Determination of interferon-gamma in Chinese hamster ovary cell culture supernatant by coupled-column liquid chromatography.
A robust tandem HPLC method coupling size-exclusion (Shodex Asahipak GS-320HQ) and reversed phase (Vydac 218TP54) columns with ultraviolet detection was developed for quantitative determination of interferon-gamma (IFN-gamma) in Chinese hamster ovary cell culture supernatant. The 2D-HPLC system was linked up by a 6-port 2-position low hold-up volume switch valve. Compared to a commercial ELISA kit for IFN-gamma, the coupled column LC approach was able to detect and quantify soluble IFN-gamma, regardless of the glycoprotein's molecular/conformational variability and sample background. Each LC-LC analysis took 90 minutes inclusive of column regeneration. The relative standard deviation of measurements (n = 5) was less than 3%. The limit of detection (LOD) was determined to be 0.35 microg IFN-gamma.
Goh
Lin-Tang
LT
Bioprocessing Technology Institute, Centros, Singapore. goh_lin_tang@bti.a-star.edu.sg
Yap
Miranda Gek Sim
MG
D000818:Animals; D016466:CHO Cells; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D017077:Culture Media, Conditioned / Q000032:analysis / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D007371:Interferon-gamma / Q000032:analysis* / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet
D017077:Culture Media, Conditioned; D007371:Interferon-gamma