在生物制品生产，如AAV载体及腺病毒载体疫苗生产中，宿主细胞DNA残留是关键质量参数之一。宿主细胞DNA主要以染色质形态存在，其中的组蛋白通过离子相互作用及疏水相互作用与DNA紧密结合，从而影响DNA的检测与酶切处理。

高盐浓度能够提高效率及产量：

SAN HQ (Bioprocessing grade)是一种新型的、耐高盐的工程化内切酶。该酶在0.5 M NaCl条件下具有最佳活性，是大规模生产及生物工艺流程中去除核酸污染的理想选择。

盐浓度是纯化工艺的重要参数之一，高盐浓度能够减少聚集、增加目标产物溶解度及提高目标产物产量。高盐浓度下，宿主DNA与蛋白质能够完全解离，从而更容易被降解。

优势：

1. 高盐条件下，DNA清除效率更高；

2. 高盐抑制AAV载体聚集，提高载体产量；

3. 无需透析、脱盐操作，简化工艺，节省时间；

4. 减少酶用量，降低直接成本。

特性：

在治疗性药物（如抗体、病毒载体药物等）的生产及工艺流程中，核酸杂质的去除至关重要。US FDA指南要求：治疗用重组生物制品终产品中，核酸杂质含量低于100 pg/dose。SAN HQ独特的耐高盐特性、合规的生产体系标准，让其成为大规模生产工艺中核酸处理的理想选择。

Figure 1. 在高盐条件下SAN HQ酶活优于其他内切核酸酶。不同浓度的NaCl（0 M、0.25 M和0.5 M）和温度（6 ℃、25 ℃、37 ℃）下，两种市售内切核酸酶和SAN HQ的活性比较。

Figure 2. SAN HQ在不同浓度Na+和K+条件下的活性变化。SAN HQ在0.5 M的NaCl条件下具有最佳的活性，且在较宽的NaCl和KCl范围内都有较高活性。酶活检测体系是25 mM Tris-HCl缓冲液（pH 8.5，5 mM MgCl2，不同浓度的NaCl和KCl），最高活性设置为100%。

Figure 3. SAN HQ ELISA Kit用特异性单抗对SAN HQ进行检测及定量分析。检测下限（LOD）是0.4 ng/ml，线性检测范围是0.4 – 25 ng/ml。

质控体系：

ArcticZymes致力于提供高质量产品，具有好的批间一致性、稳定可靠的质量。ArcticZymes所有产品的开发、生产及销售等都符合ISO 13485:2016质量管理体系标准。鉴于治疗用生物制品更严格的质量要求，厂家对SAN HQ (Bioprocessing Grade)的生产及质控，在符合ISO13485:2016体系基础上，增加了cGMP相应要求。

SAN HQ (Bioprocessing Grade)的生产用原辅料是Non-animal和Non-plant来源的，终产品经过0.22 µm过滤除菌，放行检测包括微生物、真菌及内毒素检测等，所有标准符合USP-EP要求。作为SAN HQ (Bioprocessing Grade)和M-SAN HQ (Bioprocessing Grade)的生产商，ArcticZymes厂家管控整个供应链及生产流程，协助客户进行文件审计及现场审计。

部分质控参数：

高pI——通过离子交换 (IEX) 易去除：

细胞基因治疗常用产品的pI值如表格所示。与其他核酸酶相比，SAN HQ更容易去除。

Figure 4. SAN HQ紧密结合在SP-sepharose柱子上（体系是0.2 M盐浓度，pH 9.0）。将混有SAN HQ (pI=9.6)的BSA（pI=4.7）溶液过SP-sepharose柱子，两种物质分离很容易且彻底。

应用实例：AAV生产：

北美某病毒载体研究中心，在AAV生产过程中比较了SAN HQ和市售一款全能核酸酶的性能。每组用150*10^6 HEK293细胞，裂解后加入指定量的两种酶（0K, 2K, 3K, 4K, 5K, 6K），在各自最适反应条件下，37℃酶切1hr；用Quant-iT dsDNA High Sensitivity Assay Kit (Invitrogen)检测Genomic DNA含量，以加酶0K实验组数据为100%作图。

该中心Director表示，SAN HQ独特的耐高盐特性，能够提高AAV下游纯化效率，缩短纯化时间，且不影响AAV载体产量及活性；且这种特性对不同血清型均有效果。

酶学特征：

来源：Pichia pastoris

分子量：该酶是糖蛋白，不含糖基化的分子量是26 kDa

酶比活：≥1.75 x 10^5 Units/mg

酶活定义：在37℃，25mM Tris-HCl，pH8.5 (25℃)，5mM MgCl2，500mM NaCl反应体系中，一个单位的SAN HQ在30分钟内消化50 µg/ml的小牛胸腺DNA，产生OD260nm处1A的吸光值变化。

特异性：非特异性内切核酸酶，能够将单链及双链的DNA及RNA，消化成5个碱基为主的寡核苷酸oligos。
酶活条件：

对常用添加剂的耐受性：

References：

Allen WE, Kauvar IV, Chen MZ, Richman EB, Yang SJ, Chan K, Gradinaru V, Deverman BE, Luo L, Deisseroth (2017). Global Représentations of Goal-Directed Behavior in Distinct Cell Types of Mouse Neocortex. Neuron 94 (4) 891-907, doi.org/10.1016/j.neuron.2017.04.017

Adams B, Bak H, Tustian AD (2020). Moving from the Bench Towards a Large Scale, Industrial Platform Process for Adeno-Associated Viral Vector Purification. Biotechnology & Bioengineering, doi:10.1002/bit.27472

Chan, K, Jang, M, Yoo, B (2017). Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat Neurosci 20, 1172-1179, doi.org/10.1038/nn.4593

Levy JM, Yeh WH, Pendse N (2020). Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. Nature Biomedical Engineering, 4(1):97-110, doi:10.1038/s41551-019-0501-5

Q&A：

【Q1】How can I inactivate or remove SAN High Quality after use?

【A1】There are various ways of inactivating or removing the protein after use. Due to the high pI of the protein (pI 9.6), it can be removed by the use of cation exchange chromatography. Irreversible inactivation can be achieved by the use of reducing agents (e.g. TCEP, DTT) combined with moderate temperatures. Alternatively, the activity of the enzyme can be inhibited by chelators.

【Q2】Could SAN High Quality be used for purification of viral vectors, e.g. AAV based vectors?

【A2】Yes, this endonuclease is suited for viral vector purifications. The purity of virus vectors is pivotal prior to in vivo transductions of cells or animals. Salt is often an important component to achieve highly purified vectors by minimizing aggregation and increasing target yield. SAN High Quality is highly compatible with the use of high salt conditions.

【Q3】Can I replace my current endonuclease with SAN High Quality in all applications?

【A3】SAN High Quality has a different activity profile and tolerance for certain buffer components compared to other endonucleases. SAN High Quality can replace your current endonuclease in most cases and is a superior nuclease in high salt conditions.

【Q4】What is the advantage of SAN High Quality compared to other nucleases?

【A4】Other nucleases can generally not tolerate salt very well. SAN High Quality has an optimal activity at high salt concentrations, enabling high salt conditions throughout the workflow.

【Q5】What is the lowest reaction temperature I can use for SAN High Quality?

【A5】The optimal reaction temperature of SAN High Quality is 37°C. At lower temperatures, the reaction will be slower. Activity at 4°C is about 5-10% compared to the activity at 37°C. You may compensate by increasing the reaction time or increasing the amount of enzyme.

【Q6】What salt concentrations and pH-values are optimal?

【A6】Optimal conditions of NaCl are between 0.4-0.6 M and optimal pH-values are between 8.5-9.5. Low concentrations of salt can be compensated for by using a higher pH. Low pH can be compensated for by using a higher concentration of salt.

【Q7】Does the product contain BSA?

【A7】No.

【Q8】How stable is SAN High Quality?

【A8】SAN High Quality is a robust enzyme which is stable at room temperature for weeks. The product should be stored at -20°C or 4°C for best storage stability.

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