Download to read offline
More Related Content
Similar to Stabilization of for protein production of protein drug for clinical trial-2-1
Stabilization of for protein production of protein drug for clinical trial-2-1
- 1.
- 2. RAMSHEENA PAYAMBROT 3RD SEMESTER DPTOF MSC. BIOTECHNOLOGY SIAS Stabilization of protein- Production of protein drug for clinical trial
- 3. Contents Introduction Recombinantprotein drugs Importance of insulin Recombinant insulin approaches Human insulin production Chain A & B method Pro-insulin method Applications Conclusion References
- 4. Introduction “Protein whichare engineered in the laboratory for pharmaceutical use are referred to as therapeutic proteins” Proteins which are absent or low in individuals with an illness such as Cancer ,Infectious diseases, Hemophilia BC, etc. are artificially synthesized on large scale through genetically modified host cells and delivered
- 5. This therapeuticapproach in treating diseases using proteins and peptides is termed protein therapeutics Protein therapy is similar to gene therapy, but unlike gene therapy, protein therapy delivers protein to the body in specific amounts (as would be ordinarily present), to help repair illness, treat pain or remake structures In 1920’s Human insulin is considered to be the first therapeutic proteins
- 6. What is Recombinantprotein? • Recombinant protein are protein that are artificially made through the recombinant DNA technology • It provides a more efficient method to obtain large amount of proteins • Protein can be used in many areas such as diagnostic tools, vaccines, therapeutics, detergents , cosmetics, food production
- 7. Recombinant protein drugs Hormonepeptide drug Human hematopoitic factor Human cytokines
- 8.
- 9. Production of recombinantinsulin • Initial attempts for the production of recombinant insulin started in the late 1970s • First trial of human trial of recombinant insulin was conducted in 1980 Insulin was the first recombinant DNA technology product administrated to humans Trial was successful and which lead to exploration of r DNA technology for the pharmaceutical products
- 10. Insulin Is apolypeptide hormone produced by the B cells of the islets of Langerhans in the pancreas Its main function is enabling the cells to take up glucose (providing it with energy it needs) Its required for normal glucose homeostasis
- 12. Diabetes mellitus DMis a genetically linked disease which is commonly found in general population Elevated blood glucose level is the characteristics of this disease Its due to the inefficient production of insulin Lack of efficient insulin , glucose accumulate in blood Elevated glucose levels, exceeding 180mg dL can result in the excretion of glucose in urine
- 13. Importance of insulin Without insulin, the blood glucose builds up in the blood and the cells are starved of their energy source Some of the symptoms that may occur include
- 14. The cellswill begin to use fat ,the energy source stored for emergencies When this happens for too long a time the body produces ketones, chemical produced by the liver Ketones can poison and kill cells if they build up in the body over an extended period of time. This can lead to serious illness and coma
- 15. complications Nephropathy Retinopathy Neuropathy Heart disease etc
- 16. Recombinant insulin approaches Cows & pigs Immunogenic Yeast cells Costly Plant cell Not fully developed • E . Coli - Simple, well understood genetics -Its very easy to manipulate -Culturing cost is minimal -Fermentation easy to scale up -Ease of inclusion bodies purification •
- 17. Industrial production ofinsulin There two methods for the production of recombinant human insulin from genetically modified bacterial culture I. Two chain method (chain A & B method) II. Pro insulin method
- 18. 1- Chain A& B Method This method consists chemically synthesizing two oligonucleotide which encode the 21 amino acid A chain 30 amino acid B chain individually in two different E. coli
- 20. Upstream processing Step1: Obtaining of insulin gene
- 21. Step 2:Insertion of cDNA of both chains into plasmids
- 23. Step 3:Transfection Recombinant plasmids enter the bacteria in a process known as transfection Cacl2 treatment and electroporation can be used These cells are later known as transformed cells
- 24. Step 4:Media and equipment preparation The LB broth is prepared using LB powder Its autoclaved and ampicillin and lactose are added Inoculation is done by adding the transformed bacteria into the media Preparation of the bioreactor
- 25. Step 4- Fermentation: This stage consists of small scaling( culture in shake flask) to large scaling (fermentor) Two chain are grown separately The small scaling(early stage) uses shake flasks to do the enrichment culture method for selecting the desired type of E. coli for fermentation
- 26. The largescale where transfected bacterial cells are transferred from small flask and replicated under optimal condition such as temperature, pH in fermentation tank Monitoring & control step The bacterial cell process turn on the gene for human insulin chain and then insulin chains are produced in the cell
- 27. Down stream processing When the products are subjected to series of processes including separation and purification which are collectively known as Downstream processing • It is also known as product recovery step 6: Isolation of crude product Cells are removed from tank and are lysed using different methods such as enzyme digestion, freezing etc. The enzyme lysosome is used to digest the outer layer of the bacterial cells
- 28. Step 7:purification of crude product Centrifugation is conducted to helps to separate the cell components Stringent purification must be taken to remove any impurities Several chromatographic methods such as gel filtration and ion exchanges are used
- 29. Step 8- Obtainingof insuin chain The protein isolated after lysis consists of the fusion of B – galactosidase and insulin chain Its due to there is no termination or disruption Cyanide bromide is used to split the protein chain at methionine residues, allowing the insulin chains to be obtained
- 30. Step 9 –synthesis of active insulin
- 31. Step 10– PR- HPLC to obtain highly purified insulin: Its performed lastly to remove almost all the impurities to produce highly purified insulin The insulin then can be polished and packaged to be sold in the industries
- 33. Synthesis ofthe DNA containing the nucleotide sequences of the A& B polypeptides chains of insulin Plasmid + restriction endonuclease – insertion of the insulin gene into plasmid ( circular DNA) Restriction enzymes cut plasmidic DNA DNA ligase agglutinates the insulin gene and the plasmidic DNA .ie, plasmid + insulin gene
- 34. Introduction ofrecombinant plasmids into bacteria : E. coli E.coli = factory for insulin production Using E.coli- mutatants to avoid insulin degradation Bacterium reproduces—the insulin gene replicates along with plasmid E. Coli
- 35. Formed proteinpartly of a byproduct of A or B chain of insulin Extraction and purification of A and B chain Connections of A and B chain by reaction forming disulfide cross bridges results pure synthetic human insulin
- 36. 2- Pro insulinmethod Insulin is naturally synthesized as pre- pro insulinin the pancrease Its converted to proinsulin with the N- terminal signal peptide enzymatically removed The proinsulin coding sequence is inserted into the non – pathogenic E . coli bacteria Bacteria undergo fermentation , where they replicate and produce proinsulin
- 37. The connectingsequence between the A &B chain is then spliced away with an enzyme and resulting insulin is prepared The different downstream process is required for the proinsulin process as compared to the chain A & chain B process Enzymatic proteolysis is a unique step for the proinsulin production
- 40. Approval of recombinantinsulin The approval for using recombinant human insulin was given in 1982 In 1986, Eli Lilly received approval for the manufacturing and marketing of recombinant human insulin The recombinant human insulin was marketed under the trade name Humulin
- 41. APPLICATIONS Several proteins arecreated from recombinant DNA (recombinant proteins) and are used in medical applications. Hematopoietic growth factor. Interferon's Hormones Recombinant protein vaccines Tissue/bone growth factors and clotting factors Biological response modifiers Monoclonal/Diagnostic/Therapeutic antibodies Recombinant proteins is extensively used in biotechnology, medicine, and research
- 42. conclusion Protein andpeptide bases pharmaceuticals are rapidly becoming a very important class of therapeutic agents and are likely to replace many existing organic based pharmaceuticals in the very future Peptide and protein drugs will be produced on a large scale by biotechnology processes and will become commercially available for therapeutic use
- 43. References 1. Tripathi NK,Shrivastava A. Recent developments in bioprocessing of recombinant proteins: expression hosts and process development. Frontiers in Bioengineering and Biotechnology. 2019;7. 2. Cheung AT, Dayanandan B, Lewis JT, Korbutt GS, Rajotte RV, Bryer-Ash M, Boylan MO, Wolfe MM, Kieffer TJ. Glucose-dependent insulin release from genetically engineered K cells. Science. 2000 Dec 8;290(5498):1959- 62. 3. Glick BR, Patten CL. Molecular biotechnology: principles and applications of recombinant DNA. John Wiley & Sons; 2017 Jun 1.
- 44.