Reliable quantification and efficient purification of osteopontin from bovine milk using anion-exchange and membrane chromatography.

Osteopontin (OPN) is a bioactive milk protein with important nutritional and immunological functions, yet its accurate quantification and efficient isolation from bovine milk remain challenging due to its low concentration, structural heterogeneity, and the complexity of dairy feedstocks. In this study, an anion-exchange HPLC method was developed for reliable OPN quantification by exploiting its highly anionic nature. An OPN standard was prepared via consecutive anion-exchange chromatography followed by size-exclusion chromatography and was used to establish the calibration curve with good linearity (R2 = 0.9988). The isoelectric point of OPN was experimentally determined to be below 3.5, which was lower than the theoretical value. Using the WPI-OPN model solution, membrane chromatography outperforms column chromatography, achieving higher OPN purity (98.2%), greater dynamic binding capacity (50.4 mg mL-1), and a markedly shorter processing time. Furthermore, application of membrane chromatography to industrial whey increased OPN purity from 0.3% to 82.6%, demonstrating its practical application prospects.