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‐γ (IFN‐γ) 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‐γ, the coupled column LC approach was able to detect and quantify soluble IFN‐γ, 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 μg IFN‐γ.