Determination of residual dextran sulfate in protein products by SEC–HPLC

• Residual dextran Sulfate is quantitated in high concentration protein products by SEC–HPLC.
• Protein sample is precipitated by trichloroacetic acid and dextran sulfate is released.
• Sample is centrifuged and the supernatant is neutralized.
• Dextran sulfate in the supernatant is quantitated using a post column reaction of dextran sulfate in the sample and dimethylene blue dye.

Dextran sulfate is a polyanionic derivative of dextran, produced by esterification of dextran with chlorosulphonic acid. Dextran sulfate with an average molecular weight of 8000 Da can be added to the cell culture to inhibit binding of proteins to cells, increasing cellular growth and productivity. Residual dextran sulfate levels must be monitored during the purification process development to insure clearance. A size-exclusion chromatography based HPLC assay has been developed for the separation and quantitation of dextran sulfate in a highly concentrated purified protein drug substance sample. Trichloroacetic acid (TCA) was used to precipitate the protein and separate the dextran sulfate. Detection and quantitation of dextran sulfate was achieved by post column reaction with dimethylene blue to form a metachromatic complex that absorbs visible light at 530 nm. The quantitation limit (LOQ) was determined to be 1.5 μg/mL dextran sulfate in high concentration protein samples.