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biotechnology final.pptx
- 1. Biotechnological aspects of product Development , Concepts and technologies’ PRESENTED TO: DR. ASAD ULLAH MADNI PRESENTED BY: MISBAH HAROON SAFINA SOHAIL
- 2. biotechnology concept History Drugs from biotechnology Route of administration Process of development of biotechnological drugs Techniques for biotechnological drugs Applications of biotechnological drugs
- 3. Concept Biotechnology is the use of biology to develop new products, methods and organisms intended to improve human health and society Biotechnology is used in various fields including agriculture, food science and pharmaceuticals
- 4. Definition Biotechnology “Biotechnology is the use of biological process, organisms or systems to manufacture products, intended to improve the quality of life .” Pharmaceutical Biotechnology companies use recombinant DNA technology, which includes genetic modification of cells, or monoclonal antibody for making their biotechnological products
- 5. What are biotech. drugs The products or the drugs produced by biotechnology are also called biological, biotechnological drugs ,biological drugs or biopharmaceuticals. True biotechnological products are manufactured in live biological systems known as expression systems.
- 6. The modern practice of biotechnology draws from various disciplines of science and technology , including the following :molecular biology Chemistry Bionics Genetic engineering Informatics Nanotechnology Genomics
- 7. History Based on the work of the scientists, genetic engineering was developed in 1973. This method is the foundation of modern biotechnology practices and recent advances. It enabled the first direct manipulation of plant and animal genomes, which is the complete set of genes present in a cell.
- 8. Examples of Biotechnological drugs Examples of Classes of Protein-based Biotech drugs are, Erythropoietin(EPO) Blood factors(Factor VII) Human growth hormone, HGH, Somatotropin Cytokines i)Interlukins ii)interferon
- 9. Route of administration The route of administration of biotechnology drug from a traditional drug taken as capsule or pill. these drugs are given intravenously, subcutaneously or intramuscularly. There are also biotech drugs given to patient 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 version so that they Can be self- administered at home with an auto injector device e.g Insulin
- 10. Process of Development of Protein Biotech Product: Isolation of gene of interest. Introduction of gene to expression vector. Transformation into host cells Growth of cells isolation&purifi cation of protein Formulation of protein product
- 11. Isolation of gene of interest: Genes are pieces of DNA which store information for specific proteins that control specific traits. Genes are present in nucleus chromosomes which code for one polypeptide. The double strand of DNA are cut at specific site by special enzymes which are known as restriction endonucleases. Ligases enzymes used to joined pieces of DNA through covalent bond.
- 12. Through these enzymes DNA containing genes of interest is cut and joined with vector. A vector, as related to molecular biology, is a DNA molecule (often plasmid or virus) that is used as a vehicle to carry a particular DNA segment into a host cell as part of a cloning or recombinant DNA technique
- 14. 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 extraction Purification Fragmentation
- 16. • 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 hybid DNA is then introduced into a chosen cell for reproduction and synthesis.
- 17. Transfer of Gene to Expression Vector: Different organism may be used to express a target protein. The most commonly used organism is Bacterium and 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 in Baculovirus Mammalian cell culture Plants Animals
- 18. Transformation into host cell: Vector containing gene of interest is transformed into host cells.
- 19. 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): For growth on small scale incubators are used. The most commonly used is shake flask incubator For productions (large scale): Fermentors and Bioreactors are used.
- 20. 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
- 21. Fermentors and Bio reactors Bioreactor or fermentor is a container in which substrate is turned into product where gene of interest is mass produced. Types of production process are: Batch Continuous Fed batch
- 22. 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
- 23. Advantages and disadvantages of batch operations Advantages: Ease in operating Genetic stability of organism could b 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
- 24. 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.
- 25. 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
- 26. 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 run Possible to control the rate of growth of the microorganisms or the concentration of the biomass by controlling the fee parameters Most commonly used process in industry
- 27. Isolation & Purification of Protein: Protein is isolated from microorganism and purified. For isolation of extracellular products destruction of cell is not necessary. For intracellular cell disruption is needed which is done through following methods, Detergents lysis Enzymatic lysis Osmotic lysis Freeze-thaw cycles Ultrasonication Homogenization
- 28. Formulation of protein products: Freeze drying or lyophillization is the common technique used for protein product formulation. Finally protein biotech drug are prepared.
- 29. Techniques of biotechnology Monoclonal antibody gene therapy
- 30. PRODUCTION OF MONOCLONAL ANTIBODIES Monoclonal antibodies are produced by Hybridoma technology. HYBRIDOMA TECHNOLOGY: Hybridoma technology involves fusion of plasma B cells and myeloma cells resulting in production of hybrid cells.
- 31. STEPS INVOLVED IN MONOCLONAL ANTIBODY PRODUCTION Immunization Of Mice Screening Of Mice For Antibody Production Fusion of Myeloma Cells with Immune Spleen Cells Separation Of fused Hybridoma Cell and Screening Cloning of Hybridoma cells
- 32. Step 1: - Immunization of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells The first step in making a hybridoma is to generate antibody producing B cells. This is done by immunizing a mouse against the antigen of interest. IP (intra peritoneal injections) are more commonly used route of administration for immunization.
- 33. Step 2: - Screening Of Mice for Antibody Production After several weeks of immunization, tests are performed and examined for the presence of antibodies If the host is producing the desired antibody, the spleen is removed and dissociated in culture medium to release the resident B cells.
- 34. Step 3:- Fusion of Myeloma Cells with Immune Spleen Cells Plasma cells producing antibodies are fused with myeloma cells to produce hybridoma cells. Hybridoma cells take the advantage of both cells to mass produce antibodies of interest. Fusion can be enhanced using various methods; Chemical agents e.g. PEG (Polyethylene glycol) Physical agents e.g. Electro fusion
- 35. Step 4:- Isolation Of Fused Hybridoma And Screening After fusion three types of cells are present in mixture; Un fused myeloma cells Un fused spleen cells Fused (hybridoma ) cells It is necessary to separate hybridoma cells from un fused myeloma and plasma cells. Selection of hybridoma cells is performed by using HAT (hypoxanthine, aminopterin, thymidine) medium.
- 36. Step 5: - Cloning of Hybridoma Cell Lines Clone each positive culture Test each supernatant for antibodies Expand positive cultures either In vitro low concentrations are produced (1-10 microgram/ml) In vivo high concentrations are produced (1-10 mg/ml.
- 37. Gene therapy Gene therapy: It is an experimental technique for correcting defective genes that are responsible for disease development. Types of gene therapy: Somatic gene therapy Germ line gene therapy
- 38. Somatic gene therapy Somatic gene therapy involves transfer of a section of DNA to somatic cells of the body. Not passed to future generation. Short lived Appropriate and acceptable for many disorders such as: Cystic fibrosis Muscular dystrophy cancer Infectious diseases
- 39. Types of somatic gene therapy Ex vivo gene therapy: Cells are modified outside the body and then transplanted back into the body. In- vivo gene therapy: Genes are changed in cells when the cells are still in the body.
- 40. Germ line gene therapy Germ line gene therapy is transfer of a section of DNA to germ line cells. Effects of gene therapy Result in permanent changes. Effects passed to future progeny. Possibility of eliminating some diseases from a particular family.
- 41. Issues in biotech products Issues related to the use of protein based drugs: Drug delivery Denaturation or chemical alteration Rapid liver clearance antigenicity Foreign protein may induce allergic reactions Stability Denaturation leads to loss of 3D conformation of protein Covalent bond breaking at high pressure and low pH
- 42. Sterility consideration of biotech drugs It is impossible to sterilize the end product therefore it is important that a) All the raw material should be sterilized b) All the equipments should be sterilized c) processing should be carried out in aseptic environment
- 43. Quality control Viral decontamination There is no well determined mean to detect viruses in cell culture Bacterial decontamination Filtration sterilization of the final product by bacterial filter “0.22 micro meter membrane filter Pyrogen removal
- 44. Applications of biotechnological products Vaccines used to stimulate the immune system against a particular disease Recombinant DNA vaccine eg Hepatitis A and hepatitis B vaccine and lyme disease vaccines are commonly available. Monoclonal antibodies Trastuzumab used to treat metastatic breast cancer
- 45. Gene therapy The potential scope of gene therapy is enormous. Following are examples of potential gene therapies Immune deficiencies Hereditary blindness Hemophilia Parkinson’s disease Cancer Diabetes
- 46. Forensic applications: DNA fingerprinting is classic example of forensic application. It is most widely used for law enforcement and crime scene investigation