Provis Biolabs offers Provinase®, a GMP-grade recombinant endonuclease engineered for efficient residual DNA removal in AAV, lentiviral and cell therapy manufacturing workflows.
The cell and gene therapy (CGT) sector is expanding rapidly, with hundreds of clinical programmes advancing toward commercialisation worldwide. As viral vector manufacturing scales from clinical batches to commercial production, one of the most critical downstream processing challenges remains the efficient removal of residual host cell DNA (hcDNA). Regulatory agencies require that hcDNA levels in the final drug product be reduced to 10 ng per dose or less, with fragment sizes below approximately 200 base pairs, to mitigate theoretical risks including oncogenicity and immunogenicity.
Provis Biolabs addresses this challenge with Provinase®, a GMP-grade recombinant endonuclease specifically engineered for nucleic acid clearance in biopharmaceutical downstream processing. Available through us in the BeNeLux and Europe, Provinase® offers a reliable, animal-origin-free solution for researchers and manufacturers working across AAV, lentiviral vector, vaccine and cell therapy production.
During the production of adeno-associated virus (AAV) vectors, lentiviral vectors and other viral-based therapeutics, host cells are lysed to release the viral particles into the culture medium. This process inevitably introduces large quantities of host cell DNA, residual plasmid DNA and other nucleic acid impurities into the product stream. Other nucleic acid removal techniques such as chemical precipitation often do not yield the desired results. Endonucleases, by contrast, use only nucleic acids as their substrate and do not interact with proteins or other biomolecules, ensuring high product quality and recovery.
Residual hcDNA impurities are associated with several theoretical risks, including insertional mutagenesis that may dysregulate oncogenes or tumour suppressor genes, inflammatory responses through the cGAS-STING pathway, and immunotoxicity. These concerns are particularly pronounced in AAV-based gene therapies produced using human-derived cell lines such as HEK293 and HeLa, where the presence of transforming factors in the host genome demands stringent DNA clearance.
Endonuclease treatment during downstream processing has therefore become an established and essential step in viral vector manufacturing. It is widely applied after cell lysis to degrade free-floating nucleic acids before the purification stages of chromatography, ultracentrifugation and tangential flow filtration.
Provinase® is a recombinant endonuclease overexpressed in E. coli from the Gram-negative enterobacterium Serratia marcescens. It is a non-specific endonuclease that hydrolyses both double-stranded and single-stranded DNA and RNA with high specific activity. The protein consists of two subunits (homodimer), each with a molecular weight of approximately 30 kDa, forming an active molecule stabilised by two disulphide bonds. Provinase® has an isoelectric point (pI) at pH 6.85.
The enzyme attacks and digests all forms of nucleic acids (single-stranded, double-stranded, linear and circular) into short oligonucleotides of 3 to 5 bases in length. It is functional between pH 6 and 10 and from 0 °C to above 42 °C, with optimal performance at 37 °C, pH 8-9 and in the presence of 1-2 mM Mg²⁺ as a required co-factor. This broad operational window makes Provinase® adaptable to the diverse buffer systems and conditions encountered in viral vector production.
Key Features of Provinase®
| Feature | Description |
| Substrate specificity | All DNA and RNA forms (ss, ds, linear, circular) |
| Digestion products | Oligonucleotides of 3 to 5 bases in length |
| Molecular structure | Homodimer (2 × 30 kDa subunits, two disulphide bonds, pI 6.85) |
| Specific activity | >250 U/µl |
| Purity | ≥99% by SDS-PAGE |
| Optimal conditions | 37°C, pH 8–9, 1–2 mM MgCl₂ as co-factor |
| Operating range | Functional from pH 6–10 and 0°C to >42°C |
| Animal-origin-free | Yes — TSE/BSE free, no animal-derived raw materials |
| Stability | At least 18 months at −20°C; stable after 4 freeze-thaw cycles |
| Available grades | RUO and GMP grade |
Provinase® is tested in HEK293 cell cultures for removal of nucleic acids, with a recommended usage range of 10 to 50 U/ml of cell culture harvest depending on cell density, viscosity and other interfering components. Beyond nucleic acid clearance, Provinase® also reduces viscosity in protein extracts, prevents cell clumping and improves resolution on gel electrophoresis by eliminating bound nucleic acids.
| Application | Role of Provinase® |
| AAV vector manufacturing | Degrades residual host cell DNA/RNA released during cell lysis to meet regulatory limits for hcDNA clearance |
| Lentiviral vector production | Supports downstream purification by reducing nucleic acid load prior to chromatography and filtration |
| CAR-T and cell therapy | Enables safe and reproducible processing by clearing contaminating nucleic acids from cell-based products |
| Vaccine manufacturing | Removes nucleic acids from viruses and virus-like particles in vaccine production for cleaner formulations |
| Protein biologics | Minimises host cell DNA in monoclonal antibody and recombinant protein preparations to enhance purity |
| Analytical & QC laboratories | Prepares samples for electrophoretic and chromatography techniques; reduces cell lysate viscosity |
When integrating Provinase® into a manufacturing workflow, process scientists should consider several important operating parameters. The enzyme requires Mg²⁺ as a co-factor, with optimal activity at 1–2 mM MgCl₂. Importantly, EDTA at concentrations as low as 1 mM can reduce endonuclease activity by over 85%, so chelating agents should be excluded from the treatment step. Sodium chloride concentrations above 100 mM and phosphate buffer above 25 mM also show reduced activity, and these buffer conditions should be considered during process design.
Provinase® demonstrates good compatibility with common bioprocessing additives: polysorbate 80 up to 1% and antifoam up to 4% have no significant effect on enzymatic performance. The enzyme is stable for at least 18 months at −20 °C and retains activity after up to four freeze-thaw cycles, which supports practical supply chain and inventory management.
After nucleic acid treatment, Provinase® can be cleared from the product stream through standard downstream steps including affinity chromatography, ion exchange, hydrophobic interaction or size exclusion chromatography, as well as ultrafiltration. Residual Provinase® levels can be quantified using a validated immunoenzymetric assay with a limit of quantification of 0.31 ng/ml.
A significant practical advantage of Provinase® is that it is available in both Research Use Only (RUO) and GMP grades across multiple pack sizes from 100,000 U to 5,000,000 U per vial. This dual-grade availability enables researchers and process development scientists to adopt the same endonuclease platform from early-stage R&D through to clinical and commercial manufacturing, avoiding the need for process changes and bridging studies when transitioning between development phases.
Provis Biolabs manufactures Provinase® in ISO 9001:2015 and GMP-certified facilities with ISO class 7 and 8 clean rooms, following ICH Q7 GMP principles. The product is formulated in USP-grade excipients, filled in non-pyrogenic USP Class VI compliant containers under laminar air flow, and shipped at −20 °C with temperature loggers. Comprehensive quality documentation includes Certificates of Analysis, Certificates of Conformance and vendor qualification packages, supported by Halal and Kosher certifications.
As the CGT industry matures and more therapies transition from clinical trials to commercial launch, the demand for qualified, GMP-grade nucleases is growing significantly. Supply chain security and supplier qualification have become key considerations for manufacturers, since endonuclease products are integrated into validated production processes where switching suppliers requires extensive re-qualification.
Provinase® from Provis Biolabs offers manufacturers an additional qualified source of GMP-grade endonuclease with affordable pricing and faster delivery of 2 to 3 weeks, supporting supply chain diversification and reducing dependency on single-source suppliers. With commercial operations now established in both India and the USA, Provis Biolabs is positioned to support global manufacturing needs.
Efficient nucleic acid clearance is a cornerstone of safe and compliant viral vector manufacturing. Provinase® by Provis Biolabs delivers a robust, animal-origin-free endonuclease solution with high purity (≥99%), high specific activity (>250 U/µl) and no detectable protease activity, supporting the full spectrum of cell and gene therapy production—from early process development through commercial-scale GMP manufacturing.
Provinase® and the complete Provis Biolabs portfolio of animal-origin-free recombinant proteins and enzymes are available through out webshop for researchers and manufacturers in BeNeLux and Europe.
Interested in Provinase® or other Provis Biolabs products? Contact your Bio-Connect account manager or reach out to technical support for expert guidance.
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