At-line multi-angle light scattering detector for faster process development in enveloped virus-like particle...

Area of Science:

• Biotechnology
• Bioprocessing
• Analytical Chemistry

Background:

• Virus-like particles (VLPs) are crucial in vaccine development and gene therapy.
• Efficient downstream processing is essential for VLP production scalability.
• Real-time monitoring methods are needed to optimize VLP purification.

Purpose of the Study:

• To develop and validate an at-line monitoring strategy for fluorescent VLP purification.
• To enable rapid identification of product-containing fractions and streamline process development.
• To assess the efficiency and scalability of the developed purification process.

Main Methods:

• Production of fluorescent Human Immunodeficiency Virus-1 Gag VLPs in HEK 293 cells.
• At-line coupling of multi-angle light scattering and fluorescence detectors to anion-exchange chromatography.
• Purification using a single-column anion-exchange chromatography with salt gradient elution.
• Characterization of purified VLPs using nanoparticle tracking analysis, SEC-MALS, and TEM.

Main Results:

• Successful at-line monitoring of fluorescent VLPs during purification.
• Rapid identification of product-containing fractions, reducing process development time.
• Achieved 66% total recovery and 50% yield in the main product peak.
• Concentrated VLPs 17-fold to 4.45 × 10^10 particles/mL.
• Demonstrated suitability for large-scale applications due to simple buffers and operation.

Conclusions:

• At-line static light scattering and fluorescence monitoring is effective for real-time tracking of fluorescent VLPs.
• This approach significantly accelerates downstream process development and optimization.
• The developed anion-exchange chromatography method is efficient, scalable, and suitable for industrial VLP production.