Solid Substrate for High Throughput Protein Chromatography
- Author(s): Alvarez, Adrian
- Advisor(s): Kummel, Andrew C
- et al.
Protein A chromatography is the industry standard method for the purification of monoclonal antibodies. In this work, an emulsion templated sol-gel reaction is used to synthesize rigid, millimeter-scale SiO2 particles with hierarchical porosity, presenting an alternative to existing protein A chromatography resins for high throughput purification. An average particle diameter of 0.61mm allows for increased packed bed length/width ratios and high mobile phase flow rates to be used with minimal pressure drop. FITC conjugated BSA and fluorescence microscopy are used to show that micron-scale, spherical pores (average diameter = 4.69µm) allow for unhindered diffusion of protein throughout the particles. High temperature calcination (1000°C) is used to induce SiO2 reflow and increase intraparticle wall density (decreasing wall thickness from 1.48µm to 1.04µm, p = 0.014), significantly increasing the overall particle strength. A BCA assay and fluorescence spectroscopy are used to show that treating the particles with concentrated H2O2 increases the immobilized protein ligand density from 6.8 to 24.0 mg protein/mL particles due to an increase in SiO2 surface hydroxyl density. Initial protein separation testing shows poor column efficiency with early protein breakthrough, suggesting mobile phase channeling between particles and a need to sieve the particles to decrease the size distribution.