For the nucleic acids removal in biopharmaceutical production

• Molecular Biology Grade (MBG) and Good Manufacturing Practice (GMP)-grade
• Highly active in a broad range of temperatures (>20% at 15–55°C)
• Maximum nucleolytic activity at high salt concentrations (optimal concentration for NaCl or KCl is 450–600 mM) and other buffer additives
• Highly active in the typical buffers and grow media
• Requires ≥1 mM Mg²⁺ to activate and shows a broad spectrum of pH activity (optimum at pH 8.5, working pH range >with enzyme activity ≥20% 6.8–9.3)

Saltonase is a recombinant endonuclease derived from psychrophilic bacteria and produced in E. coli. It is highly active across a wide range of salt concentrations and pH levels, making it effective for digesting both DNA and RNA substrates under various buffer conditions and temperatures. Saltonase demonstrates robust activity in challenging environments, including those with high salt content and a broad pH range. These characteristics make Saltonase an invaluable tool for eliminating undesired nucleic acid contamination during protein purification processes in both laboratory and biomanufacturing workflows. 

It is supplied with 20 mM Tris (pH 7.1 w 25°C), 620 mM NaCl, 26 mM MgCl₂ and 50% (v/v) glycerol. 

One unit is defined as an increase in absorbance at 260 nm of 1.0 in 30 minutes at 37°C in 50 mM Tris-HCl buffer, pH 8.5 (25°C) supplemented with 500 mM NaCl, 5 mM MgCl₂, 0.1 mg/mL BSA and 0.5 mg/mL herring sperm DNA as a substrate. 

*Disclaimer for Drug Manufacturers  

“GMP-grade” is a marketing term used by QIAGEN for enzymes manufactured in compliance with established quality standards, including ISO 13485, US QSR (21 CFR 820) and ISO 9001. These enzymes are biopharma-suitable, meet stringent purity requirements and are free from animal-origin materials (AOF) used in the manufacturing processes and final product. They are produced in a dedicated lane and with AOF-compliant equipment. The “GMP-grade” enzymes do not contain beta-lactam antibiotics and have low endotoxin and bioburden levels. When incorporated into drug manufacturing processes, they are considered ‘inactive ingredients’ and are not intended to remain in the final drug product. It is the customer’s responsibility to ensure the safety and compliance of the final end-use product, including adherence to all relevant legal and regulatory requirements, as well as to verify that the enzymes perform and function as specified.  

While QIAGEN adheres to rigorous quality management systems as a medical device manufacturer, it does not produce Active Pharmaceutical Ingredients (APIs) or Excipients, nor does it comply with Current Good Manufacturing Practice (cGMP) regulations for pharmaceuticals. The drug manufacturer is responsible for evaluating the suitability of QIAGEN “GMP-grade” enzymes within their specific production processes and ensuring compliance with all applicable legal and regulatory requirements. QIAGEN makes no representations or warranties except as explicitly stated in its terms and conditions of sale. 

Challenges in biomanufacturing: the need for efficient host cell DNA removal 

Biomanufacturing, especially in viral vector production, faces several critical challenges in achieving high purity and compliance with regulatory requirements. One of the biggest concerns is the removal of host cell DNA, which can introduce risks such as oncogenicity and infectiousness if left in the final product. The structure of chromatin, where DNA is tightly bound around histones, creates significant barriers for endonucleases to access and degrade the DNA efficiently. Additionally, high DNA concentrations can lead to increased solution viscosity, which hampers enzyme activity, reduces process efficiency, and complicates downstream purification steps. These challenges demand highly specialized solutions that not only perform consistently under diverse conditions but also maintain high activity to meet the ever-growing demands of the biopharmaceutical industry. 

The role of salt in cell lysis and viscosity reduction 

Salt is crucial in optimizing cell lysis and enhancing viral vector production. While many bioprocesses rely on physiological salt concentrations, viral vector purification often benefits from using high salt concentrations during the cell lysis step. Salt helps to reduce the aggregation of viral particles, ensuring a higher yield of a product. It aids in breaking down the viscosity created by released DNA, allowing endonucleases to work more effectively. By reducing viscosity, high salt concentrations enable smoother downstream processing and improve overall workflow efficiency. Saltonase, a salt-active endonuclease, is specifically designed to perform optimally under high salt conditions, facilitating efficient DNA cleavage into 3–5 nucleotide fragments, making it a safe solution in viral vector production.

Quality Control

Enzyme activity is measured in a 1.575 mL reaction volume containing herring sperm DNA as a substrate, incubated for 30 minutes at pH 8.5 at 37°C with an enzyme that degrades DNA to acid-soluble oligonucleotides. A 1 U of the enzyme causes an increase in absorbance at A260 of 1.0.  

The purity of Saltonase MBG is ≥ 95%, as evaluated by SDS-PAGE electrophoresis. The purity of Saltonase GMP-grade is ≥ 99%, as evaluated by HPLC and SDS-PAGE electrophoresis. 

Protease activity is measured by incubation of 10 µg of BSA with 10 µg of enzyme for 20 hours at 37°C. Results are visualized on a polyacrylamide gel. No detectable degradation of the protein mixture as determined by SDS-PAGE with Coomassie Blue detection.  

Bioburden is measured in accordance with P.h. Eur 2.6.12. Microbiological Examination of Non-Sterile Products (Total Viable Aerobic Count).   

Endotoxins – automated LAL reaction using CHARLES RIVER Endosafe®nexgen-PTS™ system. The test for bacterial endotoxins is used to detect or quantify endotoxins from gram-negative bacteria using amoebocyte lysate from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). The chromogenic technique is based on the development of color after the cleavage of the synthetic peptide-chromogen complex. 

Usage

The optimal final concentration of Saltonase in a reaction depends on several factors: 

• level of nucleic acid contamination
• incubation temperature
• time of incubation
• salt concentration
• and other compounds present in the reaction mixture. 

We recommend setting the enzyme concentration at 20–50 U/mL, with the following conditions considered optimal:

• NaCl: 500 mM (Note: Saltonase requires a minimum of 150 mM NaCl to boost its activity)
• MgCl₂: 5 mM (Note: Minimum 2 mM Mg²⁺ ions are required to activate Saltonase)
• pH: 8.5 (or lower, such as 7.4)
• Temperature: 37°C (or lower, such as 23°C)

This is used for applications such as:

• Gene therapy viral vector production (e.g., Adenoviruses, Adeno-associated viruses, lentiviruses, oncolytic viruses)
• Production of Virus-Like Particles (VLPs)
• Purification of recombinant proteins and enzymes
• Reduction of viscosity and prevention of DNA aggregates during the cell lysis step
• Removal of undesired nucleic acid contamination in molecular biology reagents in demanding systems