Assignment on:

Production of biopharmaceutical product.

Submitted to:

Mir. Md. Abdullah Al Mamun

Assistant professor, Dept. of pharmacy

Nothern University Bangladesh

Submitted by:

Md. Mizanur Rahman

Batch no: 8B

Roll no: 130101065

Date: 04/03/2017

What is Biotechnology ?

The term biotechnology was coined in 1917, by Hungarian engineer, karl erky, to describe a process for large scale production of pigs. Biotechnology can be defined as application of technology Using the living organisms to obtain useful products.

Available Definitions of Biotechnology:

Biotechnology is the application of biological organisms systems or process to manufacturing and service industries ( British biotechnologists).

Biotechnology is integrated use of biochemistry, microbiology and engineering sciences in order to achieve technological applications of microbes, cultured cells and parts there of (European federation of biotechnologist).

Biotechnology is the controlled use of biological agents, such as microbes or cellular components. (U.S.National science foundation).

Development of Biotechnology:

Ancient Biotechnology: Early history relates to food and shelter, includes domestication.

Classical Biotechnology: Fermentation food production and medicine.

Modern Biotechnology: Manipulates genetic information in an organism. Eg. genetic engineering.

Genetic engineering:

It is in vitro DNA technology used to isolate genes from an organism manipulate them in laboratory as per desire and insert them into other cell or system for specific character. It is also called gene cloning.

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Fields of Biotechnology:

Cell and Molecular Biology • Microbiology • Genetics • Physiology • Biochemistry • Immunology • Virology • Recombinant DNA Technology.

List of biotechnology products ?

According to the Biotechnology Industry Organization, there are more than 250 biotechnology products available, including medicines, vaccines, fertilizers, pest-resistant crops, bio-fuels and bio-defense products. Some specific bio-tech products include an anthrax detection device, non-browning apples, snake venom antitoxin, insulin, ethanol and Treethanol, which is made from tree cellulose.

Recombinant DNA technology :

Technology involving removing, modifying and inserting the gene of interest .

rDNA technology requires :

Vectors

Plasmids Restriction

Enzyme

DNA Requence and templates

Living organism.

Producing Recombinant molecules:

Isolate the gene

Insert it in a host using a vector

Produce as many copies of the host as possible

Separate and purify the product of the gene

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Insulin:

Insulin is a hormone that lowers the amount of glucose, or sugar, in the blood. A low level of insulin always exists in the bloodstream, but the pancreas secretes more after a meal is eaten. Insulin is made and secreted in the islet beta cells of the pancreas.

Insulin decreases blood glucose levels by stimulating fat, muscle and red blood cells to take in glucose from the blood.

Blood glucose levels are measured in milligrams per deciliter, or mg/dL. Normal levels of blood glucose lie between 70 mg/dl and 100 mg/dl. If blood glucose levels rise significantly above 100 mg/dl, then insulin is released from the pancreas. If glucose levels fall below 70 mg/dL, the secretion of insulin from the pancreas decreases.

Insulin has been used in the treatment of diabetes mellitus since 1922 when Leonard Thompson became the first human to receive an injection of man-made insulin.

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Production:

1. Human insulin is extracted from pancreas cells and an insulin-producing gene is isolated.

2. A plasmid DNA is extracted from a bacterium and cut with restriction enzyme, forming plasmid vector.

3. Insert human insulin-producing gene into the bacterial plasmid vector to form the recombinant DNA of human insulin-producing gene.

4. Introduce this recombinant DNA into a bacterial cell to form the recombinant bacterium.

5. The recombinant bacteria multiply in a fermentation tank and produce human insulin.

6. Insulin is extracted, purified and bottled. It is then ready to be injected into diabetic patients.

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Vaccine:

Vaccines protects our body from life threatening diseases. They give us a healthy defense. The vaccine treatment for a particular infection is called “vaccination”.

History:

Use of cow pox virus against small pox in 1796 by Edward Jenner.‘Vacca’ meaning Cow.Rabies vaccine by Louis Pasteur in 1885. Vaccines laws were passed.

Production:

Step 1: Generation of the antigen

Step 2: Isolation of the antigen

Step 3: Purification

Step 4: Addition of other components

Step 5: Packaging.

Vaccine preparation:

Vaccine production has several stages. First, the antigen itself is generated. Viruses are grown either on primary cells such as chicken eggs (Influenza), or on continuous cell lines such as cultured human cells (Hepatitis A). Also bacteria are grown in bioreactors (Haemophilus influenzae).

Alternatively, a recombinant protein derived from the viruses or bacteria can be generated in yeast, bacteria, or cell cultures. After the antigen is generated, it is isolated from the cells used to generate it.

A virus may need to be inactivated, possibly with no further purification required. Recombinant proteins need many operations involving ultrafiltration and column chromatography.

Finally, the vaccine is formulated by adding adjuvant, stabilizers, and preservatives as needed. The adjuvant enhances the immune response of the antigen, stabilizers increase the storage life, and preservatives allow the use of multidose vials.

Combination vaccines are harder to develop and produce, because of potential incompatibilities and interactions among the antigens and other ingredients involved.

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Protropin (Human Growth Hormone) Production:

Growth hormone inadequacy (GHI) occurs when the pituitary gland, at the base of the brain,

fails to produce enough growth hormone to stimulate growth.

According to the Human Growth Foundation, an estimation of 15,000 children in the United States have GHI.

Protropin:

Protropin was approved for treating human growth hormone deficiency in children in May 1985. It has also been approved to be used in 67 countries where it is marketed by licenses.

It is the first biotechnology-derived human growth hormone treating thousands of children with GHI.

Protropin (Synthetic versions by the trade names of Somatrem and Somatropin etc.) is administered by injection during childhood to stimulate and regulate body growth.

Production of Protropin:

1. A gene that produces growth hormone in humans is isolated.

2. The growth hormone production gene is inserted into the DNA of E.Coli bacteria.

3. The bacteria recognise the inserted DNA as its own DNA and begin to produce human growth hormone.

4. The bacteria multiply and produce the growth hormone in a culturing media.

5. The growth hormone is extracted and purfied and is then ready to be injected into children with GHI.

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Monoclonal Antibodies:

The technique of monoclonal antibodies (Mabs) becomes an increasingly important tool for diagnosing diseases.

Besides pregnancy testing and cancer screening, medical uses of Mabs are also applied in the diagnosis of hepatitis B, cystic fibrosis and sexually transmitted diseases including AIDS.

Advantages of Monoclonal Antibody as Diagnostic Tool

1. Rapid testing

2. High sensitivity

3. Less material needed to be taken from patients

4. Involvement of inexpensive equipment.

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ELISA Test - A Common Method used in Monoclonal Antibody:

ELISA stands for enzyme-linked immunosorbent assay.

In an ELISA test, a micro-well is pre coated with an antibody.

The sample to be tested is then added and any antigen may attach to the precoated antibody.

A second antibody, labelled with an enzyme, is then added. It will be specifically immobilised to antigen, which has bounded to the antibody, in the well.

Unbound reactants are then washed away and a colourless enzyme substrate is added. Colour develops as the enzyme acts on the substrate.

The colour intensity is measured with a photometer. The higher the intensity, the higher the antigen content is in the sample.

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Antibodies?

An antibody is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target. Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell.

Polyclonal antibodies are antibodies that are derived from different cell lines. They differ in amino acid sequence.

Characters of Monoclonal Antibodies:

Monoclonal antibodies (mAb) are a single type of antibody that are identical and are directed against a specific epitope (antigen, antigenic determinant) and are produced by B-cell clones of a single parent or a single hybridoma cell line. A hybridoma cell line is formed by the fusion of a one B-cell lymphocyte with a myeloma cell. Some myeloma cells synthesize single mAb antibodies naturally.

Production of monoclonal antibodies:

Since 1975, when Keller and Milstein developed hybridoma technology,

technological strides towards the production of antibodies have been made.

The first success in generating human mAbs (hmAbs) with predefined specificity was conducted in 1980 through ,

the fusion of human spleen cells from patients with human myelomas.

Since then, several major methods have been established to generate hmAbs, including

1) immortalization of antigen-specific human B cells;

2) acquisition of antigen-specific human B cells via phage display technology;

3) the production of hmAbs from transgenic mice;

4) single human B cell cloning techniques to directly clone and express immunoglobulin (Ig) genes in vitro from antigen-specific B cells.

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Conventional hybridoma technology :

Step 1: Immunization of mice :

Mice are immunized with an antigen (attached to adjuvant).

The antigen can be whole cells, membrane fragment, or complex molecules. Mice serum’s are screened using various techniques such as ELISA. When sufficient titre is reached the mice are euthanized and spleen is removed as a source of cells for cell fusion.

Step 2: Preparation of Myeloma Cells:

Myeloma cells are immortalized cells that are capable of dividing indefinitely. These cells are treated with 8-azaguanine to ensure sensitivity to HAT

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Step 3: Fusion of myeloma cells with Spleen cells:

Spleen cells harvested from mice are fused with myeloma cells. ,through co-centrifuging in polyethylene glycol. Cells are plated in selection medium .Hypoxanthine-aminipterin-thymidine (HAT) selection medium –blocks nucleotide synthesis. Only fused cells with grow on HAT. Cells are distributed on feeder cells (murine bone-marrow) to promote growth of the hybridomal cell

Step 4: Cloning of Hybridoma cells:

A mouse is inoculated with the cell and thereby becomes a factory for producing the MAbs. Ascites fluid collected from the mouse

Step 5: Antibody is screened and purified:

Antibodies are screened using specific Ag binding. Advantage of in vivo

Step 6: Desired antibodies are cloned:

This is done in vitro on culture bottle.

Mechanism of action:

Monoclonal antibodies are proteins that bind to other proteins. In cancer treatment, monoclonal antibodies can be used to bind to proteins that are on the surface of cancer cells. The result is that the cancer cells can be killed by the immune system, or even by the antibody binding process itself. The image below provides a nice picture of what monoclonal antibodies look like and how they bind to the surface of cancer cells.

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Some lists of monoclonal antibodies :

Thank you !

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