title: Development of a sensitive size exclusion HPLC method with fluorescence detection for the quantitation of recombinant human erythropoietin (r-HuEPO) aggregates.
abstract: Human erythropoietin produced by recombinant DNA technology, is now marketed worldwide for the treatment of anemias associated with chronic renal failure and chemotherapy. No sensitive methods, which can determine r-HuEPO dimer or oligomer aggregate content in formulated products, have been published to date. This report describes the development and validation of a sensitive size exclusion high performance liquid chromatography (HPLC) method for the quantitation of r-HuEPO aggregates in formulations containing 0.03% polysorbate 80. A Waters Alliance 2690 HPLC system connected to a TosoHaas TSKgel G3000 SWxl (7.8 mm x 30 cm, 250 A pore size, 5 microm particle size) column and a Waters 474 fluorescence detector was used. The mobile phase for the SEC-HPLC method consists of isopropyl alcohol-potassium phosphate (0.1 M)/potassium chloride buffer (pH 6.8+/-0.1, 0.2 M) (25:75, v/v). The flow rate was 0.3 mL/min and the method run time was 60 min. The SEC-HPLC method presented here was shown to be specific for r-HuEPO total aggregates (dimer and oligomers) and allows for their quantitation at 80 ng/mL or 4 ngs/injection, in the presence of r-HuEPO monomer and the pharmaceutical excipients, glycine (5 mg/mL), sodium chloride (4.3 mg/mL), and 0.03% polysorbate 80. The finalized method is stability-indicating and is suitable for determining r-HuEPO aggregates between 0.2 and 0.5% levels in the formulated product of r-HuEPO. This method offers a robust way to measure total aggregates on a routine basis with a high sensitivity for use in product quality control.
authors.affiliation: Pharmaceutical Sciences, GBSC, Centocor Inc., 1001 Route 202 South, Raritan, NJ 08869, USA. sgunturi@cntus.jnj.com
authors.forename: Srinivas R
authors.initials: SR
authors.lastname: Gunturi
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Ghobrial
authors.forename: Basant
authors.initials: B
authors.lastname: Sharma
mesh_terms: D019840:2-Propanol / Q000737:chemistry; D002021:Buffers; D002626:Chemistry, Pharmaceutical; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D004921:Erythropoietin / Q000032:analysis*; D006801:Humans; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D012997:Solvents; D013050:Spectrometry, Fluorescence
chemical_list: D002021:Buffers; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D012997:Solvents; D004921:Erythropoietin; D019840:2-Propanol