Background
With the rapid development of the biopharmaceutical field, the variety of biological products is increasing. At the same time, biological products have strict quality control systems, with residual nucleic acids being a key focus for regulatory agencies both domestically and internationally. Products such as AAV vectors, antibody drugs, vaccines, and recombinant protein drugs are produced using continuously passaged cells, and despite meticulous purification processes, residual host nucleic acids may still be present. These residual nucleic acids can pose risks such as pathogenicity and tumorigenicity. Hence regulatory agencies have strict guidelines for the amount of residual nucleic acids in biological products.
In addition, in the CAR-T cell therapy industry, large-scale purification of lentiviruses, protein extraction, and other fields also require strict control of nucleic acid residues. Currently, methods for removing nucleic acids mainly include enzymatic degradation, polyethyleneimine (PEI) precipitation, and ion exchange chromatography. Among these, enzymatic methods using nonspecific nucleases are highly favored for their efficiency, cost-effectiveness, and ease of use.
GMP Endonuclease to Degrade all DNA & RNA
KACTUS has developed a GMP-grade universal nuclease—MaxNuclease™, which degrades all DNA and RNA into 2-5 base oligonucleotides. It has been developed and validated according to GMP manufacturing standards to ensure the traceability of raw materials throughout the production process. This product has been registered with the U.S. FDA Drug Master Files (#036799), providing documentation on aspects such as production processes and control, and material control.
High-efficiency, enzymatic nucleic acid degradation for biologics manufacturing.
What is MaxNuclease™?
MaxNuclease™ is identified from Serratia marcescens and is genetically engineered and expressed in E. coli under cGMP manufacturing standards. MaxNuclease™ is a non-specific nuclease with high activity and specificity that degrades all forms of nucleic acids including single- and double-stranded, linear and circular nucleic acids.
How does MaxNuclease™ work?
MaxNuclease™ is a homodimer of two 30 kDa subunits containing two disulfide bonds that are essential for activity and stability. It hydrolyzes internal phosphodiester bonds between nucleotides in nucleic acids to produce 5'-monophosphate oligonucleotides of 2-5 bases in length.
What is MaxNuclease™ good for?
Products such as lentiviral or AAV vectors, antibody drugs, vaccines, and recombinant protein drugs are expressed and produced by continuously passaged cells. Even after a fine purification process, host nucleic acids may remain in the products, and the residual nucleic acids may cause pathogenicity, tumorigenesis, etc. Residual nucleic acids need to be removed to a safe level in the final drug product.
Product Features
Manufactured in a GMP-compliant facility
Raw materials free from animal-derived components
Low Endotoxin: ≤ 0.01 EU/kU
Strict quality management for clinical manufacturing
FDA Drug Master Files: DMF #036799
Product Specifications & Quality
Product Specifications
| Parameter | Specification |
|---|---|
| Catalog No. | GMP-NUC-SE101 |
| FDA Drug Master Files | #36799 |
| Source | E. coli with endonuclease gene from Serratia marcescens |
| Molecular Weight | Approximately 27.8 kDa |
| Formulation | 20mM Tris-HCl, 20mM NaCl, 2mM MgCl2, 50% Glycerol, pH 8.0 |
| Storage | Store at -20±5°C. Avoid repeated freeze-thaw. |
| Activity | ≥250 U/µL analyzed by degradation of Herring Sperm DNA |
| Unit Definition | One unit corresponds to the amount of enzyme required to produce a change in absorbance at 260 nm of 1.0 in 30 minutes, at 37°C and pH 8.0. |
Quality Control Criteria
| Assay | Specification |
|---|---|
| Activity (Dissolve herring sperm DNA) | ≥ 250 U/µL |
| Purity (Bis-Tris) | ≥ 95% |
| Purity (SEC-HPLC) | ≥ 99% |
| Residual Protease | Negative |
| Residual Host Protein | ≤ 10 ppm |
| Endotoxin | ≤ 0.01 EU/kU |
| Sterility | Negative |
| Residual Heavy Metal | ≤ 10 ppm |
| Mycoplasma | Negative |
Product Validation Data
Host Cell DNA Removal
Virus harvest solution was treated with 25U/mL and 50U/mL endonuclease at 37°C for 2 hours, respectively. Detection of Host Cell DNA (HCD) residue was analyzed. MaxNuclease™ has higher degradation activity versus Competitor B demonstrated by lower HCD residue for both 25U/mL and 50U/mL working concentrations.
Plasmid DNA Removal
Virus harvest solution was treated with 25U/mL and 50U/mL endonuclease at 37°C for 2 hours, respectively. Detection of plasmid DNA (pDNA) residue was analyzed. MaxNuclease™ has higher degradation activity versus Competitor B demonstrated by lower pDNA residue for both 25U/mL and 50U/mL working concentrations.
Degradation of PCR Product, Genomic DNA, and Plasmid DNA
MaxNuclease™ added to PCR product, genomic DNA, and plasmid DNA shows comparable degradation activity of nucleic acids to leading competitors.
GMP Compliance
Our MaxNuclease™ has been developed and verified with reference to cGMP production standards. The manufacturing process is in accordance with GMP production standards to ensure the traceability of raw materials in the production process. At present, this product has also been filed with the U.S. FDA Drug Master Files (DMF #036799).
MaxNuclease™ is available in long-term bulk supply with batch-to-batch consistency to ensure suitability for industrial applications.
Quality Management System
→ ISO13485 Accreditation
→ Digital Manufacturing Execution System (MES)
→ Process and Analytical method validation
→ Batch-to-batch stability and consistency
→ Free from antibiotic residues and raw materials of animal origin
→ Comprehensive records for batch production
→ Pharmaceutical Class A & C Clean Room
→ Validated and maintained equipment
MaxNuclease™ ELISA Kit
After nucleic acid removal using MaxNuclease™, the residual enzyme in the product or sample must be evaluated. For residual MaxNuclease™ detection, KACTUS has developed a highly sensitive and specific sandwich ELISA quantification kit, with a sensitivity of up to 23 pg/mL.
Frequently Asked Questions
About MaxNuclease™
MaxNuclease™, a broad-spectrum nuclease, is derived from Serratia Marcescens. It is expressed by genetically engineered E.coli and purified, all in a GMP-Grade environment. This enzyme can degrade all forms of DNA and RNA, including single-stranded, double-stranded, linear, circular, native, and denatured nucleic acids. It digests them into 5'-monophosphate oligonucleotides of 3-5 bases in length, and has no base recognition specificity. MaxNuclease™ can maintain high stability and enzyme activity under a wide range of conditions and is suitable for removing nucleic acid residues in samples and improving product purity in pharmaceutical industries such as viral vaccines, viral vectors, and recombinant proteins.
- Removal of DNA/RNA from biological products: The US FDA requires that the nucleic acid content of each dose of biological products for therapeutic use be less than 10pg. MaxNuclease™ can remove nucleic acids in industrial biological products such as vaccines, polysaccharides, and proteins, allowing the final nucleic acid content of biological products to meet regulatory requirements and improve their efficacy.
- Purification of cell culture-derived products: Nucleic acids easily adhere to the surface of cell-generated particles such as virus-like particles (VLP), virus particles, and inclusion bodies. This changes the size and charge of the particles and causes aggregation of these particles, such as the agglomeration phenomenon during the storage of peripheral blood single cells (PBMC). MaxNuclease™ can effectively degrade nucleic acids, avoiding the influence of nucleic acids on cell products and purification, and improving the purification efficiency of cell products.
- Reduce the viscosity of lysed cells: MaxNuclease™ can degrade all forms of nucleic acids, reduce the viscosity of cell lysate, increase protein yield, improve separation effect, make it easy to filter (especially ultrafiltration), and facilitate downstream chromatographic purification operations.
- Preparation of samples for biochemical analysis: In analyses such as ELISA, chromatography, two-phase electrophoresis, and footprinting analysis, treatment of protein samples containing nucleic acids with MaxNuclease™ can improve resolution and recovery.
KACTUS' MaxNuclease™ ELISA detection kit uses a double-antibody sandwich method to determine the content of MaxNuclease™ enzyme. Both the detection antibody and the coating antibody are recombinantly expressed monoclonal antibodies. The detection antibody is pre-coupled with HRP, which simplifies the operation steps and saves time. The kit has a sensitivity of 23.44 pg/mL and a linear range of 46.88 pg/mL-3000.00 pg/mL.
Usage
We have tested the stability of MaxNuclease™ at 25°C and 37°C. It was found that MaxNuclease™ can maintain >90% of the enzyme activity under both these temperatures for 7 days, which proves that the enzyme is very stable in a suitable storage buffer.
Store at -20°C. Avoid repeated freezing and thawing. The enzyme is valid for at least 3 years. We recommend shipping on dry ice.
MaxNuclease™ can be used for a variety of purposes (see “What are the main applications of MaxNuclease™?”). Its usage and dosage vary according to the purpose of use. To produce biological products such as viral vaccines, viral vectors, recombinant proteins, etc., MaxNuclease™ is generally added after harvesting and before purification.
This depends on your application scenario and experimental condition. Generally, 25-50U/mL is suitable in the cell lysis step of an AAV production process. Due to different buffers and application scenarios, there will be relatively large differences in enzyme activity and optimal concentrations. For example, if the temperature is lower than 37°C during use, the enzyme activity will decrease. At 4°C, MaxNuclease™ retains approximately 22% of its enzyme activity compared to its activity at 37°C. When the temperature is lower than 37°C, the same nucleic acid removal effect can be achieved by appropriately extending the reaction time.
Temperature, nuclease concentration, and time are the three conditions that mainly affect enzyme activity, and these three aspects can be optimized in practical applications. If you would like assistance in the usage of MaxNuclease™, please contact us at support@kactusbio.us.
1-2mM Mg2+ is necessary to maintain enzyme activity. The reaction system can be adjusted by adding additional Mg2+ to maintain enzyme activity.
Monovalent cations can inhibit enzyme activity, such as >300mM Na+, K+, or >100mM ammonium sulfate precipitation agent. Additionally, >1mM EDTA will inhibit enzyme activity because EDTA will chelate Mg2+ ions necessary for enzyme activity. Denaturing agents such as urea, as well as proteases, will also inactivate the enzyme.
MaxNuclease™ is easily removed in downstream purification with techniques such as depth filtration and tangential flow filtration (TFF). In pharmaceutical manufacturing, endonucleases are often removed by ion exchange chromatography. The PI of MaxNuclease™ is 6.85, and anion exchange chromatography is generally used to make the MaxNuclease™ flow through or be eluted.
In addition, residue detection of MaxNuclease™ is also part of the quality control analysis of pharmaceutical products. Generally, an ELISA kit can be used to detect MaxNuclease™ (including active and inactive) remaining in the product. The MaxNuclease™ detection kit developed by KACTUS uses a double antibody sandwich method to accurately quantify the residual MaxNuclease™ in the product, with a sensitivity of 23.44 pg/mL and a linear range of 46.88 pg/mL-3000.00 pg/mL.
One unit of activity corresponds to the amount of enzyme required to change the absorbance at 260 nm by 1.0 under the condition of excess herring sperm DNA at 37°C and pH 8.0 for 30 minutes (equivalent to the amount of enzyme required to completely digest 37µg of DNA substrate). The verified enzyme activity of MaxNuclease™ is ≥ 250 U/µL.
Quality
Yes, MaxNuclease™ is manufactured in our dedicated GMP-Grade production facility and undergoes strict quality release testing. Additionally, we provide customizable documentation for regulatory filings. MaxNuclease™ has also been submitted to the FDA Drug Master Files (DMF #036799).
Yes, with our prokaryotic fermentation system and supernatant expression, there is no need for renaturation, which allows us to avoid mixing intermediate renaturation products. Additionally, high expression levels allow for large-scale individual batches to meet manufacturing production requirements.
Documents such as product specification, COA, COO, MSDS, TSE/BSE declaration can be provided. This product has passed the FDA DMF Type II record, with DMF #036799.
60 Hickory Drive
Waltham, MA 02451
United States
Universal Nuclease (GMP-grade) | MaxNuclease™ for Biologics Manufacturing | KACTUS