Viruses are essential raw materials used to genetically modify cells for viral vaccines or as viral vectors for gene therapy manufacturing. For intracellular viruses, the host cells used to manufacture virus particles must be lysed during the virus harvest step. This results in a release of virus particles, along with host DNA and RNA fragments, into the culture media. The presence of host nucleic acids in suspension with virus particles can negatively impact the purification process by fouling of separation media, decreasing resin binding capacity, and threatening product safety, which all impact virus product yield and purity. A recent video produced by Millipore Sigma entitled Efficient DNA Clearance: Better, Faster Virus Production describes the value-added benefits of adding Benzonase® endonuclease to downstream virus purification protocols to address these issues. During the virus purification process, it is imperative to remove free nucleic acid impurities to comply with regulatory guidelines, described in the video, from the World Health Organization and the US FDA as they pose a risk to patient safety.
Benzonase® endonuclease from MilliporeSigma is a genetically engineered endonuclease from Serratia marcescens, which can completely hydrolyze all forms of DNA and RNA (single-stranded, double-stranded, linear, and circular) into smaller oligonucleotides. The video details the utility of this endonuclease to improving the efficiency and yield of the virus product throughout the different stages of virus purification from clarification, tangential flow filtration (TFF), and chromatography. Additionally, the residual Benzonase® is easily removed during TFF, which speaks to how easy it is to incorporate into existing workflows.
Overall, the video highlights the effectiveness of adding Benzonase® endonuclease for downstream virus purification process intensification improving the speed of purification, reducing cost, improving product purity, and yield while also meeting regulatory requirements.
We were fortunate to be able to interview MilliporeSigma on virus production and the use of Benzonase® endonuclease:
What process difficulties arise from lysing host cells and the release of virus particles during virus harvest?
Process challenges are caused by release of host cell DNA. This could cause changes in virus characteristics due to formation of virus-nucleic acid complexes leading to loss of virus yield, DNA could lead to fouling of expensive downstream equipment and shorten life-time of chromatography columns and filtration units. DNA mediated viscosity increase could lead to challenges in liquid handling.
How has the release of virus particles during virus harvest been handled in the past?
For the depletion of nucleic acids from the virus harvest multiple methods could be used, physical degradation by shear stress, chromatography based on charge and/or hydrophobicity, normal flow filtration with positively charged resin binder and/or diatomaceous earth, selective precipitation and enzymatic degradation. Wherein the enzymatic degradation is widely used.
What led to the discovery of the benefit of adding Benzonase® endonuclease to improve the virus purification process?
Benzonase® endonuclease is standardly used in DNA digestion in R&D laboratories. Researchers found that Benzonase® endonuclease is not only working in small R&D scale but has the probability to be scaled up to industrial virus purification in vaccine and viral vector manufacturing.
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