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 3438 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 Launch 1998 Vial Price (Rs.) 900.00 1450.00 3000.00 5000.00 890.00 1690.00 550.00 820.00 1090.00 1590.00 590.00 890.00 1190.00 1390.00 798.00 1550.00 799.00 1551.00 323.00 798.00 1298.00 1498.00 3998.00 882.00 1750.00 800.00 1550.00 750.00 1200.00 1400.00 2750.00 LG Espogen Inj. LG Life Sciences India 2000 i.u./ml 4000 i.u./ml 10000 i.u./ml 20000 i.u./ml 2000 i.u./ml 4000 i.u./ml 2000 i.u./ml 3000 i.u./ml 4000 i.u./ml 6000 i.u./ml 2000 i.u./ml 3000 i.u./ml 4000 i.u./ml 6000 i.u./ml 2000 i.u./0.5ml 4000 i.u./1ml 2000 i.u./ml 4000 i.u./ml 2003 1000 i.u./2ml 2000 i.u./2ml 3000 i.u./2ml 4000 i.u./2ml 10000 i.u./2ml 2000 i.u./ml 4000 i.u./ml 2000 i.u./ml 4000 i.u./ml 2000 i.u./ml 3000 i.u./ml 4000 i.u./ml 10000 i.u./ml 10000 i.u./ml 40000 i.u./ml 2000 i.u./ml 4000 i.u./ml 2000 i.u./ml 4000 i.u./ml Eprex Johnson & Johnson 1995

Vintor

Emcure Pharma

2001

Epofer

Emcure(Shweiz)

2001

Wepox

Wockhardt

2001

Epotin

Claris life sciences

Zyrop

Zydus Biogen

Hemax

Hindustan Antibiotics

2000

Shanpoietin

Shantha Biotechnics

2005

Ceriton

Ranbaxy

2003

Erykine 10k Erykine 40k Erypro

Intas Pharmaceuticals

2005

BIOCON

Epofit 2k Epofit 4k

Intas Pharmaceuticals

2005

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 mL 300 mcg/1 mL 500 mcg/1 mL (Prepare in both Polysorbate Solution and albumin solution)

2009, Maulik P. Suthar

Table: 3 Indian market growth for Erythropoetin

2001 2005 Growth between 2001-05 2007 Growth in 2007

Bio-generic Molecule

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

Host cell
Transfection Transformation

Plasmid construction

Host cell

Erythropoietin Expression from Transfected Cell Lines. Strain generation

EPO Production
Up-stream Down-stream

Inoculum Preparation and Fermentation Fermentation

Harvesting and Cell Separation

Anion exchange chromatography (as capture)

Hydrophobic Interaction Chromatography (HIC) Affinity chromatography Anion exchange Chromatography (Final Purification) 2009, Maulik P. Suthar

BIOASSAY
Bioassay on the mouse for determination of the specific activity in vivo of EPO from human cell lines. Result: EPO from cell line HeLa S3 (Sample 1) HeLa S3 (Sample 2) Specific Activity U/mg 100,000 110,000
2009, Maulik P. Suthar

PATENTS
Sr. No Patents title US 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. 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. 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-1,2ethanediyl) 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

Recombinant human erythropoietin with altered biological activity

6489293

03/06/2000

Arthur J. Sytkowski, Jennifer Grodberg.

Pharmaceutical compositions of erythropoietin

6696056

04/07/2000

Basant Sharma, Wing K. Cheung, Selima Begum, Els Vercammen, Jaya Natarajan, Marilyn Sanders.

 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. 165169, January 1986

2009, Maulik P. Suthar