本产品是纤维素微球直接硫酸酯化后形成的亲和层析分离介质，与琼脂糖基质微球一样同属于天然高分子来源的“软胶”。与琼脂糖相比，纤维素微球的原料有标准牌号，纤维素骨架不含阴离子基团，纤维素微球的非特异性吸附是琼脂糖微球的1/10~1/3，例如本产品的纤维素基球对细胞色素C的非特异性吸附量可低至2 μg/mL。经过硫酸酯化后的介质对多种活病毒、灭活的或结构分裂的病毒、病毒或微生物抗原和肝素结合蛋白具有亲和力。
同时，硫酸酯是一种优良的耐盐型阳离子交换配基，可作为阳离子交换填料，对溶菌酶的结合量可达180 mg/mL，且在50~200 mM NaCl盐浓度下仍结合良好。
阿拉丁硫酸酯修饰纤维素亲和层析介质储存在20%乙醇中，凝胶和保护液的体积比为1:1，我司产品规格为实际凝胶的体积。
参数 | 指标 | 基质 | 高度交联纤维素 | 配基 | 硫酸酯，160~200 μmol H+/mL | 粒径范围 ① | 45~165 μm | 平均粒径 | ~90 μm | 结合能力 ② | ≥120 mg/mL | 推荐工作流速 | 60~150 cm/h | 最大流速与压力 ③ | 300 cm/h，0.3 MPa | 使用pH | 4~8.5（推荐的工作pH），3~12（长期稳定）；2~14（短期稳定） | 化学稳定性 | 在以下溶液中稳定：常用的水相缓冲液、0.5 mol/L氢氧化钠等 | 储存 | 20%乙醇，2~8℃ | 保质期 | 5年 |
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注：
① 90%体积以上的微球在此粒径范围；
② 测试方法为孵育2小时，该结果与DBC10%接近;
③ 10 cm 柱高下的最大测试流速。
使用说明
1、色谱柱装填
以下阐述与层析系统连接时，填料的色谱柱装填方法。
（1）所有需要用到的材料的温度要与色谱操作的温度一样，液体最好做脱气处理。
（2）填料用量计算：通过沉降定量填料体积，需要的沉降填料体积=柱体积×压缩比（也称压缩因子）。沉降体积是指填料在20%乙醇保存液中自然沉降完全读取的稳定体积。硫酸酯修饰纤维素亲和层析介质的压缩比为~1.15。为使达到压缩比，可通过柱头下压的方法，也可以通过高流速压柱。
（3）填料清洗：将填料悬液充分摇匀后量取相应体积，抽去液体，并用约3 倍填料体积的纯化水洗涤，重复3 次，以除去保存液。
（4）装柱填料悬液准备：将填料转移到适当的容器中，加入适量的装柱液，制成50~75 v%的装柱填料悬液，使用前搅匀。
（5）装填前准备：在清洗干净的层析柱下端加入装柱液，以除去下垫片及层析柱下端的空气，在柱内保留少量的蒸馏水，拧紧下堵头，调整柱子使其垂直于地面。
（6）装填：将搅匀后的填料悬液一次性缓慢倒入层析柱内（必要时使用装柱器），为避免引入气泡，应使之沿层析柱内壁自然流下。将所有填料加入后，用装柱液将装柱器加满，拧紧装柱器上盖，将柱子与层析系统连接。
（7）压柱：使柱内填料自然沉降（或50~150 cm/h 的低流速下沉降），待填料沉降完全后（填料与液体的界面清晰），去除装柱器，装上上柱头，并将柱头下降至界面处；通过高流速（推荐流速见下表，注意柱压不超过0.3 MPa），继续压柱至界面清晰稳定，标记界面稳定时的柱高。停泵，打开柱头上的阀门/堵头，关闭柱底的阀门/堵头，下压柱头至标记位置下方与压缩比对应的位置，旋紧柱头，装柱完成。装柱完成后，需用高流速平衡柱内的填料。
装柱条件 | 硫酸酯修饰纤维素亲和层析介质 | 压缩比 | 1.15 | 装柱流速 | 150~300 cm/h |
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2、柱效测定和评价
完成装柱后、使用前可通过柱效测定和评价可以确认层析柱装填质量。柱效通常用理论塔板高度（HETP）和非对称因子（As）来评价。柱效测定可以采用丙酮或者NaCl 作为样品进行，按照下表配制样品溶液和流动相。
样品 | 1.0%丙酮 | 0.8~1.0 M NaCl | 样品体积 | 1.0%柱体积 | 1.0%柱体积 | 流动相 | 纯水 | 0.4 M NaCl | 流速 | 30 cm/h | 30 cm/h | 检测器 | UV-280 nm | 电导 |
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根据UV或者电导率曲线计算理论塔板高度（HETP）、理论塔板数（N）和非对称因子（As），公式如下：
- HETP = L / N
- N = 5.54 × (VR / Wh )2
- As = a / b
其中：L为柱高； VR为保留体积；Wh为半高峰宽；a为在10%峰高处的第一个半峰宽；b为在10%峰高处的第二个半峰宽。
一般来说，HETP的数值应小于填料平均粒径的三倍（即HETP/D50<3，D50为填料的平均粒径），As应在0.8~1.5之间。
3、平衡与上样（Equilibration & Loading）
在上样前，可用初始缓冲液A在操作流速下平衡层析柱，观察检测器的变化，直到电导、pH等参数不变。根据应用不同，本产品的缓冲液体系可为磷酸盐、柠檬酸盐和Tris-HCl等，pH为4~9居多，例如10 mM PB + 50~150 mM NaCl，pH7.5。
4、洗脱 （Elution）
洗脱过程则可以进一步提高缓冲液A中的盐浓度，添加0.5~2 M的NaCl可将目的蛋白有效洗脱。在前期工艺筛选时建议用线性梯度洗脱以确定合适的洗脱浓度，在工艺优化和确定时建议用阶跃梯度洗脱，以节省洗脱液体积和提高效率。
5、再生（Regeneration）和在位清洗（CIP）
通常上述的再生过程可将填料彻底清洗。若发生柱压升高，或为了避免交叉污染，可采用以下溶剂进行再生与在位清洗：1~2 mol/L NaCl，0.5 mol/L NaOH、70%乙醇或30%异丙醇等。在位清洗可有效去除介质上的杂质，反向效果更佳。清洗后需用3~5 柱体积的纯水除去上述溶剂。
CIP 过程 | 目的 | 1~2 mol/L NaCl，0.5~2 CV；水，3~5 CV。 | 除去通过离子相互作用结合的杂质 | 0.5 mol/L NaOH，1~2 h的接触时间，若除去内毒素则需更长时间；缓冲液A，5~10 CV；水，3~5 CV。 | 去除蛋白沉淀、疏水性蛋白或去除热源等其他杂质 | 30%异丙醇或70%乙醇，5~10 CV；水，3~5 CV。 | 去除紧密结合的脂蛋白和脂质体等 |
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6、填料保存
填填料的初始保存液为20%乙醇，使用过后可继续用20%乙醇保存。保存温度在2~8 ℃为宜，不可冻存。
This product is an affinity chromatography resin prepared by direct sulfate esterification of cellulose microspheres. Like agarose matrix microspheres, it belongs to natural polymer-derived "soft gels". Compared with agarose, cellulose microspheres use raw materials with standard grades, and the cellulose backbone contains no anionic groups. The non-specific adsorption of cellulose microspheres is 1/10 to 1/3 of that of agarose microspheres. For example, the non-specific adsorption of cytochrome C on the cellulose base matrix of this product can be as low as 2 μg/mL.
After sulfate esterification, the resin exhibits affinity for a variety of live viruses, inactivated or split viruses, viral or microbial antigens, and heparin-binding proteins.
Meanwhile, sulfate ester is an excellent salt-tolerant cation exchange ligand. It can also be used as a cation exchange medium, with a binding capacity for lysozyme up to 180 mg/mL, and remains well-bound at NaCl concentrations of 50–200 mM.
Aladdin Sulfate-modified Cellulose Affinity Resin is stored in 20% ethanol with a gel-to-storage solution volume ratio of 1:1. The product specification indicates the actual gel volume.
Product Parameters
Parameter | Specification | Matrix | Highly cross-linked cellulose | Ligand | Sulfate ester, 160–200 μmol H + /mL | Particle size range ① | 45–165 μm | Mean particle size | ~90 μm | Binding capacity ② | ≥120 mg/mL | Recommended operating flow rate | 60–150 cm/h | Maximum flow rate and pressure ③ | 300 cm/h, 0.3 MPa | Working pH | 4–8.5 (recommended); 3–12 (long-term stable); 2–14 (short-term stable) | Chemical stability | Stable in common aqueous buffers, 0.5 mol/L NaOH, etc. | Storage | 20% ethanol, 2–8 °C | Shelf life | 5 years |
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Notes：
①More than 90% of microspheres by volume fall within this range;
②Test method: 2-hour incubation; result is close to DBC₁₀%;
③ Maximum tested flow rate at 10 cm bed height.
Instructions for Use
1. Column Packing
The following describes column packing for connection to a chromatography system.
(1)Equilibrate all materials to chromatography temperature; degas liquids if possible.
(2)Calculate medium volume: Settled medium volume = Column volume × Compression factor. Settled volume is the stable volume after full sedimentation in 20% ethanol storage solution. Compression factor of sulfate-modified cellulose affinity chromatography medium: ~1.15. Achieve compression by lowering the adapter or high-flow packing.
(3)Wash medium: Mix medium suspension thoroughly, measure required volume, remove liquid, and wash with 3× medium volume of purified water (repeat 3 times) to remove storage solution.
(4)Prepare packing suspension: Transfer medium to a suitable container, add packing buffer to make 50–75 v% suspension; mix well before use.
(5)Pre-pack preparation: Add packing buffer to the bottom of clean column to eliminate air under the bottom frit; leave a small volume of distilled water, tighten bottom plug, and set column vertically.
(6)Packing: Pour mixed suspension slowly and in one batch into the column (use a packing reservoir if needed), running down the inner wall to avoid bubbles. Fill reservoir with packing buffer, seal top, and connect to system.
(7)Compaction: Let medium settle naturally (or at 50–150 cm/h). After complete settling (clear gel–liquid interface), remove reservoir, install top adapter, and lower adapter to interface. Compact at high flow (see table below; do not exceed 0.3 MPa) until interface is stable; mark bed height. Stop pump, open top vent/plug, close bottom valve/plug, lower adapter to marked position corresponding to compression factor, tighten adapter. Equilibrate column at high flow after packing.
Packing condition | Sulfate-modified Cellulose Affinity Resin | Compression factor | 1.15 | Packing flow rate | 150–300 cm/h |
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2. Column Efficiency Test & Evaluation
Column efficiency can be measured and evaluated to confirm the packing quality after packing and before use. Column efficiency is usually evaluated by Height Equivalent to a Theoretical Plate (HETP) and Asymmetry factor (As).
Column efficiency can be determined using acetone or NaCl as samples. Prepare sample solutions and mobile phases according to the table below.
Item | 1.0% Acetone | 0.8–1.0 M NaCl | Sample volume | 1.0% column volume | 1.0% column volume | Mobile phase | Pure water | 0.4 M NaCl | Flow rate | 30 cm/h | 30 cm/h | Detector | UV-280 nm | Conductivity |
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Calculate HETP, Number of theoretical plates (N) and As based on UV or conductivity curves using the following formulas:
- HETP = L / N
- N = 5.54 × (Vr / Wh)2
- As = a / b
Where:L = column height; Vr = retention volume; Wh = peak width at half height; a = first half-peak width at 10% peak height; b = second half-peak width at 10% peak height.Generally, HETP should be less than three times the mean particle size of the resin (i.e., HETP/D50 < 3, D50 = mean particle size of the resin), and As should be between 0.8 and 1.5.
3. Equilibration & Loading
Equilibrate column with starting buffer A at operating flow rate until conductivity, pH, etc., are stable.Common buffers: phosphate, citrate, Tris–HCl, pH 4–9 (e.g., 10 mM PB + 50–150 mM NaCl, pH 7.5).
4. Elution
Elute target protein by increasing salt concentration in buffer A, typically with 0.5–2 M NaCl.
Process screening: linear gradient elution to determine optimal salt concentration.
Process optimization: step gradient elution to save buffer and improve efficiency.
5. Regeneration & Cleaning-in-Place (CIP)
Standard regeneration cleans medium thoroughly. For elevated backpressure or cross-contamination prevention, use:1–2 mol/L NaCl, 0.5 mol/L NaOH, 70% ethanol, or 30% isopropanol. CIP removes impurities effectively; reverse flow is preferred.Wash with 3–5 column volumes (CV) of pure water after cleaning.
CIP procedure | Purpose | 1–2 mol/L NaCl, 0.5–2 CV; water, 3–5 CV | Remove ionically bound impurities | 0.5 mol/L NaOH, 1–2 h contact time (longer for endotoxin); buffer A, 5–10 CV; water, 3–5 CV | Remove protein precipitates, hydrophobic proteins, or pyrogens | 30% isopropanol or 70% ethanol, 5–10 CV; water, 3–5 CV | Remove tightly bound lipoproteins and liposomes |
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6. Resin Storage
The initial storage solution for the resin is 20% ethanol. After use, it can still be stored in 20% ethanol. The suitable storage temperature is 2–8°C; do not freeze.
ZJ26F0636593
危险品化学品经营许可证（带存储）