Post on 30-Oct-2014
© 2009, Maulik P. Suthar
Case study-Erythropoietin production
Maulik P. Suthar
© 2009, Maulik P. Suthar
INTRODUCTION
• Erythropoietin (EPO) is a 30,400-dalton glycoprotein that regulates red cell production.
• EPO acts primarily to rescue erythroid cells from apoptosis (programmed cell death) to increase their survival.
• Miyake et al. reported purification to homogeneity of human EPO.
• The kidney was proven to be the primary site of production of EPO.
• Loya et al. using hypoxic transgenic mice, reported a tubular epithelial cell site for EPO production.
© 2009, Maulik P. Suthar
Erythropoietin
• Erythropoietin, or its alternative erythropoetin or EPO, is a glycoprotein hormone that controls erythropoiesis, or red blood cell production. It is a cytokine for erythrocyte (red blood cell) precursors in the bone marrow.
• Also called hematopoietin or hemopoietin, it is produced by the kidney, and is the hormone that regulates red blood cell production. It also has other known biological functions.
• For example, erythropoietin plays an important role in the brain's response to neuronal injury. EPO is also involved in the wound healing process.
• When exogenous EPO is used as a performance-enhancing drug, it is classified as an erythropoiesis-stimulating agent (ESA).
• Exogenous EPO can often be detected in blood, due to slight difference from the endogenous protein, for example in features of posttranslational modification.
© 2009, Maulik P. Suthar
Erythropoietin
• Erythropoietin (EPO) is a glycoprotein hormone of 34–38 kDa which stimulates proliferation and differentiation of erythroid precursor cells (CFU-E, BFU-E) to more mature erythrocytes.
• EPO is primarily produced in adult kidney and fetal liver cells. Cells responsive to EPO have been identified in adult bone marrow, fetal liver or adult spleen.
• In cultures of erythropoietic progenitor cells, EPO stimulates the proliferation and differentiation of these cells to more mature red blood cells.Erythropoietin (EPO) regulates the level of erythrocytes in response to the level of oxygen in the blood. When tissues meet hypoxic conditions, the EPO level in the blood increases, and the elevated EPO level triggers differentiation of progenitor cells in bone marrow and release of erythrocytes from bone marrow into the blood.
© 2009, Maulik P. Suthar
Erythropoietin - structure
© 2009, Maulik P. Suthar
Erythropoietin - structure
© 2009, Maulik P. Suthar
PHARMACOLOGICAL PROFILE OF ERYTHROPOIETIN
• Mechanisms of Erythropoietin Action
© 2009, Maulik P. Suthar
EPO Receptor
• EPO Receptor
© 2009, Maulik P. Suthar
Erythropoietin – mechanism
© 2009, Maulik P. Suthar
Erythropoietin – mechanism
• Anemia and erythropoietin synthesis.• (A) The healthy kidney maintains the balance between erythrocyte production and erythrocyte loss. In anemia
because of (B) iron deficiency or (C) hemolysis, the kidney senses a reduction in hemoglobin level and increases erythropoietin synthesis in an attempt to stimulate erythrocyte production. (D) In the diabetic kidney, sensing of hemoglobin level is uncoupled from erythropoietin synthesis
© 2009, Maulik P. Suthar
Erythropoietin – mechanism
© 2009, Maulik P. Suthar
ERYTHROPOIETIN MARKET
© 2009, Maulik P. Suthar
Indian Market of Erythropoetin Brand name Company Year of
LaunchVial Price
(Rs.)
LG Espogen Inj. LG Life Sciences India 1998 2000 i.u./ml4000 i.u./ml10000 i.u./ml20000 i.u./ml
900.001450.003000.005000.00
Eprex Johnson & Johnson 1995 2000 i.u./ml4000 i.u./ml
890.001690.00
Vintor Emcure Pharma 2001 2000 i.u./ml3000 i.u./ml4000 i.u./ml6000 i.u./ml
550.00820.00
1090.001590.00
Epofer Emcure(Shweiz) 2001 2000 i.u./ml3000 i.u./ml4000 i.u./ml6000 i.u./ml
590.00890.00
1190.001390.00
Wepox Wockhardt 2001 2000 i.u./0.5ml4000 i.u./1ml
798.001550.00
Epotin Claris life sciences 2000 i.u./ml4000 i.u./ml
799.001551.00
Zyrop Zydus Biogen 2003 1000 i.u./2ml2000 i.u./2ml3000 i.u./2ml4000 i.u./2ml10000 i.u./2ml
323.00798.00
1298.001498.003998.00
Hemax Hindustan Antibiotics 2000 2000 i.u./ml4000 i.u./ml
882.001750.00
Shanpoietin Shantha Biotechnics 2005 2000 i.u./ml4000 i.u./ml
800.001550.00
Ceriton Ranbaxy 2003 2000 i.u./ml3000 i.u./ml4000 i.u./ml10000 i.u./ml
750.001200.001400.002750.00
Erykine 10kErykine 40k
Intas Pharmaceuticals 2005 10000 i.u./ml40000 i.u./ml
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Erypro B I O C O N - 2000 i.u./ml4000 i.u./ml
-
Epofit 2kEpofit 4k
Intas Pharmaceuticals 2005 2000 i.u./ml4000 i.u./ml
--
© 2009, Maulik P. Suthar
US Erythropoetin market
Brand name Generic name Company Year of Launch
Vial/tablet Price(USD)
Epogen/ Procrit Epoetin alfa Amgen, Inc. 1989 2,000 unit/ml 3,000 unit/ml 4,000 unit/ml 10,000 unit/ml (1 vial, 2ml) 10,000 unit/ml (10 vials, 1 ml) 40,000 unit/ml (10 vials, 1 ml) 20,000 unit/ml (10 ml)
$26.75$39.59$52.44
$267.56
$1,264.03
$5,242.35
$2,711.83
Aranesp Darbepoetin alfa (Albumin)
Amgen, Inc. 2002 200 mcg/1 mL300 mcg/1 mL500 mcg/1 mL (Prepare in both Polysorbate Solution and albumin solution)
© 2009, Maulik P. Suthar
Table: 3 Indian market growth for Erythropoetin
Bio-genericMolecule
2001 2005
Growth between 2001-05
2007
Growth in 2007
Market Size Market Size Market Size
INR USD INR USD INR USD
(Crores) (Millions) (Crores) (Millions) (Percent) (Crores) (Millions) (Percent)
Erythropoietin (EPO)
35.2 7.26 283.2 58.39 68 412 84.95 21
© 2009, Maulik P. Suthar
Rising EPO market
© 2009, Maulik P. Suthar
© 2009, Maulik P. Suthar
ERYTHROPOIETIN PRODUCTION
© 2009, Maulik P. Suthar
EPO Production
Plasmid construction
Transformation
Host cell
Transfection
Erythropoietin Expression from Transfected Cell Lines.
Fermentation
Inoculum Preparation and Fermentation
Harvesting and Cell Separation
Up-stream
Down-stream
Anion exchange chromatography (as capture)
Hydrophobic Interaction Chromatography (HIC)
Affinity chromatography
Anion exchange Chromatography(Final Purification)
Strain generation
Host cell
© 2009, Maulik P. Suthar
BIOASSAY
• Bioassay on the mouse for determination of the specific activity in vivo of EPO from human cell lines.
Result: Specific Activity EPO from cell line U/mg HeLa S3 (Sample 1) 100,000 HeLa S3 (Sample 2) 110,000
© 2009, Maulik P. Suthar
PATENTSSr.No
Patents titleUS Patent
No.Filling date Applicant Outcomes
1 Production of erythropoietin
5621080 06/06/1995 Lin Fu-Kuen. Genomic DNA, cDNA and manufactured DNA sequences coding for part or all of the sequences 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 procaryotic or eucaryotic host cells such as bacteria, yeast or vertebrate cells in culture.
2 Recombinant human erythropoietin with altered biological activity
6489293
03/06/2000 Arthur J. Sytkowski,Jennifer Grodberg.
DNA encoding modified, secretable erythropoietin proteins whose ability to regulate the growth and differentiation of red blood cell progenitors are different from the wildtype recombinant erythropoietin and to methods of modifying or altering the regulating activity of a secretable erythropoietin and using modified secretable erythropoietin proteins.
3 Pharmaceutical compositions of erythropoietin
6696056 04/07/2000 Basant Sharma,Wing K. Cheung,Selima Begum,Els Vercammen,Jaya Natarajan,Marilyn Sanders.
The present invention provides aqueous pharmaceutical formulations of erythropoietin that are free of human serum blood products, stabilized with a quantity of an amino acid and a sorbitan mono-9-octadecenoate poly (oxy-1,2-ethanediyl) derivative and also provides aqueous stable, preserved pharmaceutical formulations of erythropoietin that contain an antimicrobial quantity of cresol and a quantity of an amino acid.
© 2009, Maulik P. Suthar
© 2009, Maulik P. Suthar
SUMMARY
• The past decade has been characterized by a growing awareness of the role of anemia with respect to impairment of cancer patients.
• Novel technology has recently been expanded by the recent improvements in upstream and downstream processing in commercial production of EPO.
• Now a day, erythropoietin market size in India is approximately 21 % that indicate faster EPO market growth. The erythropoietin therapy with maintaining
normal Hb levels may improve treatment outcomes, including overall survival and disease-free survival in anemic cancer patients.
© 2009, Maulik P. Suthar
© 2009, Maulik P. Suthar
References
• JUDITH B. SHERWOOD*, Continuous production of erythropoietin by an established human renal carcinoma cell line: Development of the cell line, Proc. Natl. Acad. Sci. USA, Vol. 83, pp. 165-169, January 1986