Bioprocessing with Salt Active Nucleases - High Salt Conditions
Bioprocessing with Salt Active Nucleases - High Salt Conditions
OverView
for SAN HQ and SAN HQ ELISA kit
Applications
- Purification of biologics from residual nucleic acids in biopharma manufacturing
- Purification of recombinant proteins and enzymes for research and diagnostic use
- Removal of unwanted nucleic acids contamination in molecular biology reagents in challenging conditions
- Reduction of viscosity in biological samples during production and automation
- Vaccine manufacturing and viral vector preparation
- DNA removal in high-salt lysates
SAN HQ - Peak performance at high salt conditions
Salt Active Nuclease High Quality (SAN HQ) is a Bioprocessing Grade nuclease developed as the most efficient solution for removal of both single and double stranded DNA and RNA, at high salt conditions.
This nonspecific endonuclease has peak activity at salt concentrations between 400 – 650 mM (Fig. 1)
Non-enveloped viruses like Adenoviruses and Adeno-Associated Viruses (AAV’s) are inherently more robust with two distinct advantages. 1( They exhibit higher tolerance to additives like salt and detergents and 2) their production often involves the lysis of host cells, allowing for harvesting non-secreted vectors.
For Adeno-Associated Viruses (AAVs), which are often harvested from crude cell lysate, the high salt tolerance of SAN HQ is particularly beneficial. Salt is typically added to such lysates to reduce viral aggregation, facilitating more effective nuclease action to digest residual DNA.
SAN HQ's is engineered for optimum activity in these high salt environments ensuring that you achieve unparalleled DNA removal without compromising the integrity of these robust viral vectors.
Key Benefits
- Optimized Residual DNA Removal: Ensures efficient degradation of residual DNA in high-salt conditions, meeting stringent quality requirements for biologics and vaccines.
- Boosted AAV Vector Purification: Enhances the purification process for adeno-associated viral vectors in high-salt conditions, improving quality and yield.
- Streamlined Workflow: Eliminates the need for desalting stages, simplifying the bioprocessing protocol and saving time and resources.
- Enable High-Throughput Processes: Facilitates scale-up and automation by working effectively in high-salt environments, increasing operational throughput.
- Potential Surge in Virus Yield: Operates under conditions that may boost the titer yield of AAV production, potentially enhancing overall viral yield.
- Economized Enzyme Usage: Reduces the need for excess enzyme and additional process adjustments, resulting in significant cost savings.
- Minimized Risk of Process Disruptions: Offers reliable performance in various high-salt bioprocessing conditions, reducing the likelihood of disruptions due to enzyme inhibition.
- Reliability: Provides consistent enzyme activity in challenging high-salt conditions, adding a layer of predictability and dependability to your operations.
- Broader Applicability: Versatile enough to be used in a wide range of viral vector systems, expanding your research and production capabilities.
- Enhanced Viral Stability: High-salt levels stabilize viral vectors, and SAN HQ operates effectively in these conditions, maintaining high yield and quality.
- Host Cell Lysis: Facilitates efficient lysis of host cells in high-salt conditions, optimizing the harvest of both secreted and non-secreted viral vectors.
Key Features
- High purity (≥ 98%)
- No protease detected
- Supplied with extended product documentation
- Compatible with SAN HQ ELISA
The Challenge in Removing Host Cell Chromatin Impurities
In bioprocessing, the primary role of a nuclease is to efficiently digest and fragment host-cell DNA into sufficiently small pieces, facilitating its removal during downstream processing. While most nucleases can effectively degrade naked DNA into tiny fragments under optimal conditions—as demonstrated by M-SAN HQ and SAN HQ, which can digest dsDNA into fragments smaller than 8 nt—the reality in bioprocessing is more complex. (See fig. 5)
The DNA targeted for removal often exists as chromatin, embedded in a complex matrixcontaining remnants of the lysed host cell as well as large amounts of the therapeutic product.The product may or may not have an affinity for the chromatin you aim to remove.
High salt is often applied to mitigate issues like aggregation. The real challenge lies in anuclease's ability to efficiently fragment chromatin under these more complicated, high-salt, conditions—not merely degrading naked DNA under ideal circumstances.
SAN HQ ELISA Kit
SAN HQ ELISA kit is developed for the detection and quantification of SAN HQ. The kit is designed as a classical sandwich ELISA, with two monoclonal antibodies specific towards SAN HQ nuclease (fig 6)
Features
- Sensitive: 0.4 – 25.6 ng/ml
- Precise: RSD = 15%
- Accurate: 100% ± 15%
- Stability: 12 months when stored between +2°C to +8°C
Figures
SAN High Quality has optimum activity between 400 - 650 mM NaCl but can be used in a broad range of NaCl-concentrations. The activity was measured at 37°C in a 25 mM Tris-HCl buffer, pH 8.5, 10 mM MgCl2 with varying concentrations of NaCl. Maximum activity was set to 100%. Similar results were obtained using KCl instead of NaCl (results not shown).
SAN HQ performs best in the 8.2 - 8.8 pH range. A working range starting at pH 7.3 allows use in most biological buffers. The activity was measured at 37°C in a 25 mM Tris-HCl or Bis-Tris Propane buffer buffer at various pH, 5 mM MgCl2 and 500 mM NaCl. Maximum activity was set to 100%.
SAN HQ originates from a cold-adapted organism, which allows excellent performance at the temperature ranges commonly used in bioprocessing. For temperature sensitive products, DNA digestion overnight at 4°C is also possible. The activity was measured in a 25 mM Tris-HCl buffer at pH 8.5 (@ measurement temperature), 5 mM MgCl2 and 500 mM NaCl. 37°C was set to 100% as this is the temperature used for defining the activity in Units.
SAN HQ shows excellent stability during use at 37°C, which is the typical incubation temperature in bioprocessing. After incubation at relevant DNA digestion conditions for 1 hour at 37°C, no significant loss of activity was observed compared to control sample kept on ice in enzyme dilution buffer. Buffer compositions: Lysis buffer (50 mM Tris-HCl pH 8.0 @ 25°C, 5 mM MgCl2, 0.5 % Triton X-100, 500 mM NaCl), DMEM (DMEM supplemented with 10 % FBS, 5 mM MgCl2 and 300 mM NaCl), Lysis buffer in DMEM (Lysis buffer and DMEM 1:1).
Gel analysis showing that M-SAN HQ effectively reduces residual host cell DNA to under 8 nucleotides.
The SAN HQ ELISA kit is designed as a classical sandwich ELISA. Two SAN HQ-specific antibodies recognising different epitopes are used to respectively capture and detect SAN HQ in test samples. The detection antibody is linked to Biotin, which is detected by Streptavidin in the next step. Streptavidin is linked to a Peroxidase which catalyses a reaction that can be quantified colorimetrically by a spectrophotometer measuring OD at 450 nm.
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