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题名	按需设计的琼脂糖层析介质——制备、表征和应用
作者	赵岚
学位类别	博士
答辩日期	2012-06-05
授予单位	中国科学院研究生院
导师	苏志国
关键词	琼脂糖 层析介质 配基脱落 疏水层析 乙型肝炎表面抗原
其他题名	Tailor-made Agarose-based Chromatography Media: Preparation, Characterization and Application
学位专业	生物化工
中文摘要	层析技术在生物分离纯化领域占有非常重要的地位。作为该技术主要组成部分，层析介质的结构与性能极大地影响最终分离纯化效果。但是，市售商品介质大多存在结构不可调节以及配基脱落等问题。本论文以琼脂糖层析介质为对象，旨在开展介质的按需设计（tailor-made）研究，考察tailor-made介质的表征和应用，探索配基脱落机制，并提出解决策略，主要进展如下：确定了琼脂糖微球的最佳合成工艺。通过优化琼脂糖种类、乳化温度和乳化转速等参数，确定了琼脂糖微球的最佳制备路线，制得微球形貌规整，粒径分布均匀，产率达到60%以上。自行设计和开发了一套层析介质自动合成装置，采用该装置可大大缩短合成工时，工艺时间缩短一半以上，且介质产率超过80%。开发了新型高流速琼脂糖微球的制备工艺。以溴丙烯为交联剂，采用预交联方法制备交联琼脂糖微球，通过调整交联方法、交联剂用量及溶剂种类等，反应效率大大提高，制备的琼脂糖微球最大线性流速可达商品介质的两倍以上。探索了琼脂糖性质和交联程度等对琼脂糖微球孔形貌和分离性能的影响规律，通过增加琼脂糖溶液粘度和改变交联程度，初步实现了微球的孔结构可调。从介质自身结构出发，推测了免疫球蛋白G（immunoglobulin G，IgG）亲和介质的配基脱落机制并提出解决策略。通过反相色谱（RP-HPLC）、电泳（SDS-PAGE）、酶联免疫吸附法（ELISA）、红外光谱（FT-IR）、高效分子排阻色谱-多角度激光散射（HPLC-MALLS）等多种手段证实IgG介质在储存和使用过程中存在配基泄露，确定泄漏物主要由IgG配基及其片段、连有配基的基质碎片以及未连配基的基质碎片三部分组成。其中，基质碎片是脱落物的主要组成部分，配基脱落量占总脱落量的比例极低（pH 3.0时小于0.3%，pH 11.0时小于7%）。通过进一步交联基质的方法，可以有效控制配基脱落，酸性和碱性环境下配基脱落量分别减少约27%和10%，糖脱落量分别减少30%和17%。开展了琼脂糖疏水层析介质的tailor-made研究。通过调节间臂长度和配基密度，初步实现了丁基硫疏水介质的疏水性可控并纯化了乙型肝炎表面抗原（HBsAg）。当介质间臂为C8、配基密度为9.18 μmol/g湿胶时，中国仓鼠卵巢（Chinese hamster ovary，CHO）细胞表达的HBsAg回收率达90%以上，纯化倍数96.5。对于酵母表达的HBsAg，介质间臂为C3、配基密度为13.80 μmol/g湿胶时，纯化效果较好，疫苗活性回收率为92.1%，纯化倍数为3.8。通过理论计算分析了间臂和配基密度对疫苗纯化过程的影响。
英文摘要	Chromatography has been playing an important role in bioseparation. As one of the main components of chromatography, the structure and properties of chromatography media have great effects on purification results. However, commercial chromatography media have many problems such as unchangeable structure and ligand leakage. This paper is mainly focused on tailor-made study of chromatography media based on agarose beads. Characterization and application of this tailor-made chromatography media were investigated. Also, the mechanism of ligand leakage was studied and the resolution strategy was proposed. The following results were obtained: (1) The optimum conditions of agarose beads preparation were determined. By optimizing agarose type, the emulsion temperature, the emulsification speed, etc., agarose beads were prepared with regular shape and a narrow distribution with productivity of more than 60%. Automation system was self-designed for chromatography media preparation. Using this system, agarose beads were prepared with a productivity of more than 80% and process time shortened to 1/2 of the original. (2) A new method was developed to prepared agarose beads with a high flow velocity in column. Agarose beads were crosslinked using allyl bromide. By optimizing the reaction conditions such as the crosslinking procedure, the amount of the crosslinking agent and the type of the solvent, the reaction efficiency was greatly improved and crosslinked agarose beads were obtained with the maximum flow velocity of about two times as high as that of the commercial products. (3) The effects of agarose type and the degree of crosslinking on the pore morphology and on the gel permeation properties were studied. By using agarose with a high viscosity or varying the crosslinking degree, agarose beads with a controllable pore structure were obtained. (4) Ligand leakage from IgG Sepharose 6FF was studied and the resolution strategy was studied. The leakage was analyzed by RP-HPLC, SDS-PAGE, ELISA, FTIR and HPLC-MALLS. The leakage consists of three parts: (i) the ligand and its fragments; (ii) ligand plus polysaccharide in which the ligand is chemically attached to the polysaccharide fragments, and (iii) polysaccharide fragments from the adsorbent matrix. The decomposed matrix was the main component of the leakage, and the amount of the detached ligands was very low, i.e., no more than 0.3% of the total leakage at pH 3.0, and 7% at pH 11.0. A high degree crosslinking could reduce 27% ligand leakage at pH 3.0, and 10% at pH 11.0, and could reduce 30% polysaccharide fragments at pH 3.0, and 17% at pH 11.0. (5) Tailor-made study was performed on Butyl-S-agarose, a hydrophobic chromatography medium, with its hydrophobicity well controlled by introducing various lengths of spacer arm and ligand densities. Practical separation of rHBsAg from cell culture was performed with this tailor-made chromatography medium and the purification results were greatly improved. For CHO-rHBsAg, the best purification result was achieved using C8 spacer arm with a ligand density of 9.18 μmol/g suction-dried wet gel. The rHBsAg recovery was more than 90% and the purification factor was 96.5. For Hans-rHBsAg, the best purification result was achieved using C3 spacer arm with a ligand density of 13.80 μmol/g suction-dried wet gel. The rHBsAg recovery was more than 92.1% and the purification factor was 3.8. Theoretical calculations indicated that the length of spacer arm and ligand distribution had great effects on the interaction of rHBsAg with the adsorbent.
语种	中文
公开日期	2013-09-25
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/1786]
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	赵岚. 按需设计的琼脂糖层析介质——制备、表征和应用[D]. 中国科学院研究生院. 2012.

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