摘要
液相层析技术是生物分离过程中的一个重要单元,具有分辨率高、操作设备简单和有利于保持生物大分子的活性等优点,是一种最有效、最普遍采用的生物分离方式之一。而层析技术的关键就是层析介质,随着科技的进步和对生物产品需求的不断增长,层析介质的性能也得到了不断的提高。本文以刚性陶瓷/琼脂糖复合层析介质的制备、功能化及应用为目标,以复合介质的制备为中心,希望开发一种具有新型结构的层析介质,主要开展了以下几方面的工作:
(1)制备多孔氧化硅陶瓷微球。以纳米氧化硅粉体为骨架材料,PEG10000为致孔剂,海藻酸钠溶液为胶黏剂,采用反相悬浮法制备陶瓷微球生胚,再通过高温烧结得到一种孔隙率高、机械强度大、球形度好的多孔陶瓷微球。经氮气吸附实验测定,其比表面积达28.75 m2/g,BJH平均孔径为33.3 nm,BJH孔容为0.246 m3/g;并且其流通性能良好,在764 cm/h的高操作流速下柱背压只有0.18 MPa。
(2)制备了刚性陶瓷/琼脂糖复合层析介质。采用热渗透法将琼脂糖凝胶填充到多孔陶瓷微球中,再通过研磨、筛选和流化床淘洗的方式去除微球表面的琼脂糖凝胶,得到一种复合介质。其湿真密度为1.70 g/mL,平均粒径为65.3μm,经性能评估可知复合介质在耐受柱压、柱效、装柱速度等方面均优于传统琼脂糖基填料6FF,较适宜用于制备型蛋白质层析。
(3)将复合介质功能化为阳离子交换剂并考察其蛋白质吸附性能。首先,采用二次交联法对复合介质进行化学修饰并偶联氯乙酸,制备得到CM-复合介质,其离子交换容量为72.4μmol/mL。然后,以溶菌酶为模型蛋白考察了其等温吸附曲线、吸附动力学曲线和不同流速下的穿透曲线。结果表明其静态饱和吸附容量为55.9 mg/mL,符合二级吸附动力学模型;在高操作流速时依然可对蛋白质进行有效吸附。
(4)将复合介质功能化为具有谷胱甘肽功能基团的亲和层析介质,并实现了对谷胱甘肽转移酶融合蛋白的纯化。经优化,复合介质的环氧基团活化密度为13.1μmol/mL,GSH配基密度为10.2μmol/mL,偶联效率达77.8 %;之后考察了洗脱方式、上样量和操作流速对介质吸附性能的影响。结果表明,谷胱甘肽亲和作用具有专一性好的特点,目标蛋白可被完全吸附;线性洗脱或梯度洗脱都能达到目标纯度;并且复合介质具备较高的吸附容量,可在较高操作速度下应用,较适合融合蛋白的纯化。
Liquid chromatography, as an important unit in bioseparation process, is the most effective and widely used technology with the advantages of high resolution, simple operating equipment and high bioactivity preservation. The chromatographic media is the key of this technology. With the advance of technology and the increase of requirements for biological products, the performances of chromatographic packing materials have been continuously improved. The present work was aimed at developing a new chromatographic media with novel structure, mostly focused at preparation, functionalization and application of rigid ceramic/agarose composite adsorbents for liquid chromatography. The main works were carried out as follows:
(1) Preparation of porous silica beads. Firstly, the ceramic pulp was fabricated by silica powders as skeleton, PEG10000 as porogenic agent and sodium alginate solution as binder. Later, the ceramic pulp was prepared into elemental silica particles by the method of oil emulsion and immobilized by calcium chloride. Then, the final porous silica beads with high porosity, strong bonds and good sphericity were obtained through high temperature sintering. Nitrogen adsorption-desorption analysis given a BET surface area of 28.75 m2/g, a BJH mean pore size of 33.3 nm and a porosity of 0.246 m3/g, respectively. The ceramic beads had good hydromechanics with the back pressure of 0.18 MPa at the flow velocity of 770 cm/h.
(2) Preparation of porous ceramic/agarose composite beads. The porous of ceramic beads were filled with agarose solution. After mildly grinding with water in a mortar, the gel outside of the beads was removed by screening with standard sieve and elutriation in a fluidized bed. The composite particles had a wet density of 1.70 g/mL and a mean size of 65.3μm. The composite beads were superior to the traditional agarose based beads of 6FF in hydromechanics, column efficiency and column packing by the performance evaluation. Above all, it was appropriate for preparative chromatography.
(3) Functionalization of the composite beads as cation ion-exchanger. Firstly, the beads were double cross-linked with glycol diglycidyl ether and epichlorohydrin. Then, carboxymethyl (CM) groups were coupled to the composite beads and the total ionic capacity reaches 72.4μmol/mL. Finally, The adsorption isothermal, adsorption dynamics, breakthrough curves were determined by using lysozyme as model protein. The saturated adsorption capacity was 55.9 mg/mL. The adsorption dynamics dates were well fitted with the pseudo second-order kinetic mode, and the composite beads could be operated at high velocity without loosing binding capacity.
(4) Functionalization of the composite beads as GSH-adsorbents and purification of GST-ADAM15. Firstly, The BDGE activation efficiency and GSH ligand density reached 13.1 and 10.2μmol/mL, respectively. And the final coupling efficiency reached 77.8 %. Then, the chromatographic process of the GSH-adsorbents as function of elution mode, sample injection and operating velocity were evaluated. The results indicate that the GSH affinity adsorbents were provided with high specifity. The target protein could be adsorbed completely and target purity could be obtained with both of linear gradient and 100 % B solution elution. The composite adsorbents prepared were suitable for purification of fusion protein at high velocity.
引文
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