Bioprocessing with Salt Active Nucleases - High Salt, Easy Separation

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For SAN High Quality 2.0 and SAN High Quality 2.0 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 High Quality 2.0 - Peak performance with easier removal

SAN High Quality 2.0 is the second generation of our SAN HQ enzyme. The primary difference with SAN HQ 2.0 is its easier removal and wider compatibility with downstream processes used in bioprocessing workflows while exhibiting the same salt tolerance and other biochemical characteristics as SAN HQ.

The downstream compatibility improvements are of particular importance if used with anionic exchange media or using size exclusion chromatography / TFF below 100 kDa pore size.

Easy Separation

Due to its high isoelectric point (9.55), SAN HQ 2.0 binds strongly to cation exchange media, even at elevated pH and salt concentrations, where few other proteins binds. SAN HQ 2.0 does not typically bind to anion exchange media. SAN HQ 2.0 does not have a His-tag, and consequently does not bind to IMAC media. This allows easy separation if the protein of interest contains a His-tag.

For detailed information, see Figure 1.

Description

SAN High Quality 2.0 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 (Figure 1).

SAN High Quality 2.0 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, simplifies the bioprocessing protocol, and saves 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 enzymes 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 2.0 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 (≥ 99%)
No protease detected
Supplied with extended product documentation
Compatible with SAN HQ 2.0 ELISA

SAN High Quality 2.0 ELISA Kit

SAN HQ ELISA kit 2.0 is developed for the detection and quantification of SAN HQ 2.0. The kit is designed as a classical sandwich ELISA, with two monoclonal antibodies specific towards SAN HQ 2.0 nuclease (Figure 5).

Features:

Sensitive: 0.3 – 12.8 ng/ml
Precise: RSD = 15%
Accurate: 100% ± 15%
Stability: 12 months when stored between +2°C to +8°C

Figures

Figure 1. Easy separation

Figure 2. Optimum activity in solutions with high salinity

SAN High Quality 2.0 has optimum activity between 400 - 650 mM NaCl but can be used in a broad range of NaCl-concentrations, near identical to the characterisitics of SAN HQ (pale green). 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).

Figure 3. Optimum performance at slightly alkaline conditions

SAN HQ 2.0 performs best in the 8.2 - 8.8 pH range. A working range starting at pH 7.3 allows use in most biological buffers. SAN HQ 2.0 demonstrate close to indentical pH tolerance as SAN HQ (pale green). The activity was measured at 37°C in a 25 mM Tris-HCl or Bis-Tris Propane buffer at various pH, 5 mM MgCl₂ and 500 mM NaCl. Maximum activity was set to 100%.

Figure 4. Suitable for DNA removal in the ambient to 37°C range

SAN HQ 2.0 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 temperature tolerance is near identical to that of SAN HQ (pale green ). The activity was measured in a 25 mM Tris-HCl buffer at pH 8.5 (@ measurement temperature), 5 mM MgCl₂ and 500 mM of NaCl. 37°C was set to 100% as this is the temperature used for defining the activity in Units.

Figure 5. Robust performance at 37°C

SAN HQ 2.0 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).

Figure 6. SAN HQ 2.0 ELISA kit is ideal to quantify residual SAN HQ 2.0

The SAN HQ ELISA 2.0 kit is designed as a classical sandwich ELISA. Two SAN HQ 2.0 specific antibodies recognising different epitopes are used to respectively capture and detect SAN HQ 2.0 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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Properties

Source

Recombinantly produced in Pichia pastoris

Molecular Weight

26 kDa

Protein Purity

> 99% by SDS-PAGE analysis

Isoelectric Point

9.55

Unit Definition

One unit is defined as the amount of enzyme that causes a A260 = 1.0 in 30 minutes at 37°C in 25 mM Tris-HCI pH 8.5 (@25°C), 5 mM MgCl₂,500 mM NaCl, and 50 µg/ml calf thymus DNA.

Specificity

Nonspecific endonuclease cleaving single and double stranded DNA and RNA. The enzyme degrades DNA vs RNA in a 10:1 ratio.

Working Ranges

Temperature: 7 – 38°C, 4°C overnight, optimal: 30 - 38°C
Salt concentration (NaCl / KCl): 100 – 900 mM, optimal: 400 – 650 mM
Mg2+: >1 mM is required for activity, optimal: 5 - 50 mM
pH: 7.3 – 9.2, optimal: 8.2 - 8.8

Note: The working range is defined as above 10% activity and optimal range as above 80% activity.

Tolerance to Typical Buffer Additives

Triton X-100: No reduction in activity (tested up to 15%)

Quality Control

Recommended Protocols

Ordering Info

Quantity

Product Name

SKU

Pack Size

Concentration

Price

T7 RNA Polymerase

74100-201

10 000 U

50 U/µl

$ 145.00 USD

Bioprocessing with Salt Active Nucleases - High Salt, Easy Separation