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Purification of High Yield Extracellular Vesicle Preparations Away from Virus

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Published on: September 12, 2019

Purification of a non-enveloped virus using sequential aqueous two-phase extraction.

Natalie M Nold¹, Seth A Kriz¹, Sheridan Waldack²

¹Department of Chemical Engineering, Michigan Technological University, USA; Health Research Institute, Michigan Technological University, USA.

Journal of Chromatography. A

|March 20, 2025

Summary

This study optimized a two-step aqueous two-phase system (ATPS) for purifying viruses. The improved method enhances virus recovery and impurity removal, crucial for cost-effective vaccine manufacturing.

Keywords:

Downstream processing Electrostatic interactions Hydrophobic interactions Liquid-liquid extraction Salting-out

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Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

Published on: July 27, 2018

Area of Science:

Biotechnology
Bioprocessing
Virology

Background:

Virus-based vaccines and therapies require scalable and cost-effective purification.
Aqueous two-phase systems (ATPS) with polyethylene glycol (PEG) and citrate offer high recovery but pose downstream processing challenges.
Viruses are often partitioned into viscous PEG-rich phases or interfaces in ATPS.

Purpose of the Study:

To develop a second ATPS step for efficient virus extraction into the citrate-rich phase.
To optimize ATPS parameters for improved virus purification and downstream compatibility.
To enable industrial-scale, cost-effective viral vector manufacturing.

Main Methods:

Investigated ATPS performance by varying PEG molecular weight, phase component concentrations, volume ratios, salt type, pH, and glycine addition.
Focused on optimizing parameters for the extraction of non-enveloped porcine parvovirus (PPV).
Implemented a two-step ATPS process with adjusted chemical conditions between steps.

Main Results:

Achieved 66% recovery of infectious PPV with 2.0 log removal of host cell proteins and 1.0 log removal of host cell DNA.
Optimized parameters included pH shift, reduced phase component concentrations, and increased citrate-rich phase volume ratio.
An 8 kDa PEG molecular weight facilitated a pH shift without precipitation; glycine and phosphate salts showed no significant recovery improvement.

Conclusions:

The optimized two-step ATPS significantly improves virus recovery and impurity removal.
This method addresses downstream processing challenges associated with traditional ATPS.
The process shows promise for continuous, low-cost manufacturing of viral vectors for vaccines and therapies.