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  • 1. Production of Biopharmaceuticals – An Overview
    K.R.S.Sambasiva Rao
    Department of Biotechnology
    Acharya Nagarjuna University
  • 2. What are Biopharmaceuticals
    Biologically significant compounds like hormonesand proteinsuseful for treatment of variety of human health disorders, usually called as Biopharmaceuticals or Biotherapeutics or Biologicals, usually obtained from biological source and produced through industrial biotechnology
  • 3. Microorganisms as tools for production of various Biotherapeutics
    From genes to processes
    Gene resources
    Gene diversity
    Methods of gene discovery
    Expression of genes and production of gene products
    Use of organisms and enzymes as catalysts
    Environmental processes
    Fermentation processes
  • 4. From Genes to Processes
    Gene discovery
    Production and scale-up
    Engineering the catalyst
  • 5. Important Biotherapeutics
    Insulin- hormone which lowers blood sugar; used by diabetics
    Interferon- class of cytokines effective vs viral infections
    Factor VIII- blood protein necessary for clotting; missing in hemophiliacs
    Streptokinin - bacterial enzyme to dissolve blood clots in coronary arteries
    Beta endorphins- pain suppressors
  • 6. Microbes as Factories for Biotherapeutics
    Production of Biotherapeutics
  • 7. Why we have to choose this technology
    • The technology is relatively simpler compared with the other systems
    • 8. In Vitro maintenance does not require special components
    • 9. Their unicellular nature
    • 10. Their genomes are simpler
    • 11. Their ecological distribution is very diverse
  • Potential of a Microbes
    Microorganisms are capable of growing on a wide range of substrates and can produce a remarkable spectrum of products
  • 12. Which Microbes are useful
       Several species belonging to the following
    categories of microorganisms are useful
     PROKARYOTIC  Unicellular:     Bacteria, Cyanobacteria
        Multicellular: Cyanobacter
     EUKARYOTIC    Unicellular:      Yeasts, Algae
                             Multicellular:    Fungi, Algae
  • 13. Some potential microbes used in Microbial Technology
  • 14. Bacillus Sp.
    Actinimycetes Sp.
  • 15. Eschericia coli
  • 16. Saccaromyces cerevisiae
  • 17. Coprinus cinereus
  • 18. Biologicals vs Conventional Drugs
    Conventional Drugs
    • Protein or carbohydrate based product
    • 19. Extracted from living organism
    • 20. Complex physicochemical structure
    • 21. Less well-defined
    • 22. Macromolecule (> 500 kd)
    • 23. Tertiary structure
    • 24. Location, extent and type
    of glycosylation
    • Heat- & Shear- sensitive
    • 25. Synthetic, organic compounds
    • 26. Defined structure, physical & chemical characteristics
    • 27. Chemical synthesis
    • 28. Micromolecules
    • 29. Stable
  • More than 30 recombinant therapeutics have been approved globally for commercial use and several are on the way
    In India, 12-15 of these are presently being marketed. Many of these are being imported (excepting few like Hepatitis B vaccine, Insulin etc.) and consumed and now several are underway for indigenous production
    Globally approved recombinant therapeutics are broadly categorized into blood factors, hormones, growth factors, interferons, interleukins, vaccines and other miscellaneous therapeutic products
  • 30. Some approved Therapeutics
    The products of rDNA technology
    Growth hormone, Insulin, Calcitonin, FSH
    Interferons (Interleukins), EPO, CSF, Neurotrophic factors
    Clotting factors
    Factor VIII, Factor IX
    Hepatitis B, acellular pertussis vaccine (Bordatella pertussis, whooping cough)
    Monoclonal antibodies
  • 31. Therapeutic products approved in India
    • Granulocyte macrophage
    colony stimulating factor
    • Alpha-interferon,
    • 37. Gamma-Interferon,
    • 38. Blood factor VIII
    • 39. Follicle stimulating
    • Granulocyte colony stimulating factor (GCSF),
    Shantha Biotechnies Pvt. Ltd., Bharat Biotech International, Wockhardt, Reddy Labs and Panacea Biotech, Biological E limited, Virchow Biotechniques etc. have initiated the production of several of these therapeutics and some growth factors in our country
  • 40. Commercial production of Therapeutics
    The commercial production of recombinant
    Biologicals and therapeutics now became an
    important area in global industralization
    The process developed in production of
    therapeutics has to be taken to industrial scale
    for implementing these developed process in
    industrial scale and to get viable industrial
    production of these therapeutics or
    biologicals mainly through Fermentation
  • 41. And any Fermentation process should go for scale-up from
    laboratory scale
    Industrial level
    Pilot scale Fermentors
  • 42. Pilot scale Fermenter
  • 43. Industrial Scaleup process
  • 44. Production of Quorn™
    Marlow Foods, UK
    World largest (50 m tall and 155,000 litre capacity)
    airlift fermenter (1994)
  • 45. Commercial Production of Some important
  • 46. Insulin - first recombinant protein to be produced
    Insulin is an important hormone which regulates sugar metabolism
    An inability to produce insulin results in a form of diabetes, this disease can be treated by daily injections of insulin
    Historically, insulin from pigs or cows is used, but known to produce immune reactions in some patients
    Challenge: how to make human insulin to be used as a drug in cell systems or microbes?
  • 47. Recombinant Insulin overcome many problems
    Idea: take the gene of human insulin, clone into a plasmid, introduce the plasmid into E. coli or cells, and use them E.coli as “Biological Factory” for insulin production
    Amino acid sequence produced insulin (Contains 51 amino acids) and is identical to that of the “natural human protein” and it will not cause any immune reactions
    Much more economical than attempts to produce insulin by chemical synthesis
    So, how to do this?
  • 48. Strategy for insulin production
  • 49. Insulin crystals from the purification process
    Owen Mumford Ltd., UK
  • 51. Human growth hormone (hGH, or Somatotropin)
    Secreted by the pituitary gland, and is responsible for normal body growth and development, by stimulating protein production in muscle cells, energy release from the breakdown of fats and stimulates the development of bones
    These processes together are responsible for longitudinal growth. Inadequate production of GH results in short stature, defined as a below normal height for a given age
  • 52. In children and adolescents, the rate of growth in height is primarily determined by the rate at which endogenous GH is secreted
    The growth spurt during puberty is caused by increased secretion of GH
    Under normal conditions, GH secretion and growth rate remain increased until final height is reached, after which GH secretion is reduced to a steady state
  • 53. Structure of human Growth hormone
    Hormone binding to receptors
    Growth Hormone: 191 amino acids, single chain
    Teritiary structure
  • 54. Production of recombinant GH
    Isolating and constructing hGH cDNAs
    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
  • 55. Production of hGH
    Purification of recombinant human growth hormone (rhGH) from Chinese hamster ovary (CHO) cell culture supernatant by Gradiflow large-scale electrophoresis is described. Production of rhGH in using E. coli as an alternative for using CHO cells, with the advantage that rhGH is secreted into protein-free production media, facilitating a more simple purification and avoiding resolubilization of inclusion bodies and protein refolding. proteins
  • 56. Strategy for production of growth hormone
  • 57. Erythropoietin
    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
  • 58. Kidney is the principal production site of Erythropoietin
    Bones like Femur, Tibia, Vertebra, Sternum, Rib produce most RBC and WBC under the influence of EPO
  • 59. When EPO is produced
    When RBC count comes down (Oxygen level decreases) in the blood
    It will result in
    Kidney cells specifically sense the oxygen deficit in the blood and start producingErythropoietin
  • 60. Structure of EPO ( A Glycoprotein of 165 amino acids)
  • 61. EPO also has therapeutic Abuses
    Used in sports to improve endurance
    Now detected from naturally occurring EPO by protein markers produced during post injection phase
  • 62. Production of recombinant Erythropoietin
    Isolating and constructing human EPO cDNAs
    Subjecting the cDNA to PCR using primers based on the published sequence
    The PCR products will be cloned into vector for the purpose of propagation and subsequently engineered into appropriate expression vectors
  • 63. Production process….
    Genomic DNA, cDNA and manufactured DNA sequences coding for part or all the sequence of amino acid residues of EPO or for analogs thereof are incorporated into autonomously replicating plasmid or viral vectors employed to transform or transfect suitable prokaryotic or eukaryotic host cells such as bacteria, yeast or vertebrate cells in culture
    Upon isolation from culture media or cellular lysates or fragments, products of expression of the DNA sequences display, e.g. the immunological properties and in vitro and in vivo biological activities of EPO of human or monkey species origins will be tested
  • 64. Flow Chart of Production Process
    Vial Flask/ Roller Bottle
    (Cells) Spinner Bottle / Bio-reactor
    Purification Production
    Final Bulk EPO concentrate
  • 66. TPA Has Been Developed As A Drug By Genentech
    The biotechnology company Genentech has cloned human t-PA for use in treating unwanted or life threatening blood clots
    Activase (Alterplase recombinant) is the trade name of Genentech’s t-PA
    Activase is useful in treating heart attacks and strokes when administered within 5 hours of thrombosis formation or embolism lodging in the heart or brain
    The FDA approval in 1987 and medical use of Activase has a very interesting history
  • 67. Clot Dissolution
    From the Kidneys
    From Bacteria
    TPA mode of action
  • 68. Vaccines
    Vaccines effective against many viral infections anddiseases require the cultivation and mass production of the virus followed by its attenuation
    The drawback in this is that virus requires a living medium to replicate and multiply. Rather than the traditional concept- “Sacrifice one life to save many”, Animal cell culture can be employed to mass produce the virus
    Passively, Animal cell culture can be employed to reduce the virulence of particular virus strains by cultivating them on cells other than target cells, in which the virus infection followed by repeated passaging will be performed
    The cell-culture process for vaccines offers high potential as an alternative method to egg-based production. Cell culture has the capability to offer a predictable, rapid and responsive method for production of well-tolerated and effective vaccines, with low levels of adverse events similar to egg-based vaccines
    Cell-culture materials can be stored, so the production process can be initiated at any time. In addition, production can be scaled up in response to increased vaccine demand
  • 69. Recombinant Hepatitis vaccine
    The hepatitis B virus (HBV) vaccine
    Originally based on the surface antigen purified from the blood of chronically infected individuals.
    Due to safety concerns, the HBV vaccine became the first to be produced using recombinant DNA technology (1986)
    Produced in bakers’ yeast (Saccharomyces cerevisiae
    Electron micrograph of the hepatitis B virus
  • 71. Prevalence of hepatitis B
  • 72. Recombinant Hepatitis B Vaccine
    One of the most recent developments is the production of a vaccine against hepatitis B using genetically modified yeast cells
    Hepatitis B is a viral infection which attacks the cells of the liver. It can be very serious, causing chronic liver failure, liver cancer and death
    Hepatitis B can be prevented by a vaccination, and in countries like India where it is relatively common
  • 73. Problems With The Early Traditional vaccine
    For many years the vaccine was produced by growing the live virus in animals and then inactivating it by chemical treatment
    This led to the risk of infection during the manufacture, and in the delivery of the vaccine, as well as raising animal rights issues
  • 74. The Gene coding for the HsbAg is isolated and cloned into a Vector Under the control of a strong promoter
    The cloned gene is transferred to the Yeast Expression system
    The gene is allowed to express in the yeast and the recombinant protein product of the Hepatitis is obtained
    The protein is later purified and used to for vaccination
  • 75. The process
    Gene encoding the 226 amino acid hepatitis B surface antigen (HBsAg), was cloned into yeast)
    The 5' end of the HBsAg gene was replaced with another DNA segment so as to optimize synthesis in yeast
    High-cell-density fermentations of laboratory strains of yeast have been developed for the production of HBsAg
    The HBsAg (lipoprotein particle) in cell lysates has been purified to obtain homogeneity
  • 76. to respond to a human influenza pandemic.
    Vaccine Production at industry level
    to respond to a human influenza pandemic.
  • 77. Some viral vaccines currently available for human and veterinary use
  • 78. Food Additive - Aspartame
    Aspartame (L-phenylalanyl-L-aspartyl-methyl ester) is a low-calorie artificial sweetener
    It can be synthesised biocatalytically by peptide synthesis using a thermostable protease – Thermolysin® from the facultative thermophile, Bacillus thermoproteolyticus
  • 79. Production of Nicotinamide
    Nicotinamide is an essential vitamin, and is widely used in the health-food and animal food-and-feed industries. Biological production, using the same Rhodococcus biocatalyst as for acrylamide production, operates at about 5kT p.a.
    Rhodococcus whole cell biocatalyst
  • 80. Value added products
    Acrylamideis one of the most important chemical commodities, being in great demand (200 000 tons per year worldwide) as a starting material for the production of various polymers.
    Nicotinamideis a Vitamin that can also be synthesized in the same biological process
  • 81. Uses
    Absorbent polymers
    Construction material
    Nicotinic acid
    Animal feed supplement
    Human Health food supplement
  • 82. Effects of alkaloids on humans
    High biological activity
    Produce varying degrees of physiological and psychological responses - largely by interfering with neurotransmitters
    others interfere with membrane transport, protein synthesis or other processes
    In large doses - highly toxic - fatal
    In small doses, many have therapeutic value
    muscle relaxants, tranquilizers, pain killers, mind altering drugs, chemotherapy
    Production of Alkaloids from Plants
  • 83.
  • 84.
  • 85.
  • 86.
  • 87.
  • 88.
  • 89.
  • 90. Stem Cells: Elixir for the 21st Century?
    Alzheimer’s Disease Parkinson’s Disease Various Leukemias
    Hodgkin’s Lymphoma Non-Hodgkin’s Lymphomas Immune Deficiency Disease
    Liver Failure Heart Disease Diabetes
    Stroke Multiple Sclerosis Huntington’s Disease
    Osteoarthritis Rheumatoid Arthritis Coeliac Disease
    Crohn’s Disease Lupus Erythematosus Periodontal Disease
    Sickle Cell Anaemia Thalassemia Psoriasis
    Deafness Blindness Osteoporosis
    Spinal Injuries Burns Blackfan Diamond Anaemia
    Fanconi Anaemia
  • 91. Demand for large scale manufacturing of Health care products
    At present, the majority of therapeutic biopharmaceuticals has been produced using animal cell technology and include proteins used for the
    treatment of cardiovascular diseases (tissue plasminogen activator: tPA, reteplase)
    cystic fibrosis (DNases)
    anemia (erythropoietin: EPO)
    haemophilia (coagulation factors VIII and IX)
    cancer and viral infections (interferons and interleukins), multiple sclerosis (interferon-beta2) and
    dwarfism (human growth hormone: hGH)
  • 92.
  • 93. Health Care engaged by our group
  • 94. Staphylokinse
    It is a upcoming 3rd generation thrombolytic agent. It can dissolve the blood clot effectively compared to the existing agents
  • 95. The current generation of thrombolytic agents constitute t-PA, Urokinase, Streptokinase etc.
    Thrombolysins derived from eukaryotic cells (t-PA and Urokinase) can efficiently degrade the clots. But, they cannot meet the clinical requirements of the day because they pose greater problems in commercial production due to their large molecular size and expression incompatibilities
    Streptokinase having a problem of systemic hemorrhage
  • 96. In contrast, the Staphylokinase has surpassed these incompatibilities and has proved to be a better alternative
    Major bottlenecks in clot dissolution by Therapeutics
    Half life
    Production cost
  • 97. SAK as a better alternative
    • Simple protein
    • 98. Small size
    • 99. Less antigenic than the SK
    • 100. High Fibrin specificity
    • 101. New chimera with
    SAK was developed (a patent was filed)
  • 102. Streptokinase
    • Widely using thrombolytic agent
    • 103. Cheaper than all
    • 104. Not Clot specific, can lead to Systemic hemorrhage
    • 105. Even it is dangerous, it is very popular because of
    its low cost
    • Research is going on to avoid the pitfalls of the
    • Our group is engaged in developing a new fusion
    protein with SK, a patent is going to be filed
  • 106. Synthetic Antimicrobial Peptides
    As all the bacterial strains developing resistance to the conventional antibiotics, an attempt was made by our group to develop a broad spectrum synthetic antimicrobial peptide
  • 107. Biotherapeutics are delicate drugs
    Much larger and more complex than traditional pharmaceuticals
    Composed of unstable proteins with a precise structure
    Easily damaged by unfavorable temperature history during storage
  • 108. Even insulin has temperature problems
    Insulin Shelf-life
    Insulin is a very temperature stable biotherapeutic
    A graph of storage life vs temperature shows a “saw tooth” peak
    The product dies at both temperature extremes
    Temperature (C)
  • 109. Effect of temperature storage extremes
    < 0o C
    Protein denaturation
    Formation of aggregates
    Loss of functional activity
    Formation of potentially hazardous immunogenic byproducts
    > 8o C
    Chemical side reactions
    Protein denaturation
    Formation of aggregates
    Loss of functional activity
    Formation of potentially hazardous immunogenic byproducts
  • 110. Smart packaging vs dumb packaging
    Smart packaging can guard against human errors in handling, and accidental temperature abuse.
    Is traditional dumb packaging simply a relic from an earlier era? Does it pass modern failure modes analysis?
  • 111. Thank you