Biotechnological aspects of Product Development, Concept

and Technique

What is Biotechnology

• It is the use of biological organisms, systems, or processes to develop technologies and products to improve the quality of life.

• Biotechnology is used in various fields including agriculture, food science, and pharmaceuticals.

• Pharmaceutical Biotechnology companies use recombinant DNA technology, which includes genetic modification of cells, or a monoclonal antibody for making their biotechnological products.

What are Biotech. Drugs• Biotech is the term used for biotechnology or products

produced by biotechnology. These drugs are also called biologicals, biotech drugs, biological drugs, or biopharmaceuticals. True biotech products are manufactured in live biological systems known as expression systems.

• Biopharmaceuticals are physically very different from small molecule drugs generally sized at 1 kDa

• Examples of Classes of Protein-based Biotech drugs,Erythropoietin (EPO) Blood factors (Factor VII)Human Growth Hormone: HGH, Somatotropin, Cytokines

i) Interleukins ii) Interferon

Route of administration

• The route of administration of a biotech drug is very different from a traditional drug taken as a pill or capsule, and each drug is developed with a unique route of administration. These drugs are

• mainly given intravenously, subcutaneously or intramuscularly• There are also biotech drugs given to patients by intrathecal,

intraarticular, and inhalation routes. • They cannot be given orally because they would be degraded in the

gastrointestinal tract. • Most biotech drugs are given in the clinic but for some chronic• indications the trend is to develop subcutaneous versions so that they• can be self-administered at home with an auto injector device. For

example Insulin

Process of development of protein Biotech product

1. Isolation of gene of interest.2. Introduction of gene to expression vector.3. Transformation into host cells.4. Growth of cells5. Isolation & purification of protein.6. Formulation of protein product.

Isolation of gene of interest• Genes are pieces of DNA which store information for

making specific proteins that control specific traits. Genes are present in nucleus chromosomes which code for one polypeptide.

• The double strand of DNA are cut open at specific site i.e Nucleotide sequence by special enzymes which are known as Restriction Endonucleases.

• Ligases are the enzymes used to joined pieces of DNA through covalent bonds.

• Through these enzymes DNA containing genes of interest is cut and joined with vector.

DNA Recombination• DNA recombination is a process in which the pieces of DNA from

different organisms are artificially mixed to create Recombinant DNA.

• Steps involved in Recombinant DNA technology are 1. DNA extraction2. Purification3. Fragmentation.

• Fragmentation of DNA is done by specific 'restriction' enzymes and is followed by sorting and isolation of fragments containing a particular gene. This portion of the DNA is then coupled to a carrier molecule called as PLASMID.

• The hybrid DNA is then introduced into a chosen cell for reproduction and synthesis.

DNA Recombination

Transfer of gene to expression Vector

Different organism may be used to express a target protein, the expression vector used therefore will have elements specific for use in the particular organism. The most commonly used organism for protein expression is the bacterium, Escherichia coli. However not all proteins can be successfully expressed in E. coli, and other systems may therefore be used such as• Yeasts e.g commonly used for protein expression is Pichia

pastoris.• Baculovirus.• Mammalian cell culture.• Plants.• Animals.

Growth of cells

• After the transfer of rDNA into host of choice , i.e bacteria, yeast, Ecoli they are cultivated in culture medium.

• For research (small scale):– Cell culture flasks

• For productions (large scale):– Fermentors and Bioreactors

Culture Media

• It is important to provide nutritional conditions that exist in bacterial natural habitat.

• Common components:• Water• Source of carbon and energy• Source of nitrogen• Trace elements• Growth factors• Buffer

• For growth on small scale incubators are used ,the most commonly used is shake flask incubator.

Fermentors and bioreactors

• Bioreactor or fermentor is a container in which substrate is turned into product, where gene of interest is mass produced.

• Production Process:

• Types of production processes are:1. Batch2. Continuous3. Fed Batch

• The mode of feeding determines the classification of the bioreactor.

Batch Process

• In this process the bioreactor is only fed once.

• The bioreactor will be allowed to run till completion

• Very difficult to achieve in real life because there should be no input to or withdrawal from the bioreactor even for sampling.

Advantages and disadvantages of batch operations

ADVANTAGES• Ease in operating. • genetic stability of organism could be

controlled if it is genetically engineered biocatalyst.

• lower contamination risk.Disadvantages• non-productive down time.• batch to batch variability is problem.• accumulation of inhibitory product.

Continuous Process

• The bioreactor is fed continuously• The amount of feed introduced into the

bioreactor equals the removed volume.• The process is sensitive and subjected to

influence from various factors.

Advantages and disadvantages of continuous operations

Advantages • efficient, higher productivity.• Uniform quality of product.• no accumulation of inhibitory products.Disadvantages• destruction of biocatalyst.• higher contamination risk.

Fed Batch Process

• In fed Batch process , one or more nutrients are fed to the bioreactor during cultivation and in which the product remain in the bioreactor until the end of the run.

• Possible to control the rate of growth of the microorganisms or the concentration of the biomass by controlling the feed parameters.

• most commonly used process in industry.

Isolation & purification of protein.

• There is necessity for the isolation and purification of protein prepared for Biotech drugs from micro organisms.

• The proteins required may be extracellular or intracellular so for isolation of extracellular products destruction of cell is not necessary but for intracellular cell disruption is needed which is done through following methods– Detergents lysis– Enzymatic lysis– Osmotic lysis– Freeze-thaw cycles– Ultrasonication– Homogenization

Isolation & purification of protein

• Differential centrifugation(Separation of proteins or any material on the basis of their size, mass, and density)

• Dialysis (Based on osmotic pressure)• Differential solvent precipitation• Gel electrophoresis• Column chromatography

Issue in Biotech products

Issues related to the use of protein based drugs• Proteins versus low molecular weight drugs

Proper 3D structure required for biological activity

1. Drug Delivery• Denaturation/chemical alteration

Rapid liver clearance

2. ANTIGENICITY:

Foreign proteins may induce allergic reactionsI. AnaphylaxisII. loss of efficacy

3. Stability

• Denaturation leads to loss of proper 3-D conformation of proteins.

• Covalent bond breaking at high temperature and low pH.

Sterility consideration of Biotech drugs

Sterilization:It is impossible to sterilize the end product therefore it is important thata. All the raw material should be sterilized.b. All the equipment must be sterlized.c. Processing should be carried out in aseptic

enviornment.

Quality control:I. Viral testingII. Bacterial testingIII. Pyrogen testing

• Viral decontamination:• There is no well-determined mean to detect viruses in the cell

culture.• Bacterial decontamination:• Filtration sterilization of the final product by bacterial filter “0.22 mm membrane filter.

• Pyrogen Removal:• depyrogenation refers to the removal of pyrogens such a

“endotoxins” from solutions• Pyrogen testing:1. Rabbit test2. LAL TEST(LIMULUS AMEBOCYTE TEST)

• PROTEIN CONTAMINANTS:• SOURCES

1. Growth media 2. Host cells3. Ligands from affinity chromatography columns

Biotech drugs

Applications of biotechnological Products:

1.Vaccines • Used to stimulate the immune system against

a particular disease• Recombinant DNA vaccine e,g• Hepatitis A and hepatitis B vaccines and Lyme

disease vaccines are commercially available • Interferon alfa n-1 is also available for chronic

hepatitis C.

2. Monoclonal antibodies• Trastuzumab monoclonal antibody used to treat metastatic

breast cancer.• palivizumab monoclonal antobodies used to treat respiratory

syncytial viral and fetal pneumonia.• Adalimumab prepared by abbot for rheumatoid arthritis.

3. GENE THERAPY:• Exogenous genetic material is transferred into somatic cells

to correct an inherited or acquired defect.• Gene therapy has been applied to diseases such as cystic

fibrosis, hemophilia, sickle cell anemia and diabetes.• Gene therapy has been used to treat adenosine deaminase

deficiency (ADA).

• Forensic applications:• DNA fingerprinting is the classic example of a

forensic application. It is used most commonly for law enforcement and crime scene investigation.