We are removing plasmid by using a benzonase treatment. Is this what you would recommend or are there better solutions?

This question is part of the following Ask The Expert session:

Plasmid DNA Production for Cell and Gene Therapy

Answered by:

Nargisse El-Hajjami, Ph.D.

Company: MilliporeSigma

Job Title: Associate Director, EMEA Segment Development for Cell and Gene Therapy

Answer

The removal of DNA is one of the key challenges for viral vector manufacturing. In order to achieve the regulatory requirements of <10 ng DNA/dose and DNA size <200 bp, a good strategy is needed. A typical strategy is composed out of at least three different technologies:

  • DNA-digestion, using endonucleases
  • Tangential flow filtration in order to remove fragmented DNA
  • Chromatography purification

Currently, Benzonase® endonuclease is seen as the industry standard for DNA digestion in vector processing. It is an efficient endonuclease which can degrade all forms of RNA and DNA (both single and double stranded). One unit of Benzonase® endonuclease is able to degrade approximately 37 µg DNA in 30 min to as low as 3-8 base pairs. Benzonase® endonuclease is provided in three different quality grades in order to meet the widest possible range of processing and cost requirements. The highest purity grade for example is the Benzonase® endonuclease Safety Plus Emprove® Expert, which is GMP manufactured and animal origin free. As it is part of the Emprove® program, it is backed up by quality dossiers, which can help you fast track through regulatory challenges.

In order to optimize the use of Benzonase® endonuclease, it is advised to perform a small DoE experiment. The three parameters that need to be examined are concentration, incubation time, and temperature. An example of a DoE set up can be found here. It is also preferred to examine at which step in the process the use of Benzonase® endonuclease is optimal, e.g. before cell lysis, post cell lyses or after a clarification unit.

Digestion of DNA using endonucleases has several benefits:

  • Limits virus-nucleic acid complexes (makes purification unpredictable due to shift in pI and/or retention time), therefore increasing the yield.
  • Protects downstream equipment against DNA fouling
  • Reduces the viscosity

General information on Benzonase® endonuclease can be found here.

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