“PRODUTION OF HARMONES”

PRESENTED BY: SAIMA, 15SADAF, 08

SUMBAL, 19

HARMONES:

• Chemical messages which are secreted by collection of glands (endocrine system)

ENDOCRINE SYSTEMo 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.

ENDOCRINE SYSTEM

“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.

“INSULIN”

“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.

Why is insulin needed?

“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

Why synthesize human insulin?

• No immunogenicity.

• Decline in the production of animal-derived insulin.

• Need for a more reliable and sustainable method

What types of skills required?

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

“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.

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.

Figure 1 Percentage of biopharmaceuticals produced in different expression systems

“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

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

“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 *

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.

Additional recombinant hormones now approved:

• which recently gained marketing approval:

a) Glucagon

b)Thyroid stimulating hormone

c) Parathyroid hormone

d) calcitonin

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.

TRADITIONALLY:

• Extracted from pancreatic tissues of pigs and cattle, followed by multistep chromatographic purification.

• Such commercial preparation are generally sold in freeze-dried form.

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.

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.

• 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.

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.

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

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.

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.

Thanks……