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Bioprocessing with Salt Active Nucleases  – High Salt Conditions

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For SAN HQ, SAN HQ ELISA Kit, and now SAN HQ GMP

SAN HQ GMP is biochemically identical to SAN HQ but produced under GMP conditions.

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 – 700 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 6 nt—the reality in bioprocessing is more complex. (See fig. 5)

The DNA targeted for removal often exists as chromatin, embedded in a complex matrix containing 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 a nuclease’s ability to efficiently fragment chromatin under these more complicated, high-salt, conditions—not merely degrading naked DNA under ideal circumstances.

See Case Study on Efficient Fragmentation: Superior Chromatin Digestion Before Flow-Through Purification

SAN HQ ELISA Kit

SAN HQ ELISA kit is developed for the detection and quantification of SAN HQ and SAN  HQ GMP. The kit is designed as a classical sandwich ELISA, with two monoclonal antibodies specific towards SAN HQ nuclease (fig 6).

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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

For SAN HQ, SAN HQ ELISA Kit, and now SAN HQ GMP
SAN HQ GMP is biochemically identical to SAN HQ but produced under GMP conditions.

Overview

• Sequentially isolate and purify total RNA and DNA from a single sample
• Two column system: one for DNA and one for RNA
• The RNA column is for the purification of total RNA including microRNA
• No need to split the lysate, or to use phenol or precipitation methods
• Rapid and efficient spin column procedure – it takes only 30 minutes
• Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix

These kits provide a rapid method for the isolation and purification of total RNA and DNA sequentially from a single sample of cultured animal cells and tissues, blood, bacteria, yeast, or fungi. The lysate is passed over two columns: 1) a DNA column and 2) an RNA column. Total RNA of all sizes is purified, including microRNA. Both DNA and RNA are of the highest purity and yield.

These kits are ideal for researchers who are interested in studying the genome and transcriptome of a single sample, such as for studies of microRNA profiling, gene expression including gene silencing experiments or mRNA knockdowns, studies involving biomarker discovery, and for characterization of cultured cell lines. Norgen’s RNA/DNA Purification Kits are especially useful for researchers who are isolating macromolecules from precious, difficult to obtain or small samples such as biopsy materials or single foci from cell cultures, as they eliminate the need to fractionate the sample. Furthermore, analysis will be more reliable since the RNA and DNA are derived from the same sample, thereby eliminating inconsistent results. The purified macromolecules are of the highest purity and can be used in a number of different downstream applications

RNA/DNA Purification Kit (Spin Column)

Maximum column binding capacity of 50 μg for RNA and 20 μg for DNA.

RNA/DNA Purification Micro Kit (Micro)

The purified RNA and DNA fractions can be eluted in as little as 20 μL. Ideal for cell number inputs of 500,000 and as little as 5 cells. Maximum column binding capacity of 35 μg for RNA and 10 μg for DNA.

Details

Supporting Data

Figure 1 / 4

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*Average Yield will vary depending upon a number of factors including species, growth conditions used, and development stage.

**Tissue inputs of up to 40 mg may be used, however for inputs greater than the recommended 25 mg, cross-contamination of the RNA and DNA fractions is possible.

Storage Conditions and Product Stability
Store Proteinase K at -20°C upon arrival. All other solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 1 year after the date of shipment.

Overview

• Efficient depletion of bovine exosomes from Fetal Bovine Serum
• Deplete exosome-sized vesicles from versatile FBS volumes
• No protease treatment required
• No time-consuming ultracentrifugation
• No precipitation reagents required
• No overnight incubation required
• Exosome depletion confirmed by reduction of bovine miRNAs below detectable levels
• The depleted FBS provides the same cellular growth rates as the standard FBS
• Purification is based on Norgen’s proprietary Silicon Carbide resin matrix

Norgen’s FBS Exosome Depletion Kits provides a quick and easy protocol for the depletion of bovine exosomes from FBS prior to using it as a growth supplement in your culture medium. The FBS recovered from the depletion process is exosome-depleted and does not contain any quantifiable bovine miRNAs. Moreover, the exosome-depleted FBS will support the growth of your cells of interest similar to the non-depleted FBS. Norgen’s kits allow for the processing of different FBS volumes. The depletion is based on Norgen’s proprietary resin. These kits provide a clear advantage over other available kits in that they do not require ultracentrifugation, any special instrumentation, precipitation reagents or any protease treatments. More importantly, the depletion process is an inexpensive method for exosome depletion from FBS, as compared to the expensive current ready-to-use exosome-depleted media available on the market.

Background

Most culture medium used for the growth and propagation of cells in culture require the addition of fetal bovine serum (FBS) as a growth supplement to media. FBS is obtained from bovine (cow) serum, and therefore contains large quantities of bovine exosome vesicles. These exosomes may interfere with some types of studies, or may lead to unreliable results when studying the exosomes shed from your cells of interest in normal culture conditions. Therefore, the use of exosome-depleted FBS is highly recommended for many types of studies.

FBS Exosome Depletion Kit (Slurry Format)

For FBS volumes ranging from 140 mL to 280 mL.

FBS Exosome Depletion Kit (Column Format)

For FBS volumes ranging from 120 mL to 240 mL.

Details

Supporting Data

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Storage Conditions and Product Stability
All buffers should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment.