Functional gigaporous polystyrene microspheres facilitating separation of poly(ethylene glycol)–protein conjugate

A novel sulfopropyl gigaporous polystyrene (SP-GP) microsphere enhancing the separation of poly(ethylene glycol)–protein (PEGylated protein) was first presented. The SP-GP microspheres were successfully prepared by introducing sulfopropyl groups into agarose-coated gigaporous polystyrene microspheres and used as chromatography media. Compared with a commercial medium, SP-GP microspheres exhibited improved column efficiency and reduced backpressure with increasing flow velocity, which could ensure its use in high-speed chromatography. Furthermore, a higher protein recovery and purity of the PEGylated protein could be obtained, even when SP-GP was applied at a flow velocity of 1224 cm h−1. Additionally, the dynamic binding capacity (DBC) of SP-GP was significantly improved, which was higher than 10 mg mL−1 medium even at a flow velocity of 306 cm h−1. Further investigation using a laser scanning confocal microscope (LSCM) demonstrated that the static adsorption equilibrium of the PEGylated protein on SP-GP could be completed in 5 min, whereas a much longer period (ca. 60 min) was required for the commercial medium, indicating that the mass transfer of SP-GP was much faster with the gigaporous structure. All of these results strongly support that our developed SP-GP could serve as a promising cation exchange chromatography resin for high-speed separation, especially for biomolecules of high molecular weight.

Graphical abstractA novel sulfopropyl gigaporous polystyrene (SP-GP) microsphere enhancing the separation of PEGylated protein was first presented. The SP-GP microspheres were successfully prepared by introducing sulfopropyl groups into agarose-coated gigaporous polystyrene microspheres and used as chromatography media. Compared with a commercial medium, SP-GP microspheres exhibited improved column efficiency and reduced backpressure with increasing flow velocity, which could ensure its use in high-speed chromatography. Furthermore, a higher protein recovery and purity of the PEGylated protein could be obtained, even when SP-GP was applied at a flow velocity of 1224 cm h−1. Additionally, the dynamic binding capacity (DBC) of P-GP was significantly improved, which was higher than 10 mg mL−1 medium even at a flow velocity of 306 cm h−1. Further investigation using a laser scanning confocal microscope (LSCM) demonstrated that the static adsorption equilibrium of the PEGylated protein on SP-GP could be completed in 5 min, whereas a much longer period (ca. 60 min) was required for the commercial medium, indicating that the mass transfer of SP-GP was much faster with the gigaporous structure. All of these results strongly support that our developed SP-GP could serve as a promising cation exchange chromatography resin for high-speed separation, especially for biomolecules of high molecular weight..Figure optionsDownload full-size imageDownload as PowerPoint slideHighlights► We prepared a functional gigaporous microsphere enhancing PEG–protein separation. ► We demonstrated column efficiency of the microspheres in high-speed chromatography. ► We investigated mass transfer behavior of PEG–protein into the microspheres.