Osteopontin (OPN) is a multifunctional protein presented in various organs and body fluids in human body. OPN serves important roles in bone remolding, wound healing, and other biomineralization related physiological activities, which gives it great potential health benefits. Clinical statistics and laboratory researches also indicate that OPN can be a suitable diagnostic and prognostic marker for cancer progression. Its role in cell signaling remains one of the hot topics in biomedicine research. However, there was no efficient and scalable process for OPN purification from recombinant sources reported, which prevents OPN unleashing its full potential in health industry. Low expression level, acidic isoelectric point, and the lack of well-defined secondary and tertiary structure are some of the challenges that faced purification development of OPN from Escherichia coli (E. coli) lysates. Finding an adequate capture step with sufficient specificity and binding capacity that could be paired with an orthogonal purification step was a vitally important objective for this project. In recent years, mixed-modal chromatography (MMC) resins have opened up new possibilities for protein purification. The combination of high-throughput screening (HTS) platforms and design of experiment (DOE) principles could accelerate process development exponentially. In this research, seven chromatography resins including traditional ion exchange resin, hydrophobic interaction resin and 5 mixed-modal resins were screened for OPN purification. Plate based HTS was applied and quickly revealed comprehensive information about the affinity between the ligands and the target protein, specificity of the adsorption, and the binding capacity of the resins. Based on HTS results, the bind/elute processes for all candidate resins were designed, tested and optimized with small scale batch adsorption experiments. The purification factors and OPN recoveries achieved by batch adsorption experiments were verified on packed-bed columns operated by AKTA system and analyzed statistically. In terms of purification factor and yield, HEA HyperCel and Capto Q performed significantly better than the rest of the candidate resins. Two 2-step purification processes assembled with these two resins elevated OPN purity from 2% from the lysate to over 95% purity with greater than 40% yield.
