Pharmaceutical Products of Pharmaceutical Products of BiotechnologyBiotechnology

Amritha M. S.2010-09-115

What are Biopharmaceuticals • Biologically significant compounds like

hormones and proteins useful for treatment of variety of human health disorders

• Biopharmaceuticals or Biotherapeutics or Biologicals

• Obtained from biological source and produced through industrial biotechnology

Biologicals vs Conventional DrugsBiologicals

• Protein or carbohydrate based product

• Extracted from living organism

• Complex physicochemical structure

• Less well-defined Macromolecule (> 500 kd) Tertiary structure Location, extent and type of glycosylation

• Heat- & Shear- sensitive

Conventional Drugs

• Synthetic, organic compounds

• Defined structure, physical & chemical characteristics Chemical

synthesis Micro molecules Stable

A. Recombinant Human Protein• Human proteins can be cloned

– Gene recovered from human genome (as genomic DNA or cDNA)

– Placed in an expression vector using ligation techniques and restriction endonucleases

– Transform bacteria (e.g. Escherichia coli or Bacillus spp.)

– Grow in batch culture in large industrial scale vats

– Purify protein product

Table 10.1 Some recombinant proteins approved for human use ($60 billion-2006)

Protein Company DisorderFactor VIII Baxter, Bayer Hemophilia AFactor IX Genetics Institute Hemophilia BTissue plasminogen activator (TPA)

Genetech Acute myocardial infarction

Insulin Eli Lilly, Novo Nordisk Diabetes mellitusHuman growth hormone

Eli Lilly, Genetech, Upjohn, Novo Nordisk

GH deficiency in children (dwarfism)

Erythropoietin Amgen, Ortho Biotech AnemiaDNase I Genetech Cystic fibrosisVarious interferons (IFN)

Schering, Biogen, Chiron,Genetech

Hepatitis B and C, multiple sclerosis

1. Insulin Made by pancreatic beta cells Enables cells to take up glucose from the

bloodstream to use in production of ATP Insufficient insulin causes diabetes (insulin dependent

diabetes mellitus – IDDM)Must inject insulin to avoid physiological complicationsCells cannot take up glucoseInsufficient ATP is madeGlucose spills into urine (excreted by kidneys; kidney tries

to dilute glucose by excreting large amounts of water)

• Before recombinant insulin was available, insulin was obtained from cows’ or pigs’ pancreases – Cow (Bovine) = 3 amino acid differences– Pig (Porcine) = 1 amino acid difference – Amino acid differences can stimulate allergic

responses– Therefore human insulin is preferred (51 amino

acid)Humulin – Eli lilly

Strategy for insulin production

2. Human Growth Hormone (HGH)

• HGH promotes overall body growth by increasing: amino acid uptake by cells, protein synthesis and fat utilization for energy

• Dwarfism caused by insufficient production of HGH by the pituitary gland

• HGH can treat dwarfism to help undersized children reach their normal height and size

• Old method:– Purification of HGH from cadaver pituitary glands

• 8 cadavers/year for 8 – 10 years per patient

• New method– Gene production for HGH synthesis

• Protropin Genentech• Humatrope Eli Lilly

Production of recombinant HGH• 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 chromatography)

Production of hGH• Purification of rhGH from Chinese hamster ovary (CHO) cell culture supernatant by large-scale electrophoresis.

• 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

3.Erythropoietin (EPO)• Human Erythropoietin 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

• 165 amino acids in human

• widely used in AIDS for development of immunity

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

• Culture- production-purification

• Epoetin alfa- treatment of anemia due to chronic renal failure.

• “Epogen” – 1989Recombinant hormone used to alleviate severe anemia that is a complication of many kidney diseases

• “Neupogen”– Stimulates stem cells to

produce neutrophils (and other leukocytes)

4. Factor VIII

• Hemophilia A – Abnormal blood clotting in absence of Factor VIII– Before rDNA, Factor VIII was obtained from blood

• 8000 pints needed per patient per year• Risk of transmitting HIV before wide-spread screening

for HIV– In the 1980s – thousands of hemophiliacs were infected with

HIV developed AIDS

• Factor VIII genetic characteristics– Codes for 2332 amino acids

• cDNA obtained from gene sequence and cloned into Hamster Kidney Cells Factor VIII protein– E. coli NOT USED because protein needs extensive

glycosylation (25 sites where CHO-groups are added to protein in ER and Golgi)

• Factor VIII products approved by FDA– Recombinate – Genetics Institute– Kogenate – Miles Laboratories

5. Tissue Plasminogen Activator (TPA)

• TPA is used to treat coronary thrombosis (thrombolytic agent)

• Protease that attaches to blood clots and induces other blood components to break down clot

• Plasminogen Plasmin

Fibrin ---------- Degradation of fibrin

• Streptokinase once used for this purpose– Derived from Streptococcal bacteria– Must be delivered to blood vessel directly– Urokinase = alternative, but has risk of hemorrhage

• TPA does not compromise blood clotting elsewhere therefore reduces risk of internal hemorrhaging

• Transported via circulation to affected area• Produced in mammalian cell culture (not

E.coli)

TPA By Genentech

• 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

• FDA approves “Activase” – Genetech – 1987– Clot-Dissolving Agent– Fewer side effects than streptokinase and urokinase

6. Interferon

• and are induced by “dsRNA” in cells infected by viruses

• Bind to neighboring cells and send a warning signal of viruses nearby (species-specific receptors)

• Neighboring cells protect themselves by producing proteins that inhibit viral replication

• IFNs also activate Natural Killer (NK) cells

• Before rDNA 90,000 pints of blood needed to purify 1 g IFNs

• 1980 – recombinant -IFN – Mammalian cell culture (glycosylated)

• Decrease symptoms of hepatitis• Decrease spread of herpes zoster (shingles)• Shrink certain tumors

“Intron” by Swiss Biotech Firm against a particular form of leukemia and for genital

warts -IFN 1b licensed in 1993 for Multiple Sclerosis

8. Monoclonal Antibodies

• Specific antibodies produced in vitro which can bind to:– Cytokines (anticytokine Abs)– Specific subsets of cells

• Tumor cells– MoAb against cell-surface tumor Ags can be used for

diagnosis and immunotherapy– MoAb can be conjugated to toxins or radioactive isotypes to

kill tumor cells = Immunotoxins

• Attacking T cells in grafts

• MoAbs can be “humanized” to eliminate hypersensitivity reactions (serum sickness)– Replace “Fc” portion of MoAb with human “Fc”

portion– Retain “Fab” – which contains the antigen-binding

site– rDNA techniques using cDNA can be employed to

cut and splice appropriate regions, followed by transfection into myloma

Table 10.3 Some therapeutic monoclonal antibodies approved for human use

Type of antibody Company Therapeutic useMouse, Humanized

Ortho Biotech, Protein Design, Hoffmann-LaRoche

Prevents kidney transplant rejection

Chimeric Centocor Prevents blood clots

Chimeric Genetech, Hoffmann-LaRoche

Non-Hodgkin lymphoma

Humanized (Herceptin)

Genetech HER2-positive breast cancers

Humanized Am Home Prod, Celltech, Schering, Millen. Pharm.

Certain leukemias

Humanized Genetech Asthma

9. DNA Vaccines

Numerous animal models are under investigation for the use of DNA vaccines in humansMalaria, AIDS, Herpes, Tuberculosis, Rotavirus (childhood

diarrhea) Just the DNA coding for a specific component of a

disease-causing organism in injected into the body • Saline solution/hypodermic needle• DNA-coated gold beads propelled into the body using

gene guns

DNA vaccination provides long-lived immune response (boosters not need to maintain immunity)

Stable (dried or in solution) As microbial genomes of pathogens are

sequenced, the sequence information can be used for vaccine design

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

Production of DNA vaccine• 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

• to respond to a human influenza pandemic.•

to respond to a human influenza pandemic.

                                                                           

Vaccine Production at industry level

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