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Analysis of Erythropoietin According to EP Method (LW-803)


Erythropoietin is a glycoprotein composed of 165 amino acids (molecular weight about 30,600 Da). It is a hormone promotes the production of red blood cells by targeting hematopoietic stem cells in bone marrow. Erythropoietin containing drugs are used for the treatment of renal anemia etc. According to the EP method (EP 9.6), analysis of erythropoietin should be done using a column filled with hydrophilic silica gel that should be suitable for the separation of proteins with molecular weight 20,000 to 200,000. The system suitability requires to satisfy peak area ratio between the resolution solution (erythropoietin for SEC system suitability CRS, 0.2 mg/mL in eluent) and the 50x diluted resolution solution (prepared by diluting 20 uL of the original with eluent to make up 1 mL solution) to be within 1.5 to 2.5 %. Also, resolution of dimer and monomer in the resolution solution to be ≥ 1.5. An aqueous SEC (GFC) column, PROTEIN LW-803 was confirmed to satisfy all required conditions for the analysis of erythropoietin.

Sample : Resolution solution (Erythropoietin for SEC system suitability CRS 0.2 mg/mL in eluent), 100 μL
1. Dimer
2. Monomer

Column       : Shodex PROTEIN LW-803 (8.0 mm I.D. x 300 mm)
Eluent       : 1.15 g/L Na2HPO4 + 0.2 g/L KH2PO4 + 23.4 g/L NaCl aq. 
(adjusted to pH7.4 with H3PO4 or NaOH) Flow rate : 0.5 mL/min Detector : UV (214 nm) Column temp. : 25 °C

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