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.
According to the European Pharmacopoeia (EP 9.6*), analysis of erythropoietin should be carried out with a column filled with hydrophilic silica gel, suitable for the separation of proteins with molecular weight 20,000 to 200,000 and meets following requirements. The PROTEIN LW-803 confirmed the requirements were met.

System suitability requirements:
   Peak area ratio between resolution solution** and 50x diluted resolution solution***: ≥ 1.5 – 2.5%
   Resolution of dimer and monomer in the resolution solution: ≥ 1.5

*The version at the time of the application acquisition. 
**Erythropoietin for SEC system suitability CRS, 0.2 mg/mL in eluent.
***Prepared by diluting 20 uL of the original with eluent to make up 1 mL solution.


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