Removing DNA and RNA Using Benzonase^® Endonuclease During Viral Production

Whether you are developing cell and gene therapies or viral vaccines, removing contaminating nucleic acids is an important part of viral production. The simplest way to do this is to use the Benzonase® endonuclease.

Nucleic acid impurities during virus production

Where do these nucleic acid contaminants come from? To produce viruses for vaccines and cell and gene therapy, cells are genetically transformed. After the cells produce virus, they are lysed to release the viral particles, DNA, and RNA.

These free nucleic acids pose a threat to patient safety and negatively impact product yield. The World Health Organization and the U.S. FDA recommends <10 ng/dose with any remaining nucleic acids to be <200 base pairs long. To meet these guidelines, the purification process must significantly reduce the size and quantity of nucleic acids.

What is Benzonase^®?

Benzonase endonuclease is an endonuclease that’s normally expressed in the bacterium Serratia marcescens. Benzonase^® endonuclease is expressed in E. coli W3110, a mutant strain of K12. It’s non-specific and cleaves DNA and RNA into smaller fragments, acting on both single stranded and double stranded nuclei acids as well as linear and circular forms of nucleic acids. It has no proteolytic activity. Once these nucleic acids become broken down, Benzonase^® endonuclease and small DNA fragments can be easily removed by tangential flow filtration.

What is Benzonase^® used for?

Benzonase^® can be used for many applications in bioprocessing including:

• Purification of viral particles (removing DNA and RNA from proteins and other biologicals). Whether for developing viral vaccines, viral vectors for vaccine, cell and gene therapy, or oncolytic viruses, Benzonase^® endonuclease removes nucleic acid contamination from the final product to meet regulatory standards.
• Reducing viscosity caused by nucleic acids for chromatography steps. By removing nucleic acids from the sample, this reduces DNA fouling as DNA binds to the resins and ensures maximal binding capacity for virus during chromatography to get the best yields.
• Preventing cell clumping

Optimizing Benzonase^® use

Before using Benzonase^® in your process, you’ll need to optimize how much of the enzyme you will need. This is determined by a five-step process:

1. Determine the DNA concentration
2. Calculate the theoretical concentration of enzyme you need
3. Test different concentrations of the enzyme along with different incubation times and temperatures
4. Test scaling
5. Test optimal concentration at different locations in the process

Optimizing enzyme use is most critical at larger scales, where material costs become much more important as described in the cost modelling analyses. To learn more about optimizing Benzonase^® endonuclease for your process, see our application note “Optimization Strategy and Process Economics of DNA Digestion in Viral Vector Production for Gene Therapy” and data sheet “Optimization of Benzonase^® endonuclease use in virus purification.”

For more information on how to use Benzonase^®, see our Benzonase^® Nuclease Q & A.

Benzonase^® endonuclease options

For biomanufacturing, we offer three versions of our IPEG PQG GMP  Benzonase^® endonuclease to meet your regulatory concerns:

• Benzonase^® endonuclease, purity grade I (≥99%) suitable for biopharmaceutical production EMPROVE^® Expert
• Benzonase^® endonuclease Safety Plus Emprove^® Expert
• Benzonase^® Salt Tolerant endonuclease

All versions are prepared in the E. coli W3110 expression system and pose a minimal risk of viral contamination. However, both Benzonase^® endonuclease Safety Plus Emprove^® Expert and Benzonase^® Salt Tolerant endonuclease are produced from a chemically defined medium and tested for the absence of adventitious viruses and mycoplasma. To learn more about switching from the standard Benzonase^® endonuclease to the Benzonase^® endonuclease Safety Plus Emprove^® Expert, download the data sheet or read our regulatory bulletin.

Benzonase^® endonuclease intensifies downstream processes by increasing efficiency of these steps and results in higher recovery of viral particles. It also helps meet regulatory expectations by reducing the amount of free nucleic acids in the final product.