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

Nils Hillebrandt¹, Philipp Vormittag¹, Annabelle Dietrich¹

¹Institute of Engineering in Life Sciences - Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Baden-Württemberg, Germany.

Biotechnology and Bioengineering

|June 25, 2021

Summary

Developing efficient virus-like particle (VLP) disassembly is crucial for vaccine production. This study introduces a high-throughput screening method to optimize VLP disassembly, improving capsomere yield and enabling integrated purification.

Keywords:

cross-flow filtration disassembly downstream processing high-throughput screening virus-like particles

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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.