A monolith purification process for virus-like particles from yeast homogenate

Area of Science:

• Biotechnology
• Separation Science
• Chromatography

Background:

• Conventional particle-based chromatography resins face limitations in capacity and flow rates for large biomolecules such as viruses and virus-like particles (VLP).
• Monoliths, an alternative stationary phase format, offer an open pore structure enhancing accessibility and mass transport for these complex materials.

Purpose of the Study:

• To evaluate the efficacy of monolith chromatography for the purification of VLP from clarified yeast homogenate.
• To compare the performance of hydroxyl-derivatized monoliths with conventional beaded resins for VLP capture.

Main Methods:

• Hydrophobic interaction chromatography using a hydroxyl-derivatized monolith was employed for VLP capture from a recombinant Saccharomyces cerevisiae host.
• Confocal microscopy was utilized to visualize lipid contaminants and assess their impact on column performance.
• Pre-column lipid removal strategies were investigated using Amberlite/XAD-4 beads.

Main Results:

• The hydroxyl-derivatized monolith demonstrated a three-fold superior dynamic binding capacity compared to beaded resins, achieving 90% VLP recovery.
• Lipid contaminants from yeast homogenate caused column fouling, leading to increased pressure drops.
• Pre-column lipid removal using Amberlite/XAD-4 beads reduced fouling and significantly increased the monolith's dynamic binding capacity from 0.11 mg/mL to 0.25 mg/mL.

Conclusions:

• Monolith chromatography is a highly effective alternative stationary phase for bioprocessing, particularly for challenging separations involving large biomolecules like VLP.
• Addressing feed material impurities, such as lipids, is crucial for optimizing monolith performance and longevity in bioprocessing applications.