagon:
• A single-chain polypeptide of 29 amino
acid, synthesized by alpha-cells of the
islets of langerhans .
• Breakdown of glycogen , lipid and
protien.
• Biological action of glucagon is opposite
to that of insulin.
• A hyperglycemic hormone.

23. TRADITIONALLY:
• Extracted from pancreatic tissues of pigs and
cattle, followed by multistep chromatographic
purification.
• Such commercial preparation are generally
sold in freeze-dried form.

24. Through rDNA Technology:
• More recently ,glucagon is prepared by
recombinant means.
• GlucoGen is the trade name produced by
Novo Nordisk using an engineered
S.cerevisiae strain.
• Upstream processing is done in aerobic
batch-fed fermentation.
• In downstream processing PH is adjusted in
the media.
• Glucagon is then recovered and purified
from the media.

25. b) Thyroid stimulating hormone:
• A glycoprotein hormone synthesized and
secreted in the anterior pituitary gland,
which regulates the function of the thyroid
gland.
• consists of two subunits, the alpha and the
beta subunits.
• A synthetic drug called recombinant human
TSH alpha (trade name Thyrogen), is
manufactured by Genzyme Corp.

26. • Stimulates the thyroid gland to secrete
the hormone thyroxine (T4).
• T4 is converted to triiodothyronine
(T3), an active hormone
• About 80% of this conversion is in the
liver and other organs, and 20% in the
thyroid itself.

27. c) Parathyroid hormone:
• Polypeptide containing 84a.acid that function
as a primary regulator of calcium and
phosphate.
• PTH is synthesized and secreted by the
parathyroid gland which lie posterior to the
thyroid glands.
• PTH (trade name Forsteo and Forteo ) is
produced in E.coli for the treatment of
osteoporosis in post menopausal women.

28. Contd..
• Hyperparathyroidism.
• Hypoparathyroidism and is most
commonly due to damage or removal of
parathyroid glands during thyroid
surgery.
• The average PTH level is 8–51 g/ml

29. d) calcitonin:
• Polypeptide hormone of 32a.acid and
produced in humans by parafollicular cells of
thyroid.
• It decrease calcium concentration in blood.
• Calcitonin is used clinically to treat:
• Hypercalcaemia .
• Paget’s disease.

30. Contd..
• Found in fish, reptiles, birds, and mammals.
• Its importance in humans has not been as
well established as its importance in other
animals.
• Salmon calcitonin resembles human
calcitonin, but is more active.
• At present, it is produced either by
recombinant DNA technology or by chemical
synthesis.

31. Thanks……