Application fields | DENARASE®

Viral vectors for gene therapy are expressed in mammalian cells such as HEK 293. After the cell culturing step, the cells are lysed to increase the vector yield. At this stage the product is exposed to large concentrations of nucleic acids originating from the host cell as well as residual plasmids.

DNA tends to form aggregates with the product and cell debris, which makes viral vector purification challenging. Addition of DENARASE^® during or immediately after the lysis step helps to rapidly reduce the amount of host-cell and plasmid DNA, without interacting with the product. This helps to improve performance of subsequent purification steps and increases the overall vector yield.

Endonucleases such as DENARASE^® play an important role in vaccine manufacturing processes. In most cases they are used to reduce host-derived nucleic acids during the harvest step. Increased levels of extracellular DNA cause a higher viscosity and can build aggregates with the product, resulting in lower process efficiencies.

Addition of DENARASE^® during or immediately after the lysis step rapidly reduces the free nucleic acid content and so improves the efficiency of consecutive downstream processing. As endonucleases do not interact with proteins or viral particles, they can easily be implemented in vaccine production processes and are the best choice when process development needs to be fast.

DENARASE^® is used in a range of vaccine applications using various expression systems such as E. Coli, Pichia pastoris and HEK 293 cells. Examples of processes using DENARASE^® are:

Live-attenuated and inactivated vaccines
Viral Vector Vaccines (e.g, Adenovirusses)
Subunits for Virus Like Particles (VLP’s)

In various applications it has been reported that the production of viral vectors can benefit from higher salt concentrations. It has been demonstrated that the ionic strength of the solution impacts the solubility of impurities and vectors and so affects vector aggregation. Salt concentrations between 200 mM and 400 mM are reported to reduce aggregation, and so increase the vector yields and make DNA more accessible for degradation by endonucleases.

As the wild-type Serratia marcescens endonuclease is inhibited by salt concentrations above 200 mM, elevated salt levels reduce the described benefits. DENARASE^® High Salt is the first engineered Serratia marcescens endonuclease that keeps its activity also at higher salt concentrations. As the engineered enzyme also remains active over a broader pH range, DENARASE^® High Salt offers more flexibility for process developers to optimize the purification process of viral vectors.

Application Fields - DENARASE^®

Viral Vector Production for Gene Therapy

Vaccine Manufacturing

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Application Fields - DENARASE®