HPLC Analysis of Size Exclusion Separation of Proteins and PEGylated Proteins
CONDITIONS
column
Zenix SEC-150, 30 cm x 7.8 mm I.D., 3 μm particles, 150 Å, (Z777018)
mobile phase
150 mM phosphate buffer, pH 7.0
flow rate
1.0 mL/min
column temp.
Ambient
detector
UV, 214 nm
injection
20 μL, 2.0 mg/mL
sample
1. Protein with MW 70KD (from an undisclosed drug development company) 2. Protein 1 attached with 1 PEG (MW 40KD) molecule
3, Protein 1 attached with 2 PEG (MW 40KD) molecules
说明
分析说明
Pegylation of biotherapeutic drugs improves solubility, increases half life in the blood stream and reduces immunogenicity. Size exclusion chromatography (SEC) is often applied to characterize protein–polyethylene glycol (PEG) conjugates and establish the number of attached PEG chains, since SEC is a precise and robust technique that combines ease of set up and strainghtforward method develpopment.