Application fields | DENARASE®

Endonucleases are commonly applied for the removal of DNA in various bioprocessing applications. These enzymes specifically digest DNA and RNA into smaller fragments of around 3-5 base pairs leaving the therapeutic molecule intact. Because of their high efficiency and specificity for nucleic acids, endonucleases from Serratia marcescens became the current industry standard for enzymatic DNA removal. Integrated into standardized workflows for viral vector manufacturing, DENARASE^®enzymes facilitate downstream processing by reducing the viscosity of lysates, resulting in increased yield and purity:

Viral vectors for gene therapy are expressed in mammalian cells such as HEK 293. After the cell culturing step, the cells are often 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 process steps. 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)

Endonuclease inhibition: Elevated salt concentrations reduce the activity of standard wild-type S. marcescens endonucleases.
Suboptimal alternatives: Commercial salt-active endonucleases (Salt-E) exhibit low activity and unfavorable salt and pH profiles.

DENARASE^®High Salt is the first engineered S. 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.

Characteristic	DENARASE^®	DENARASE^® High Salt
Enzyme Origin	Serratia marcescens	Engineered from Serratia marcescens (few amino acid substitutions)
Production Host	Bacillus sp. (comparable production process)
Molecular Weight	27 kDa (per monomer)
Temperature Optimum	37°C
Quantification of Residual Enzyme	DENARASE^® ELISA Kit
Magnesium Optimum	1-5 mM	5-25 mM
Isoelectrical Point	pH 6.2	pH 7.83

Application Fields - DENARASE^®

DNA Removal in Bioprocessing

Viral Vector Production for Gene Therapy

Vaccine Manufacturing

Benefits of DNA Removal

When do I need DENARASE^® High Salt?

Benefits of High Salt Concentrations

Challenges of High Salt Levels

DENARASE^® & DENARASE^®High Salt - The same, but different!

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Application Fields - DENARASE®