L4 - MICROBES IN BIOPHARMING and its uses.pptx

L4 - MICROBES IN BIOPHARMING and its uses

1. Nature of microbes used as biofactories : Microbial production of insulin, recombinant proteins of therapeutic or industrial value V. Rajanbabu BTB 404 Molecular Pharming (1+1) B. Tech. (Biotech.), CPMB&B, TNAU, Coimbatore Lecture 4 1

2. Why we have to choose this technology Microbial technology is relatively simpler compared with the other systems In Vitro maintenance does not require special components Unicellular nature of microbes In Vitro maintenance does not require special components Simple genome Diverse ecosystems and ecological distribution 2

3. Potential of a Microbes Microorganisms are capable of growing on a wide range of substrates and can produce a remarkable spectrum of products 3

4. Microbes suitable for biopharming PROKARYOTIC   Unicellular:     Bacteria Multicellular : Cyanobacter  EUKARYOTIC    Unicellular:      Yeasts, Algae   Multicellular :    Fungi, Algae 4

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6. Approved rDNA technologyproducts in therapeutics Hormones Growth hormone, Insulin, Calcitonin , FSH Cytokines Interferons (Interleukins), EPO, CSF, Neurotrophic factors Clotting factors Factor VIII, Factor IX Vaccines Hepatitis B, acellular pertussis vaccine ( Bordatella pertussis , whooping cough) Monoclonal antibodies 6

7. Therapeutic products approved in India Human Insulin Streptokinase Erythropoietin Hepatitis B vaccine Human growth hormone Human interleukin Granulocyte macrophage Alpha-interferon, Gamma-Interferon, Blood factor VIII Follicle stimulating hormone Granulocyte colony stimulating factor (GCSF), colony stimulating factor 7

8. Companies involved in Therapeutic products Shantha Biotech Pvt. Ltd., Bharat Biotech Wockhardt biotech park Reddy Labs Panacea Biotech, Biological E limited, Virchow Biotech etc. Have initiated the production of several of these therapeutics and some growth factors in India 8

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11. Pilot-Scale process studies 11

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14. Microbial production of insulin 14

15. What is insulin? Hormone secreted by the pancreatic beta cells  facilitates the uptake of glucose into skeletal muscle and adipose tissue by increasing the number of glucose transporters ( GLUT 1 and GLUT 4) that facilitate glucose diffusion into these targeted cells contains 51 amino acids  DM: Type 1(IDDM or juvenile-onset) and Type 2 (NIDDM or adult onset) Diabetes  15

16. Why is insulin needed? Protein hormone produced by beta cells of islets of Langerhans in the pancreas Regulates blood sugar by allowing uptake of glucose from bloodstream into body cells Patients with diabetes have insufficient or impaired production of insulin 16

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19. Why synthesize human insulin? Patients’ immune systems do not produce antibodies against human insulin as they do with canine, bovine or porcine insulin Projected decline in the production of animal-derived insulin Need for a more reliable and sustainable method of obtaining the product 19

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21. Structure of Insulin Two polypeptide chains; one with 21 amino acids and the second with 30 amino acids Chains are linked via a disulfide bond Gene encoding the insulin protein is found on chromosome 11 21

22. Molecular mechanism of regulation of blood sugar 22

23. Recombinant DNA Technique Restriction enzymes used to cut out insulin gene and to cut a bacterial ( E. coli ) plasmid at the same “sticky ends” 23

24. Microbial biopharming for Insulin Mutant strains of E. coli used to avoid bacteria attacking “foreign” genes Insert insulin gene next to E. coli B-galactosidase gene which controls transcription Bacterial cells replicate and make copies of insulin gene 24

25. Recombinant DNA Technique Insulin protein is purified (B- galactosidase removed) Chains are mixed and disulfide bridges form Final product is Humulin - chemically identical to human insulin 25

26. Insulin purification The two chains are then mixed together and joined by disulfide bonds through the reduction- reoxidation reaction by an oxidizing agent Then insulin purifued from the mixture through chromatography http://www.madehow.com/Volume-7/Insulin.html#ixzz3LCxSsvDs 26

27. Insulin Drugs Rapid acting Short acting Intermediate acting Long acting 27

28. Insulin Injections: Rapid acting Acts within in 15 minutes after injection. 2-4 hours Insulin Aspart ( Novolog ):   proline was replaced by Aspartic acid in position B28. Insulin Lispro ( Humalog ): B28 Lys and B29 Proline Human insulin analog Insulin glulisine : asparagine at position B3 replaced by lysine, and lysine at B29 replaced by glutamic acid 28

29. Insulin Injections: short acting Starts within 30-60 min; duration:6 to 8 hours   Regular Insulin ( Humulin or Novolin ) administered via IV route Comes from Pork(porcine), beef(bovine) or Human pancreas or genetically engineered using rDNA technology from certain microorganisms( E.coli ) 29

30. Insulin Injections: Intermediate Acting Onset:1 to 2 hours; duration:10 to 16 hours Isophane Insulin Suspension or Neutral Protamine Hagedorn (NPH) ( Humulin N and Novolin N) . A sterile suspension of Zinc Insulin crystals and Protamine sulfate—a protein from the sperm of a fish ( Onchorynchus Fam. Salmonidae ) Insulin Zinc Suspension ( Lente ). A sterile suspension of insulin modified by the addition of Zinc chloride. Less allergic reactions than NPH. 30

31. Insulin Injections: Long Acting Insulins Onset:2 hours;duration:24 hours Glargine ( Lantus ): asparagine at position A21 is replaced by GLycine and an additional 30B-a-L-ARGinine-30B-b-L- arginINE .  is the only true 24-hour insulin available. Extended Insulin Zinc suspension(Ultra Lente ): insulin modified by the addition of Zinc chloride. Onset:4 to 8 hours:duration:36 hours Insulin detemir  : neutral soluble human insulin analogue in which the terminal amino acid at B30 has been replaced by myristic acid, a 14-carbon fatty acid chain. 24h. 31

32. Human growth hormone ( hGH , or Somatotropin ) Secreted by the pituitary gland Responsible for normal body growth and development Stimulates protein production in muscle cells, energy release from the breakdown of fats Stimulates the development of bones Inadequate production of GH results in short stature, defined as a below normal height for a given age 32

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35. Codons 0 24 191 HaeIII 0 24 24 191 Discard Retain Synthetic leader sequence Expression of somatotropin Lac promoter lacZ’ somatotropin gene E. coli transformation Somatotropin is synthesized Production of recombinant Somatotropin cDNA fragment 35

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38. Lac promoter lacZ’ Artificial somatostatin gene β –galactosidase segment met Somatostatin fusion protein Transformation into E. coli Cyanogen bromide Cleaved somatostatin Production of recombinant somatostatin 38

39. Production of Growth Hormones 39

40. IGF-I in bacteria as a secreted form fused to secretion leader sequences 40

41. recombinant human insulin-like growth factor-I ( rhIGF -I) in yeast ( S. cerevisiae ) rhIGF -I is a 7.5kDa protein containing 70 amino acid residues rhIGF -I is also known as Somatomedin -C is effective and its use apparently involved no special hazards widely exploited in humans 41

42. Erythropoietin (EPO) Human Eryhtopoietin is produced in kidney A glycoprotein, acts on the bone marrow to increase the production of red and white blood cells. Stimuli such as bleeding or moving to high altitudes (where oxygen is scarce) trigger the release of erythropoietin Known as EPO, MW 30400 Kda , 165 amino acids in human (192 Mouse) Has been widely used in AIDS for development of immunity 42

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44. EPO production When RBC count comes down, Oxygen level decreases in the blood Kidney cells specifically sense the oxygen deficit in the blood and start producing Erythropoietin EPO Abuse Sports to improve endurance Now detected from naturally occurring EPO by protein markers produced during post injection phase 44

45. Structure of EPO ( A Glycoprotein of 165 amino acids) 45

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47. Tissue Plasminogen Activator (t-PA)  Drug against stroke Genentech has cloned human t-PA for use in treating unwanted or life threatening blood clots Activase ( Alterplase recombinant) is the trade name Approved by FDA in 1987 Fastest drug development projects in history, with only 7 years 47

48. primary structure of t-PA 48

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50. Cysteine oxidoreductases DsbA and DsbC were expressed in the bacterial periplasm , and catalyzes disulfide bond isomerization to get active protein 50

51. producing disulfide-dependent proteins in bacteria 51

52. Recombinant Hepatitis vaccine Produced in bakers’ yeast ( Saccharomyces cerevisiae ) Based on the surface antigen purified from the blood of chronically infected individuals First vaccine to be produced using recombinant DNA technology (1986) 52

53.  Early Traditional vaccine Growing the live virus in animals and then inactivating it by chemical treatment Risk of infection during the manufacture, and in the delivery of the vaccine Animal rights issues 53

54. Recombinant HBV vaccine Gene coding for the S- HBsAg is isolated and cloned into a Vector Under the control of a strong promoter Expressed in Yeast  Recombinant protein product of the Hepatitis is obtained The protein is later purified and used to for vaccination 54

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56. Food Additive - Aspartame L- phenylalanyl -L- aspartyl -methyl ester low-calorie artificial sweetener Synthesised biocatalytically by peptide synthesis using a thermostable protease – Thermolysin ® from the facultative thermophile , Bacillus thermoproteolyticus 56

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58. Production of recombinant GH Constructing expression cassette with hGH cDNAs inserts Cultivating the recombinant clones in small scale flask/bioreactor Producing the hGH in pilot scale bioreactors Developing large scale purification procedure and process chromatography optimization(Affinity chromatography)  E. coli as an alternative for using CHO cells, with the advantage that rhGH is secreted into protein-free production media 58

59. Monoclonal Antibodies ( mAb ) Using Microorganisms as Cell Factories Monoclonal antibodies are specific antibodies which bind to the particular site of proteins known as epitope Production of the monoclonal antibodies was done from the identical immune cells Viruses and yeast as a cell factories for the production of monoclonal antibodies mAB production from the fungal species is preferred over the  E. coli  Fungal systems are extracellular which make protein separation process easier Common fungal organism is the  Aspergillus niger 59

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