brief intro of harmones specially insulin and then production of differernt harmones

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……