Frequently Asked Questions about DENARASE^® & DENARASE^® High Salt

As a rule of thumb, we recommend DENARASE^® High Salt for processes with salt concentrations above 150 mM. For applications below 150 mM salt the standard DENARASE^® is more suitable. However, conditions such as pH and the magnesium concentration also impact the activity of both enzymes. For this reason, we recommend testing for the best enzyme in applications with salt concentrations ranging from 150 to 250 mM. Please contact one of our specialists for more details on the optimal conditions for both enzymes.

There are several publications on how to use endonucleases derived from Serratia marcescens, such as DENARASE^®. We recommend starting with concentrations between 10 and 100 U/mL. Several parameters such as time, concentration and temperature are highly dependent on your process conditions and should be evaluated accordingly.

Furthermore, the release assay conditions (regarding concentrations of NaCl and MgCl₂) differ between the two enzymes:

• DENARASE^® 0 mM NaCl and 1 mM MgCl₂

• DENARASE^® High Salt: 250 mM NaCl and 5 mM MgCl₂

Therefore, the unit definitions of both enzymes cannot be directly compared. We recommend creating a process-specific activity profile for each enzyme.

Additionally, it is essential to increase the magnesium concentration in a process using DENARASE^® High Salt to ≥ 5 mM magnesium. The required range is between 5-25 mM, depending on other process parameters such as NaCl concentration and pH. For an initial test, we suggest using 15 mM magnesium.

No, both products do have their own benefits for specific applications and will co-exist in the future.

Both DENARASE^® and DENARASE^® High Salt will be available in two different quality grades, a Research and Development and a GMP-grade. From a technical perspective both quality grades are equal and the specification parameters are the same. 

DENARASE^® enzymes for Research and Development (R&D) use:
- Produced under ISO 9001 standard.
- Less strict requirements regarding documentation, storage, and distribution.

DENARASE^® enzymes for manufacturing under GMP: 
- Manufactured under EU GMP conditions.
- Provided with dedicated regulatory support for market approval e.g., via an own US FDA Drug Master File
- Distribution and storage comply with GDP (Good Distribution Practices).

DENARASE^® and DENARASE^® High Salt are both derived from the Serratia marcescens endonuclease and differ in only a few amino acids. Therefore, both enzymes can be detected using the same commercially available Serratia marcescens endonuclease ELISA kits including the DENARASE^® ELISA Kit. As the DENARASE^® ELISA Kit only contains a standard solution for the wild-type enzyme, a correction factor of 1.46 needs to be applied for readings with DENARASE^® High Salt. Alternatively, you can generate a standard solution for DENARASE^® High Salt. 

There are several agents that can inhibit DENARASE^® and DENERASE^® High Salt. Whereas approximately 600 mM NaCl are sufficient to inhibit the standard DENARASE^®, NaCl concentrations above 1 M are required to inactivate DENARASE^® High Salt. Depending on the used buffer, also potassium phosphate (200-300 mM) can be used to quench activity of both enzymes. Moreover, addition of chelating agents like EDTA (> 5 mM), resulting in the loss of free Mg²⁺ ions, will inhibit the enzymatic reaction.

There are several agents that can be used to inactivate DENARASE^®. One option is the use of Proteinase K.
For DENARASE^® inactivation a final concentration of at least 50 µg/mL Proteinase K is added and incubated at 55 °C for at least 1 hour.
Afterwards, Proteinase K can be inactivated at 95 °C for 10 minutes.
DENARASE^® activity was measured using the DENARASE^® activity assay as described in the DENARASE^® Validation Guide section 3.1. No activity could be measured after the above described procedure.
Note: The inactivation of Proteinase K was not verified

Yes, all batches for DENARASE^® and DENARASE^® High Salt must fulfill the specification set for endotoxin content (< 0.25 EU/kU) prior product release. See also the Validation Guides for more details.

c-LEcta offers comprehensive support throughout the change management process for DENARASE^®. This includes providing access to relevant documentation and technical support to ensure a smooth transition. For more information, you can request our 'Guidance on Change to DENARASE^® in Approved Biopharmaceutical Manufacturing Processes'. Request the guidance here.