超大孔聚苯乙烯微球的改性及作为生物分离介质的探索
文献类型：学位论文
| 作者 | 曲剑波 |
| 学位类别 | 博士 |
| 答辩日期 | 2009-05-30 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 过程工程研究所 |
| 导师 | 马光辉 |
| 关键词 | 超大孔聚苯乙烯微球 亲水改性 生物分离介质 快速蛋白质分离色谱 对流传质 |
| 其他题名 | Modification of Gigaporous Polystyrene Microspheres for Potential Bioseparation Media |
| 学位专业 | 生物化工 |
| 中文摘要 | 随着现代生物技术产业的迅速发展，蛋白质的大规模分离已显得越来越重要，但现有的层析介质由于孔道狭小难以进行蛋白质等生物大分子的快速分离。本实验室前期研制的超大孔聚苯乙烯 (PS) 微球有望满足蛋白质分离对介质孔道的要求，但是PS的非特异性吸附作用会导致蛋白质等生物大分子失活变性，限制了该类介质在生物大分子分离方面的应用。针对这个问题，本论文采用了两种亲水性高分子（聚乙烯醇和琼脂糖）对超大孔PS微球进行了修饰镀层，进而利用镀层衍生出一种DEAE弱阴离子交换介质，并对该介质在快速分离生物大分子色谱上的应用作了初步的探索。 利用人工合成亲水性高分子聚乙烯醇 (PVA) 镀层疏水材料可以有效降低疏水材料对蛋白质的非特异性吸附，关于物理吸附PVA改性PS微球的文献较多，在PS微球上化学偶联PVA还未见报道。本论文首先通过Friedel-Crafts反应在超大孔PS微球上成功引入活泼的氯乙酰基；然后在碱性条件下利用Williamson反应将不同分子量的PVA通过稳定的C-O-C键以侧面连接 (side-on) 方式偶联到超大孔PS微球上，并结合称重法和红外光谱定量法，建立了一种方便快捷地确定PVA偶联量的方法。实验结果表明，用PVA修饰PS微球既能够有效掩盖其疏水表面，又保持了PS微球的超大孔结构，改性后的PS微球亲水性大幅提高，对牛血清白蛋白 (BSA) 的非特异性吸附量从原来的89.55 mg/g干球下降到7.72 mg/g干球，但与商品化介质Sepharose 6 Fsat Flow (Sepharose 6FF) 相比还有一定差距（0.574mg/g干球）。 为进一步降低超大孔PS微球对蛋白的非特异性吸附，本文尝试使用天然多糖-琼脂糖对超大孔PS微球进行修饰。在琼脂糖的侧链引入疏水锚点，采用先物理吸附，后化学交联的策略，在超大孔PS微球表面覆盖了一层疏水改性琼脂糖分子(Agap)。琼脂糖链上苯氧基的引入既增强了Agap与PS微球表面的吸附作用力，又能准确定量吸附量。经过化学交联后镀层稳定性得到很大提高，室温下镀层在1M HCl 或1M NaOH中浸泡24小时，检测不到明显的镀层脱落情况。琼脂糖分子链的天然螺旋结构对掩盖PS微球的疏水表面比线性PVA分子更为有利，改性后微球对BSA的非特异性吸附量从原来的89.55 mg/g干球下降到1.18mg/ g干球，与Sepharose 6FF 接近。 以琼脂糖镀层的超大孔PS微球为基质，通过Williamson反应将DEAE基团偶联到微球上制备了弱阴离子交换介质 (DEAE-AP)。系统考察了DEAE化反应条件，做到配基密度可控。并对弱阴离子交换介质的吸附性能进行了表征，与商品化DEAE-FF介质进行了对比。微球的电镜照片表明DEAE-AP的孔径要明显大于DEAE-FF，与之对应，DEAE-AP对BSA的有效孔隙率比常规介质DEAE-FF提高了22%，显示超大孔介质在提高生物大分子传质速率方面有明显优势。阐释了介质配基密度对BSA吸附量以及洗脱率的影响：BSA在介质上的吸附属于单分子层多位点吸附，随着配基密度的增加BSA在介质上吸附形态有由side-on到end-on转变的趋势，吸附量逐渐增大，但最终状态是介于两者之间，进一步增加配基密度蛋白质饱和吸附量不变，只会增加单个BSA分子的配基结合数量，导致蛋白洗脱率下降。介质装柱后压力流速曲线实验表明：当线性流速达到3610 cm/h时，DEAE-AP柱压降仅为0.34MPa，而DEAE-FF柱在1670 cm/h时压降便会急剧升高，无法操作；在DEAE-FF柱可以操作的压力范围内，DEAE-AP柱的渗透系数仍然是它的3.34倍。 以BSA为探针蛋白考察了DEAE-AP装柱后的柱效和动态吸附容量，选取肌红蛋白、转铁蛋白、BSA作为模拟混合蛋白体系，使用DEAE-AP介质对混合蛋白进行了分离，并与DEAE-FF介质进行了对比。结果表明DEAE-AP柱的理论板高度 (HETP) 与流速没有明显关联，说明DEAE-AP介质内存在对流传质作用。与DEAE-FF柱相比，BSA在DEAE-AP柱上的穿透曲线形状属于窄陡型，前者属于宽缓型，表明超大孔的存在有效改善了传质效果。与之对应，不同流速下DEAE-AP介质对BSA的动态吸附容量明显高于DEAE-FF介质。超大孔介质DEAE-AP的分离速度和分离度均显著优于DEAE-FF介质，在2600cm/h流速下可以在3min之内将蛋白混合物分开。 研究表明，采用亲水性高分子PVA和Agap修饰超大孔PS微球是两种非常有效的亲水改性方法，尤其是后者，由于具有天然的螺旋结构，在抵制蛋白吸附方面比线性高分子更具优势。改性后微球大孔结构得到保持，对蛋白的非特异性吸附迅速降低，并且微球表面富含羟基容易衍生成各种色谱介质。作为示例，改性后微球进一步偶联配基制备了DEAE阴离子交换介质，初步蛋白分离实验结果表明超大孔介质在快速分离蛋白质色谱上具有很大的潜力和优势。 |
| 英文摘要 | With the rapid development of modern biotechnology, the technology of large-scale separation of preparative proteins became more and more important. However, it is difficult for convenient media to perform high-speed separation of proteins owing to their small pores. It is expected that the gigaporous polystyrene (PS) microspheres prepared in our previous study could satisfy the requirement of high-speed protein separation for media’s pore size. Unfortunately, the gigaporous PS microspheres are difficult to be directly used in the chromatography for proteins because of their high hydrophobic properties, which will lead to non-specific adsorption and denaturation of proteins. In this study, two hydrophilic polymers (poly(vinyl alcohol) and agarose) were used to hydrophilically modify the gigaporous microspheres. Afterwards, the agarose coated PS microspheres were further functionalized by coupling Diethylaminoethyl (DEAE) groups to prepare a weak anion exchange matrix (DEAE-AP), and the preliminary exploration of DEAE-AP as biomacromolecular high-speed separation medium was performed. Poly(vinyl alcohol) (PVA), a synthetic hydroxyl-rich polymer, could effectively decrease the non-specific adsorption of proteins on hydrophobic materials as a modifier. To the best of our knowledge, previous works mainly focused on physical adsorption, while few reports were available about covalent coupling. In present study, the microspheres were chloroacetylated through Friedel-Crafts acetylation with chloroacetyl chloride, and modified with hydrophilic PVA through Williamson reaction afterwards. The coupling amount of PVA was conveniently and efficiently determined through combining Fourier transform infrad spectra with gravimetric analysis together. The configuration of PVA chains on PS microspheres surfaces was side-on but not end-on, which could not only effectively mask the hydrophobic surfaces of PS microspheres but also maintain the gigaporous structure of PS microspheres. After modification, the adsorbed amount of bovine serum albumin (BSA) on PS microspheres decreased significantly from 89.55mg/g dry microspheres to 7.72mg/g dry microspheres. Nevertheless, compared with Sepharose 6 Fast Flow (0.574 mg/g of dry microspheres), the non-specific adsorption amount of BSA is still considerable. Agarose, a helical natural polysaccride, was occupied to modify gigaporous PS microspheres, which is expected to further decrease the non-specific adsorption amount of proteins on PS microspheres. First, the phenoxy groups were introduced on the side chains of agarose as hydrophobic anchors. The introduction of phenoxy groups onto agarose backbone could not only favor the adsorption by inducing hydrophobic force but also accurately quantify the adsorption amount of agarose. Then, the gigaporous PS microspheres were physically coated with hydrophobically modified agarose (phenoxyl agarose, Agap). After further chemical crosslinking of coated Agap, the stability of the coating was greatly improved and no apparent leakage of Agap was detected after 24 h at ambient temperature either in 1M HCl or 1M NaOH. The configuration of Agap chains onto PS microspheres surfaces was also side-on, which facilitated the maintenance of gigaporous structure of PS microspheres. It indicated that the helical structure of agarose is more effective than linear structure of PVA in protein resistance. Compared with PS microspheres, the adsorbed amount of BSA on Agap-co-PS sample decreased from 89.55mg/g dry microspheres to 1.18 mg/g of dry microspheres, which is very close to the adsorption amount of Sepharose 6 Fast Flow. Taking Agap-co-PS microspheres as base supports, the DEAE anion exchange matrix (DEAE-AP) was prepared by Williamson reaction in the third part. The ligand density could be controlled by optimizing the DEAE reaction conditions. Scanning electron microscopy (SEM) images show that the pore size of DEAE-AP microspheres was evidently larger than that of DEAE-FF resins. Compared with DEAE Sepharose Fast Flow (DEAE-FF), the effective porosity of DEAE-AP to BSA molecules increased 22%. This indicated that DEAE-AP has evident advantage in improving biomacromolecule mass transfer rate. The effect of ligand density on adsorption capacity and desorption ratio of BSA was also studied. The adsorption status of BSA on medium is single molecule layer and multi-sites adsorption. With the increase of ligand density, the configuration of BSA molecules on medium surfaces had the tendency to change from side-on to end-on, but the final configuration was between side-on and end-on. When the ligand density was further increased, the adsorbed capacity of medium did not ascend but the ligand number occupied by every BSA molecule increased further, which led to the decrease of desorption ratio of BSA. The results of flow hydrodynamics showed that the linear relationship was obtained on DEAE-AP column for flow velocity up to 3612cm/h. However, for DEAE-FF column, the linear relationship was only obtained for flow velocity up to 1668 cm/h owing to DEAE-FF resins’ low mechanical stability. Correspondingly, the value of bed permeability (K) for DEAE-AP column was 3.34 times higher than that for DEAE-FF column. Finally, the column efficiency and dynamic adsorption capacity of DEAE-AP column was evaluated by probe protein BSA. Taking the mixture of myoglobin, transferring and BSA as model system, the separation property of DEAE-AP column was also studied. The results showed that there was convective mass transfer in the DEAE-AP medium owing to the existence of through pores in the gigaporous microspheres, and the protein mixture could be separated completely by using DEAE-AP column under 2600cm/h in 3 min. The gigaporous DEAE-AP medium showed greater advantages than commercial DEAE-FF medium in respect to separation rate and resolution. The research of this thesis demonstrated that the hydrophilic modifications of gigaporous PS microspheres using PVA and agarose as modifiers are both effective methods. Notably, the helical structure of agarose has more advantages than linear structure of PVA in protein resistant compentence. After modification, the non-specific adsorption of proteins on microspheres was significantly decreased and the gigaporous structure of microspheres was remained. The hydrophilically modified microspheres are a promising chromatographic support for different types of chromatography such as ion-exchange or affinity chromatography since they were easily derivatized by classical methods. As an example, the modified microspheres were further coupled with DEAE groups to prepare DEAE anion exchange medium, and preliminary protein separation results indicated that gigaporous matrix has great potentials and advantages in high-speed protein chromatography. |
| 语种 | 中文 |
| 公开日期 | 2013-09-13 |
| 页码 | 158 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1198]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 曲剑波. 超大孔聚苯乙烯微球的改性及作为生物分离介质的探索[D]. 过程工程研究所. 中国科学院过程工程研究所. 2009. |
入库方式： OAI收割
来源：过程工程研究所
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