Optimization Strategy And Process Economics Of DNA Digestion In Viral Vector Production For Gene Therapy

By Ratish Krishnan, David Bohonak, Ellison Etnier, James Grobholz, Nikolai Stankiewicz

Benzonase^® endonuclease is employed to reduce the levels of host cell nucleic acids during production of viral vectors used for gene therapies. The use of this enzyme can reduce the levels of DNA by more than 100,000-fold while also decreasing viscosity and protecting downstream equipment from DNA fouling. The enzyme activity is strongly influenced by the matrix of the process intermediate and optimization of its use is often a crucial step in process development. 

Benzonase^® endonuclease is originated from bacteria Serratia marcescens and expressed in E.coli K12. It is nonspecific, making it highly active against all kinds of nucleic acids (DNA, RNA, circular, single or double stranded). The recombinant protein has 30 kDa molecular weight of each subunit and exists as a dimer. The isoelectric point (pI) is 6.85 and is effective in wide ranges of pH (6–10) and temperature (0–42 ºC). The presence of Mg²⁺ (1–2 mM) is required for enzyme activity. 

In this application note, we highlight an optimization strategy and process economics of DNA digestion in viral vector purification.