Process development for cross-flow diafiltration-based VLP disassembly: A novel high-throughput screening approach.

Area of Science:

• Biotechnology
• Vaccine Development
• Protein Engineering

Background:

• Virus-like particles (VLPs) are essential for vaccines and drug delivery.
• Downstream processing of VLPs often requires disassembly and reassembly to remove impurities and enhance morphology.
• Optimizing VLP disassembly conditions is challenging due to sequence and structure-dependent behavior, necessitating material-intensive experiments.

Purpose of the Study:

• To develop a low-volume, high-resolution screening method for time-resolved VLP disassembly analysis.
• To investigate the disassembly behavior of two C-terminally truncated hepatitis B core antigen VLPs.
• To optimize VLP disassembly and purification processes for improved yield and purity.

Main Methods:

• Development of a high-throughput, low-volume VLP disassembly screening assay.
• Investigation of VLP disassembly kinetics and aggregation under varying pH and urea concentrations.
• Implementation of a diafiltration-based process for VLP disassembly and purification.
• Integration of disassembly, capsomere separation, and reassembly into a filtration-based process sequence.

Main Results:

• The screening method provides time-resolved insights into VLP disassembly progress.
• Optimal capsomere yield for both VLP variants was achieved at moderately high urea concentrations and pH.
• Diafiltration-based disassembly yielded higher capsomere yields (up to 0.84) and allowed for integrated purification compared to mixing-based methods.
• The integrated filtration-based process significantly reduced high-molecular-weight species.

Conclusions:

• A novel high-throughput screening method accelerates the optimization of VLP disassembly.
• Diafiltration offers an efficient and scalable approach for VLP disassembly and purification.
• The developed process enhances VLP production by improving yield and purity, reducing undesirable byproducts.