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| Determination of isoaspartate in soluble CD95-Fc fusion protein by RP-HPLC |
| YU Lei TAO Lei BI Hua LI Xiang RAO Chunming |
| Division of Recombinant Biological Products, National Institute of Food and Drug Control, Beijing 100050, China |
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Abstract Objective To determine the content of isoaspartate (IsoAsp) in soluble CD95-Fc fusion protein. Methods IsoAsp content in samples was determined by ISOQUANT?誖 Isoaspartic Acid Detection Kit combined with reversed-phase high performance liquid chromatography. Phenomenex Synergi 4 μm Hydro RP-80 C18 column (150 mm × 3 mm, 4 μm) was used. Potassium dihydrogen phosphate-1.1 mmol/L potassium dihydrogen phosphate-10% methanol was used as mobile phase A, methanol as mobile phase B, gradient elution was performed at a flow rate of 0.43 mL/min. Results The content of IsoAsp in reference material was similar to that in three batches of raw liquor (0.30-0.32 mol/mol sCD95-Fc). The content of IsoAsp in three batches of finished products increased significantly (0.51-0.53 mol/mol sCD95-Fc). The content of IsoAsp in finished products did not change significantly after 24 hours of treatment at 25℃ and 37℃, but increased significantly after treatment at 60℃. Conclusion This method is suitable for the determination of IsoAsp in soluble CD95-Fc fusion protein. High temperature treatment can accelerate the formation of IsoAsp in samples.
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