PRODUCTION OF PHARMACEUTICALS BY GENETICALLY ENGINEERED CELLS (HORMONES AND INTERFERONS)

Prabhu Thirusangu Molecular Biomedicine Laboratory,

Sahyadri Science College,Kuvempu University

THE AGE OF BIOPHARMACEUTICALS…. The term ‘biopharmaceutical’ was first used in the 1980s and came to

describe a class of therapeutic protein produced by modern biotechnological techniques, specifically via genetic engineering.

Biomedical research continues to broaden our understanding of the molecular mechanisms underlining both health and disease.

Research undertaken since the 1950s has pinpointed a host of proteins produced naturally in the body which have obvious therapeutic applications.

Examples include the interferons, and interleukins, which regulate the immune response; growth factors such as erythropoietin, which stimulates red blood cell production; and neurotrophic factors, which regulate the development and maintenance of neural tissue.

Production of Human Insulin by r- DNA Technology….

Human insulin produced by recombinant DNA technology was first approved for general medical use in 1982.

It was the first product of recombinant DNA technology to be approved for therapeutic use in humans.

The quantity of purified insulin obtained from the pancreas of one pig satisfies the requirements of one diabetic for 3 days.

AN OVERVIEW OF RECOMBINATION PROCESS…

Route to the Production by Bacteria of Human Insulin

Overview of gene cloning. Cloning

animation

3D- Structure of engineered Insulin Recombinant Insulin (Humulin)

ENGINEERED HUMAN INSULIN…Product Company

Humulin (rh-insulin) Novolin (rh-insulin) Humalog (Insulin Lispro, an insulin analogue) Insuman (rh-insulin) Liprolog (Bio Lysprol, a short-acting insulin) NovoRapid (Insulin Aspart, short-acting rh-insulin

analogue) Novomix 30 (contains Insulin Aspart, short-acting

rh-insulin analogue (see NovoRapid) as one ingredient

Novolog (Insulin Aspart, short acting rh-insulin; see also NovoRapid entry above)

Novolog mix 70/30 (contains Insulin Aspart, short acting rh-insulin analogue as one ingredient; see also Novomix 30 entry above)

Actrapid/Velosulin/Monotard/Insulatard/Protaphane/Mixtard/Actraphane/ Ultratard (all contain rh-insulin produced in S. cerevisiae, formulated as short/intermediate/long-acting products)

Lantus (insulin glargine, long acting rh-insulin) Optisulin (insulin glargine, long-acting rh-insulin

analogue, see Lantus)

Eli Lilly Novo Nordisk Eli Lilly Hoechst AG Eli Lilly Novo Nordisk

Novo Nordisk

Novo Nordisk

Novo Nordisk

Novo Nordisk

Aventis Pharmaceuticals Aventis Pharma

PRODUCTION OF recombinant HUMAN GROWTH HORMONE (Hgh)….

Human growth hormone is a polypeptide hormone synthesized in the anterior pituitary. It promotes normal body growth and lactation and influences various aspects of cellular metabolism.

Dwarfism caused by insufficient production of HGH by the pituitary gland.

HGH can treat dwarfism – to help under sized children reach their normal height and size.

Recombination process – 1. DNA coding for growth hormone is inserted into the plasmid. 2. Introduction of recombined plasmid into E.Coli host cell 3. Engineered E.Coli cell is allowed to multiply in the fermentor.

RECOVERY OF HUMAN GROWTH HORMONE...

Cytokines as biopharmaceuticals

Cytokines constitute the single most important group of biopharmaceutical substances. As coordinators of the immune and inflammatory response, manipulation of cytokine activity can have a major influence on the body’s response to a variety of medical conditions.

Administration of certain cytokines can enhance the immune response against a wide range of infectious agents and cancer cells.

THE INTERFERONS…..

Interferons (IFNs) were the first family of cytokines to be discovered. In 1957 researchers observed that if susceptible animal cells were exposed to a colonizing virus, these cells immediately become resistant to attack by other viruses.

This resistance was induced by a substance secreted by virally-infected cells, which was named ‘interferon’ (IFN).

Humans produce at least three distinct classes, IFN-a, IFN-b and IFN-g. Biological effects, Induction of cellular resistance to viral attack. Regulation of most aspects of immune function. Regulation of growth and differentiation of many cell types. Sustenance of early phases of pregnancy in some animal species.

PRODUCTION OF INTERFERONS BY GENETIC ENGINEERING…

A DNA sequence coding for the product was synthesized and inserted into E. coli. The recombinant product accumulates intracellularly as inclusion bodies

Large-scale manufacture entails an initial fermentation step. After harvest, the E. coli cells are homogenized and the inclusion bodies recovered via centrifugation. After solubilization and refolding, the interferon is purified to homogeneity by a combination of chromatographic steps.

The final product is formulated in the presence of a phosphate buffer and sodium chloride.

It is resented as a 30 mg/ml solution in glass vials and displays a shelf-life of 24 months when stored at 2–8°C`.

Recovery OF RECOMBINANT HUMAN IFN-B…

3D – Structure of Human growth hormone

Recombinant interferons

Interferon toxicity…..

Like most drugs, administration of IFNs can elicit a number of unwanted side effects.

Minor side effects - Range of flu-like symptoms, e.g. fever, headache, chills.

Serious potential side effects – Anorexia Strong fatigue Insomnia Cardiovascular complication Autoimmune reactions Hepatic decompression

CONCLUSION….. Interferons represent an important family of

biopharmaceutical products.

They have a proven track record in the treatment of selected medical conditions, and their range of clinical applications continue to grow.

It is also likely that many may be used to greater efficacy in the future by their application in combination with additional cytokines

Prabhu Thirusangu

Reference…

Bibliography…… Biopharmceuticals (2nd Edition) By Gary Walsh Immunology By Kuby Genetics By Benzamin Webliography…… http://www.ncbi.nlm.nih.gov/pubmed/3296632 http://en. Wikipedia.com/pharmacology

Thank you