23396076 lecture9 case study erythropoietin production

© 2009, Maulik P. Suthar

Case study-Erythropoietin production

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.


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.


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.


Erythropoietin - structure

http://upload.wikimedia.org/wikipedia/commons/d/d8/EPO.png


Erythropoietin - structure


PHARMACOLOGICAL PROFILE OF ERYTHROPOIETIN

• Mechanisms of Erythropoietin Action


EPO Receptor

• EPO Receptor


Erythropoietin – mechanism

http://upload.wikimedia.org/wikipedia/commons/c/c3/EPO_H%C3%A4matopoese.png



• 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


ERYTHROPOIETIN MARKET


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

--

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

--


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)


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


Rising EPO market

http://upload.wikimedia.org/wikipedia/commons/f/fb/EPO_sales.png


ERYTHROPOIETIN PRODUCTION


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


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


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.


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.


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

23396076 lecture9 case study erythropoietin production

Documents

amino acid

market size

bone marrow

specific activity

erythropoietin

intas pharmaceuticals

company year

erythropoietin