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题名	新型高性能琼脂糖微球的制备及作为生化分离介质的探索
作者	赵希
学位类别	博士
答辩日期	2014-05
授予单位	中国科学院研究生院
导师	马光辉
关键词	琼脂糖微球 粒径均一 预交联工艺 胶团溶胀致孔 生物大分子分离介质
其他题名	Preparation of High-performance Agarose Microsphere for Potential Bioseparation Media
学位专业	生物化工
中文摘要	近年来，随着生物技术产业的迅猛发展，生物大分子的高效分离纯化受到普遍关注。琼脂糖微球是生化分离领域应用最广泛的分离介质母体，但现有琼脂糖层析介质存在微球粒径不均一、机械强度低以及介质孔径小等问题，在生物大分子分离纯化以及大规模层析操作中应用受限。本论文采用膜乳化技术制备出粒径均一的大粒径琼脂糖微球，发展新型的预交联工艺和胶团溶胀致孔法大幅度提高凝胶骨架刚性以及凝胶孔径，衍生出一种以高强度、大孔琼脂糖微球为基质的弱阳离子交换介质，并初步探索了该介质在生物大分子分离纯化中的使用效果。论文的主要研究内容如下： 1. 粒径均一的大粒径琼脂糖微球的制备及放大设备的研制。实验室在前期工作中使用微孔膜乳化技术成功制备了粒径均一可控的琼脂糖微球，但是产品平均粒径小于60 μm，限制了其在大规模纯化领域中的应用。针对常规膜乳化制备大粒径琼脂糖微球过程中压力难于控制，乳滴间容易聚并和沉淀等问题，本论文提出在操作压力为零的条件下，利用琼脂糖溶液自身重力通过SPG膜形成均一乳液，并重点考察了微孔膜孔径、乳化转速、油相组成等关键参数对琼脂糖微球尺寸与粒径均一性的影响，制备得到均一的大粒径琼脂糖微球（d0.5=92.1 μm，Rspan=1.15）。所得产品无需筛分即可应用于生化分离领域，粒径均一性优于筛分后的商品化介质Sepharose 4FF。在此基础上，进一步研制出适用于高黏度均一乳液制备的放大常规膜乳化装置，深入探讨了均一乳液的形成机制，为大粒径琼脂糖微球介质的规模化制备奠定了基础。 2. 均一的高交联琼脂糖微球的制备。为了解决传统交联方法交联效率低、凝胶强度不足等问题，本论文提出一种预交联制备高强度琼脂糖微球的新方法。通过向琼脂糖溶液中预引入具有特殊分子结构的交联剂烯丙基缩水甘油醚，大幅度提高交联剂在凝胶微球中的有效浓度（49.8 μmol/g gel），实现交联剂在凝胶微球中的均匀分布，并结合常规膜乳化技术对微球粒径均一性进行控制，成功制备了粒径均一的高交联琼脂糖微球。系统考察了交联剂用量和交联反应条件对琼脂糖微球机械强度的影响，并对制备工艺参数进行优化。在最佳工艺条件下，所得高交联琼脂糖微球球形圆整，最大流速可达19 mL/min，较商品化介质Sepharose 4FF提高了170%以上。 3. 大孔琼脂糖微球的制备。反胶团溶胀致孔是一种简便有效的制备大孔微球的新方法，但是目前该法局限于疏水性大孔聚合物微球的制备。为了满足生物大分子分离纯化对分离介质的要求，本论文提出采用新型的胶团溶胀致孔法制备亲水性的大孔琼脂糖微球。研究发现，采用离子型和非离子型表面活性剂复配的方式（HLB 值17.0），可成功制备出大孔琼脂糖微球，其中，离子型表面活性剂在稳定乳滴方面发挥了重要的作用。此外，油相性质、吸油溶胀时间和固化速率也对大孔结构的形成有显著影响。在最佳致孔条件下制备得到的琼脂糖微球呈均匀的网状大孔结构，平均孔径达到几百纳米。 4. 新型琼脂糖阳离子交换介质的研发和应用。采用预交联结合胶团溶胀致孔的工艺，制备出机械强度明显优于常规琼脂糖介质、同时具有较大孔径的新型琼脂糖微球（HMA），经氯乙酸修饰，得到阳离子交换介质（CM-HMA）。由于大孔的存在，CM-HMA介质孔内传质速度明显加快，模型蛋白在5 min之内即可从介质表面扩散进入微球内部；而对于商品化介质Sepharose 4FF来说，在吸附60 min后蛋白质分子仍未能进入介质的孔道内部。在蛋白质分离纯化方面，CM-HMA微球在较高的流速下（15 mL/min）仍然能够有效分离模型蛋白混合物，并且分离效果明显优于商品化介质。基于以上优势，所开发的高交联大孔琼脂糖微球在生物大分子快速分离领域具有广阔的应用前景。
英文摘要	As the biotechnology industries develop forward as a dramatic speed these years, a high-efficiency chromatographic separation technique attracted much attention for the purification of biomacromolecules. Agarose microspheres have been widely used as chromatographic packings in bioseparation field. However, its poor size distribution, low mechanical strength, as well as small pores present as limiting factors when a large-scaled chromatography is required. To conquer the above problems, general membrane emulsification technique was employed in this study to prepare uniform and large sized agarose microspheres, and the gel stability as well as pore structure was highly improved by novel pre-cross-linking and surfactant micelles swelling method. Afterwards, cross-linked and macroporous agarose (HMA) microspheres were further fabricated and functionalized by coupling carboxymethyl (CM) groups to prepare a weak cation exchange matrix (CM-HMA). And the preliminary exploration of CM-HMA microspheres as biomacromolecular separation medium was performed. In detail, this thesis mainly included the following four parts: 1. Large-sized agarose microspheres with narrow size distribution were prepared and the production capacity was scaled up by a self-designed membrane emulsification apparatus. To the best of our knowledge, it is difficult to obtain uniform-sized agarose microspheres by membrane emulsification when the particle diameter was higher than 60 μm. In the present study, the investigated process parameters, including the pore size of the SPG membrane, the operating pressure, the stirring rate of the continuous phase and the composition of the continuous phase, were optimized to ascertain microsphere uniformity. As a result, uniform-sized agarose microspheres with average diameter of 92.1 μm were successfully prepared, and the size distribution of the products was much narrower than that of commercial Sepharose 4FF. On this basis, we developed a novel middle-scaled general membrane emulsification apparatus to satisfy the production requirement. And the theoretical mechanism of droplet formation from micropores was discussed in detail. 2. A novel pre-cross-linking method was proposed to prepare rigid agarose microspheres. By introducing pre-cross-linker allylglycidyl ether (AGE) into agarose solution before microsphere formation, the effective content of AGE inside gel particle increased obviously (49.8 μmol/g gel) and thus a high-efficiency cross-linking was achieved. The uniformity of highly cross-linked agarose microsphere could be well controlled by general membrane emulsification. And the effects of AGE amount and cross-linking parameters on the mechanical strength of agarose gel were investigated in detail. Under the optimal conditions, rigid agarose microspheres with perfectly spheroidal shape were successfully prepared with operating flow rate reached 19 mL/min, which was 1.7 times higher than that of commercial Sepharose 4FF. 3. The preparation of macroporous agarose microspheres by surfactant micelles swelling method was studied and the feasibility of this method in hydrophilic system application was confirmed. The effects of surfactants type, continuous phase composition, oil-absorbing duration and solidification rate on the particle property were studied. The results indicated that mixed surfactant with HLB of 17.0 were suitable for the formation of macropores. And the addition of ionic surfactant was crucial on the stabilization of emulsion. Under the optimal conditions, agarose microspheres with homogeneous reticular structure and pore size of hundreds nanometers were successfully prepared. 4. Highly cross-linked and macroporous agarose (HMA) microspheres were fabricated combining pre-cross-linking and surfactant micelles swelling method. Taking HMA microspheres as base supports, the CM cation exchange matrices were further prepared. As expected, the mass transfer of proteins in self-made microspheres enhanced sig
语种	中文
公开日期	2015-07-08
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/15512]
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	赵希. 新型高性能琼脂糖微球的制备及作为生化分离介质的探索[D]. 中国科学院研究生院. 2014.

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