本产品在高刚性琼脂糖凝胶微球骨架的表面设计特定的间隔臂后，再偶联3-氨基苯硼酸形成的亲和层析介质。苯硼酸配基在弱碱性条件下可与1,2-顺式二醇通过可逆共价键形成硼酸酯，可特异性结合含1,2-顺式二醇结构的物质，包括糖蛋白（包括抗体）、酶、多糖、核苷、核苷酸和儿茶酚等多类生物分子。也可通过NADP+或ATP来辅助分离纯化不能直接与填料结合的目标分子。因特异性高，且用途广泛，使用本产品的纯化过程可单独归类为硼酸亲和层析（Boronate affinity chromatography，BAC）
阿拉丁硼酸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 | 高刚性琼脂糖 | 配基 | 3-氨基苯硼酸，~20 μmol/mL | 粒径范围 ① | 30~100 μm | 平均粒径 | ~60 μm | 结合能力② | ~10 mg h-IgG/mL | 推荐工作流速 | 30~150 cm/h | 最大流速与压力 ③ | 1500 cm/h，0.5 MPa | 使用pH | 3~10（操作过程），2~13（CIP过程） | 化学稳定性 | 在常用的水相缓冲液、0.5 mol/L氢氧化钠、8 mol/L尿素、30%异丙醇和70%乙醇等体系中稳定。 | 储存 | 20%乙醇，2~8℃ | 保质期 | 3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。
使用方法参考
硼酸亲和原理
硼酸亲和层析介质与1,2-顺式二醇结构形成的可逆共价键（硼酸酯）是层析过程的主要作用力（图1）。pH=8.5以上有利于填料与目标分子以硼酸酯的形式结合，但生物分子的分离纯化较少用到pH=9.0以上的条件；pH=4.0~6.0则有利于硼酸酯的解离。除了可以降低pH将目标分子洗脱，也可以用山梨醇、甘露醇等含1,2-顺式二醇结构的糖类进行竞争洗脱。
除了可逆共价键，配基中存在苯环这一疏水基团，可能与含苯环的物质之间存在π-π相互作用，这要求上样缓冲液的离子强度不能过高，通常在~50 mM，以降低疏水相互作用造成的非特异性吸附。硼酸酯的四面体结构带负电荷，为降低离子相互作用造成的非特异性吸附，又要求结合过程有较高的离子强度。硼酸亲和层析介质通常在50~500 mM的离子强度下与目标分子有较优的特异性结合。二价阳离子可降低离子相互作用和疏水相互作用造成的非特异性吸附，例如可在上样缓冲液中加入0~50 mM的MgCl2。Mg 2+ 也可以抑制填料与核酸等含磷酸基团物质的电荷互斥作用，加入Mg2+可以增强结合效果。
当形成的硼酸酯以不带电荷的平面三角形结构存在时，硼原子存在空轨道，可作为电荷转移相互作用的电子受体。未质子化的氨基是良好的电子供体，当氨基提供孤对电子给硼原子时，硼原子形成四面体型，促进硼酸酯的形成。但需要注意的是，当氨基附近有羟基存在时，填料与1,2-顺式二醇将无法形成硼酸酯结构。这也是为什么乙醇胺和Tris不适合作为上样缓冲液。
图1. 硼酸亲和层析介质的亲和原理（图片文字修改Nuperley基球 → 硼酸亲和层析介质 ）
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。硼酸亲和层析介质的压缩比为~1.10。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（原包装中填料体积分数为50%），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 | 硼酸亲和层析介质 | 压缩比 | ~1.10 | 装柱流速 | 600~1500 cm/h，依赖于色谱柱尺寸，以压缩比和柱效测试为准 |
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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
在上样前，可用上样缓冲液（平衡缓冲液）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。上样量根据样品的性质和层析介质的量进行选择，可在预实验中确定，例如静态吸附实验。一般需要将样品用上样缓冲液稀释，例如1：1稀释。
上样缓冲液pH通常为8.5~9.0，例如将糖蛋白从非糖蛋白中分离可用HEPES缓冲液（20 mM HEPES, 20 mM MgCl2, pH 8.5），从脱氧核糖核苷中分离核糖核苷可用醋酸铵缓冲液（0.25 M醋酸铵, pH 8.8）。本填料常用的上样缓冲体系有磷酸盐、醋酸铵、HEPES等，一般情况下应避免使用含氨基的缓冲液，例如Tris-HCl体系。
4、洗脱
用2-3个柱体积的平衡缓冲液冲洗上样后的层析柱，观察检测器的变化，直到电导、pH等参数不变，此时未结合的组分被清洗出去。
亲和层析柱的洗脱可用恒定洗脱、梯度洗脱或者阶跃洗脱。洗脱过程可以将pH降低至4~6，例如0.1 M甲酸、25 mM盐酸；也可以选择糖类进行竞争洗脱，如10~200 mM山梨醇；也可以用0~20 mM的EDTA；Tris可作为高效洗脱剂。
5、再生与在位清洗（CIP)
通常可用5 CV的洗脱液将填料再生。若有失活蛋白质或脂类物质在再生时洗不掉，可用在位清洗（CIP）除去。可采用以下选择进行在位清洗：0.5 mol/L NaOH、70%乙醇、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。具体的CIP溶液选择可参考下表。
CIP 过程 | 目的 | 0.5 mol/L NaOH，尽可能缩短清洗时间；水，5 CV。 | 去除蛋白沉淀，或去除热源等其他杂质 | 30%异丙醇或70%乙醇，4~10 CV，＜50 cm/h；水，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
填料的初始保存液为20%乙醇，使用过后可继续用20%乙醇保存。也可以用0.2 M醋酸钠、0.1 M甲酸等与洗脱、再生接近的缓冲液保存，缓冲液中可添加0.02%叠氮化钠以防腐，在偏弱酸性的环境中填料更稳定。保存温度在2~8 ℃为宜，不可冻存。
This product is an affinity chromatography medium formed by designing a specific spacer arm on the surface of a highly rigid agarose gel microsphere matrix, followed by coupling with 3-aminophenylboronic acid. Under weakly alkaline conditions, the phenylboronic acid ligand can form a boronate ester with 1,2-cis-diol through a reversible covalent bond, enabling specific binding to substances containing 1,2-cis-diol structures, including glycoproteins (including antibodies), enzymes, polysaccharides, nucleosides, nucleotides, catecholamines and other biomolecules. It can also assist in the separation and purification of target molecules that cannot directly bind to the resin with NADP⁺ or ATP. Due to its high specificity and wide application, the purification process using this product can be independently classified as Boronate Affinity Chromatography (BAC).
Aladdin Boronate Affinity Chromatography Resin is stored in 20% ethanol, with a volume ratio of gel to protective solution of 1:1. The product specification refers to the actual gel volume.
Item | Details | Matrix | Highly rigid agarose | Ligand | 3-aminophenylboronic acid, ~20 μmol/mL | Particle size range ① | 30–100 μm | Mean particle size | ~60 μm | Dynamic binding capacity ② | ~10 mg h-IgG/mL | Recommended operating flow rate | 30–150 cm/h | Maximum flow rate and pressure ③ | 1500 cm/h, 0.5 MPa | Working pH | 3–10 (operation); 2–13 (CIP process) | Chemical stability | Stable in common aqueous buffers, 0.5 mol/L NaOH, 8 mol/L urea, 30% isopropanol, 70% ethanol, etc. | Storage | 20% ethanol, 2–8 °C | Shelf life | 3 years |
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Notes:
①More than 90% of the microspheres are within this particle size range.
② Test method: 2-hour incubation; the result is close to DBC₁₀%.
③ Maximum tested flow rate at a column height of 10 cm.
Principle of Boronate Affinity
The main interaction during chromatography is the reversible covalent bond (boronate ester) formed between the boronate affinity chromatography medium and 1,2-cis-diol structures (Figure 1). A pH above 8.5 favors the binding of the resin to the target molecule in the form of boronate ester, while conditions above pH 9.0 are rarely used for the separation and purification of biomolecules; a pH of 4.0–6.0 favors the dissociation of boronate ester. In addition to eluting the target molecule by lowering the pH, competitive elution can also be performed with sugars containing 1,2-cis-diol structures such as sorbitol and mannitol.
In addition to reversible covalent bonds, the ligand contains a benzene ring as a hydrophobic group, which may have π–π interactions with benzene ring-containing substances. This requires that the ionic strength of the loading buffer should not be too high, usually about 50 mM, to reduce non-specific adsorption caused by hydrophobic interactions. The tetrahedral structure of boronate ester is negatively charged; to reduce non-specific adsorption caused by ionic interactions, a high ionic strength is required during the binding process. Boronate affinity chromatography medium usually achieves optimal specific binding to target molecules at an ionic strength of 50–500 mM. Divalent cations can reduce non-specific adsorption caused by ionic and hydrophobic interactions; for example, 0–50 mM MgCl2 can be added to the loading buffer. Mg2+ can also inhibit charge repulsion between the resin and phosphate-containing substances such as nucleic acids, and adding Mg2+ can enhance the binding effect.
When the formed boronate ester exists as an uncharged planar triangular structure, the boron atom has an empty orbital and can act as an electron acceptor for charge-transfer interactions. Unprotonated amino groups are good electron donors; when the amino group provides a lone pair of electrons to the boron atom, the boron atom forms a tetrahedral shape, promoting the formation of boronate ester. However, it should be noted that when a hydroxyl group exists near the amino group, the resin cannot form a boronate ester structure with 1,2-cis-diol. This is why ethanolamine and Tris are not suitable as loading buffers.
Figure 1. Affinity principle of boronate affinity chromatography medium
Instructions for Use
1. Column Packing
The column packing method for the resin connected to the chromatography system is described below.
(1)Equilibrate all materials to the same temperature as the chromatography operation; liquids are preferably degassed.
(2)Resin dosage calculation: Quantify the resin volume by sedimentation. Required settled resin volume = Column volume × Compression factor. The settled volume is the stable volume read after complete natural sedimentation of the resin in 20% ethanol storage solution. The compression factor of boronate affinity chromatography medium is ~1.10. To achieve the compression factor, compression can be performed by lowering the column top or at a high flow rate.
(3)Resin washing: Mix the resin suspension thoroughly, measure the corresponding volume, remove the liquid, and wash with approximately 3 resin volumes of purified water, repeated 3 times to remove the storage solution.
(4)Packing slurry preparation: Transfer the resin to a suitable container, add an appropriate amount of packing buffer to prepare a 50–75 v% packing slurry (the original package contains 50% resin by volume), and mix well before use.
(5)Pre-packing preparation: Add packing buffer to the bottom of the cleaned chromatography column to remove air from the lower frit and the column bottom, leave a small amount of distilled water in the column, tighten the lower plug, and adjust the column to be vertical to the ground.
(6)Packing: Pour the well-mixed resin suspension slowly into the chromatography column at one time (use a packing device if necessary). To avoid introducing air bubbles, let it flow naturally along the inner wall of the chromatography column. After adding all the resin, fill the packing device with packing buffer, tighten the top cover of the packing device, and connect the column to the chromatography system.
(7)Compaction: Let the resin settle naturally (or at a low flow rate of 50–150 cm/h). After complete sedimentation (clear interface between resin and liquid), remove the packing device, install the upper column top, and lower the top to the interface; continue compaction at a high flow rate (see the table below for recommended flow rate; note that column pressure does not exceed 0.3 MPa) until the interface is clear and stable, and mark the column height when the interface is stable. Stop the pump, open the valve/plug on the column top, close the valve/plug at the column bottom, lower the top to the position below the marked position corresponding to the compression factor, tighten the top, and column packing is completed. After packing, equilibrate the resin in the column at a high flow rate.
Packing Conditions | Boronate Affinity Chromatography Resin | Compression factor | ~1.10 | Packing flow rate | 600–1500 cm/h, depending on column size, subject to compression factor and column efficiency test |
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2. Column Efficiency Determination and 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 and Sample Loading
Before sample loading, equilibrate the column with loading buffer (equilibration buffer) at the operating flow rate, and observe changes in the detector until parameters such as conductivity and pH remain constant. The sample loading volume is selected according to the sample properties and the amount of chromatography medium, which can be determined in preliminary experiments (e.g., static adsorption experiment). Generally, the sample needs to be diluted with loading buffer, e.g., 1:1 dilution.
The pH of the loading buffer is usually 8.5–9.0. For example, HEPES buffer (20 mM HEPES, 20 mM MgCl2, pH 8.5) can be used to separate glycoproteins from non-glycoproteins, and ammonium acetate buffer (0.25 M ammonium acetate, pH 8.8) can be used to separate ribonucleosides from deoxyribonucleosides. Common loading buffer systems for this resin include phosphate, ammonium acetate, HEPES, etc. In general, amino-containing buffers such as Tris-HCl should be avoided.
4. Elution
Wash the loaded column with 2–3 column volumes of equilibration buffer, and observe changes in the detector until parameters such as conductivity and pH remain constant. At this time, unbound components are washed out.
Elution of affinity chromatography columns can be performed by isocratic elution, gradient elution or step elution. During elution, the pH can be reduced to 4–6 (e.g., 0.1 M formic acid, 25 mM hydrochloric acid); sugars can also be selected for competitive elution, such as 10–200 mM sorbitol; 0–20 mM EDTA can also be used; Tris can be used as a high-efficiency eluent.
5. Regeneration and Cleaning-in-Place (CIP)
The resin can usually be regenerated with 5 CV of eluent. If inactivated proteins or lipids cannot be washed out during regeneration, they can be removed by Cleaning-in-Place (CIP). The following options can be used for CIP: 0.5 mol/L NaOH, 70% ethanol, 30% isopropanol, etc. CIP can effectively remove impurities on the medium, and the effect is better in reverse flow. Refer to the table below for specific CIP solution selection.
CIP Process | Purpose | 0.5 mol/L NaOH (minimize cleaning time); water, 5 CV | Remove protein precipitates or other impurities such as pyrogens | 30% isopropanol or 70% ethanol, 4–10 CV, <50 cm/h; water, 5 CV | Remove tightly bound lipoproteins, liposomes, etc. |
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6. Resin Storage
The initial storage solution of the resin is 20% ethanol, and the resin can continue to be stored in 20% ethanol after use. It can also be stored in buffers similar to elution and regeneration, such as 0.2 M sodium acetate and 0.1 M formic acid. 0.02% sodium azide can be added to the buffer for antisepsis. The resin is more stable in a weakly acidic environment. The storage temperature is preferably 2–8 °C; do not freeze.
ZJ26F0736961
危险品化学品经营许可证（带存储）