| 题名 | 多聚亚基疫苗的稳定性及层析过程优化 |
| 作者 | 黄永东 |
| 学位类别 | 博士 |
| 答辩日期 | 2008-06-04 |
| 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 | 苏志国 |
| 关键词 | 多聚亚基疫苗 乙肝疫苗 百日咳疫苗 分离纯化 稳定性 添加剂 |
| 其他题名 | Protein stability and chromatography process optimization of multi-subunit vaccines |
| 学位专业 | 生物化工 |
| 中文摘要 | 多聚亚基蛋白质疫苗在层析中存在稳定性差、分离纯化难、收率低的问题，并对疫苗产品的稳定性和免疫原性产生很大影响。本论文以乙肝疫苗（重组CHO细胞（Chinese Hamster Ovary）表达的乙肝疫苗（CHO-HBsAg）和汉逊酵母细胞（Hansenula polymorpha）表达的乙肝疫苗（Hans-HBsAg））和百日咳疫苗（百日咳毒素（PT）和丝状血凝素（FHA））两种多聚亚基疫苗为研究对象，对其在层析中的稳定性进行了考察，并从溶液环境的调整、层析介质的设计和制备、层析工艺的设计和优化等角度出发，来提高疫苗在层析中的稳定性和分离纯化效果。本论文开展的研究工作如下： 1．蛋白质在离子交换吸附过程中发生强烈的多位点吸附，而介质配基密度的降低有利于减弱蛋白质与介质间的相互作用。为克服现有商用离子交换介质的不足，制备了新型的低配基密度DEAE介质，并将其用于CHO-HBsAg的分离纯化。实验结果表明，低配基密度DEAE介质的应用，减弱了CHO-HBsAg与介质间的多位点吸附，提高了疫苗在吸附-解吸过程中的稳定性，减少了因结构变化导致的活性损失，达到了稳定疫苗结构和提高层析效果的目的，将CHO-HBsAg的活性回收率和纯化倍数分别由原来的44.1%和9.4提高到65.6%和10.4。 2．为了减少高配基密度疏水介质对乙肝疫苗亚基结构的影响，设计和开发了新型低配基密度疏水层析介质（Butyl-S QZT-3），将Hans-HBsAg在疏水层析中的活性回收率和纯化倍数分别从79%和4.2提高到95%和10.5；同时，添加剂PEG（polyethylene glycol）的加入，将Hans-HBsAg在超滤浓缩中的活性回收率从30%提高到95%左右。进一步地，在整个纯化工艺整合中，为解决现有Hans-HBsAg分离纯化工艺操作繁琐、收率低和不易放大的难题，以及提高非糖基化Hans-HBsAg的稳定性，发展了一条基于三大层析技术（离子交换层析、疏水层析和凝胶过滤层析）和膜超滤浓缩的组合层析工艺。中试实验结果表明，该工艺具有操作步骤少、各单元操作衔接合理和容易放大的特点，而且Hans-HBsAg活性回收率达到22.0 ± 8.3%，比传统工艺提高了一倍左右，操作时间也从近100小时缩短到35小时左右。低温保存Hans-HBsAg的稳定性考察实验结果表明，采用该工艺制备的Hans-HBsAg具有良好的稳定性。 3．在百日咳疫苗分离纯化过程中，通过添加剂（如0.5~1.0mol/L NaCl或2mol/L脲）的加入，提高了PT和FHA在溶液中的稳定性。开发了一条采用自制的配基密度可控的结合珠蛋白（Hp）亲和介质制备单组分百日咳疫苗（PT）的层析工艺。实验结果表明，配基的纯化方法、配基的纯度和密度都对Hp亲和层析分离纯化PT具有重要影响。当采用DEAE离子交换层析制备Hp配基，配基纯度高于95%，配基密度为0.70 mg Hp/g wet gel时，Hp亲和介质的层析效果最好，单步层析即可制备电泳纯PT，其活性回收率为38.2%。 4．通过层析介质的筛选、添加剂的应用和层析工艺的设计，开发了一条用于双组分百日咳疫苗（PT和FHA）制备的脲伴随式组合层析工艺：硫酸铵沉淀-离子交换层析-凝胶过滤层析，实现了电泳纯PT和FHA的制备，其中PT的活性回收率和纯化倍数分别达到26.8%和37.8，FHA的活性回收率和纯化倍数分别达到37.5%和23.3。在层析工艺中，CM Sepharose FF离子交换层析纯化效果最为显著，去除了大量的杂蛋白，但未能实现PT和FHA的有效分离；而Sephacryl S-200凝胶过滤层析主要起到了分离PT和FHA的目的，同时还实现了PT和PT亚基的分离；添加剂2mol/L脲的引入，则起到提高PT和FHA在层析中的稳定性和层析效果的作用。 |
| 英文摘要 | The main drawback of scaling up multi-subunit vaccines purification process is the instability of multi-subunit protein structure, which is easily affected by the complex purification steps, resulting in the low recovery and the low immunogenicity of the final products. The stability and the impact factors of Hepatitis B vaccine (hepatitis B surface antigen expressed by recombinant Chinese Hamster Ovary (CHO-HBsAg) and Hansenula polymorpha (Hans-HBsAg)) and pertussis vaccine (including PT and FHA) purified by chromatographic process were firstly investigated to understand the mechanism of the subunit assembly structure change in the purification process. Furthermore, the chromatographic process was optimized by the strategies of regulating the solvent environment, designing and developing high efficient chromatographic adsorbents, adding the purification chaperons, such as PEG and urea. The following results were obtained: 1. One of the mechanisms of the subunit assembly structure change is assumed to take place on the surface of the adsorbents, where the adsorption of protein onto ion exchange adsorbents involved a multi-site interaction between the protein and the adsorbents, and such an interaction could be weakened by the reduction of the ligand density of the adsorbent. To overcome the disadvantage of the high binding intensity from the commercially available DEAE Sepharose FF ion exchange adsorbent, new DEAE adsorbents with controllable lower ligand densities were prepared and evaluated by purifying CHO-HBsAg. The results demonstrated that the weak multi-site interaction between CHO-HBsAg and the adsorbents with lower DEAE ligand density can efficiently improve the stability of CHO-HBsAg and therefore increase the bioactivity due to the reduced structure change. Comparing the application of commercial and developed adsorbents, the step recovery and purification-fold of CHO-HBsAg were increased from 44.1% and 9.4 to 65.6% and 0.4, respectively. 2. Based on the understanding of subunit stability, the new Butyl-S QZT-3 adsorbent with a low butyl ligand density was developed to reduce the hydrophobic interaction intensity and therefore stabilize the assemble structure of Han-HBsAg in hydrophobic interaction chromatography (HIC). The Hans-HBsAg recovery and the purification-fold of HIC were improved from 79% and 4.2 to 95% and 10.5 comparing with the commercial HIC adsorbent of Butyl-S Sepharose FF. On the other hand, it was indicated that the low concentration of purification chaperone of PEG can regulate the protein solution environment and therefore avoid the protein aggregation in the process of membrane ultrafiltration (UF) to concentrate Han-HBsAg. The step recovery was improved from about 30% to 95% by the addition of 0.5% (w/v) PEG 10000. Furthermore, to simplify the process for Han-HBsAg purification and therefore improve the final purification recovery, the integrated chromatographic process was developed and then optimized, which integrated ion exchange chromatography (IEC), HIC, gel filtration chromatography (GFC) and ultrafiltration, and the integrated process was scaled up from lab bench to pilot scale. The results indicated that this process was simple and easy to scale up. The Hans-HBsAg recovery and the operation time of the whole purification process were 22.0 ± 8.3% and about 35 hours, respectively, which were superior to those of the traditional purification process. The stability during cold storage was further investigated and demonstrated that Hans-HBsAg purified by this integrated procedure was very stable. 3. To develop the purification process of pertussis vaccine, the stability of the main components of pertussis vaccine PT and FHA in bulk solution was investigated and then improved by the addition of 0.5~1.0 mol/L NaCl or 2 mol/L urea which was therefore applied in the purification process. Haptoglobin (Hp) affinity adsorbents with controllable ligand density were synthesized, and an Hp affinity chromatography process for the purification of PT was developed. The effect of the affinity ligand Hp from different purification methods and the ligand density on the purification of PT was further investigated. The resuls demonstrated that the optimum chromatography efficiency was obtained when the Hp ligand was prepared by IEC to obtain a purity above 95% and the ligand density was 0.70 mg Hp/g wet gel. After a single process of Hp affinity chromatography, the purified PT was obtained with a recovery of 38.2%. 4. Finally, a urea (2.0 mol/L) accompanied integrated chromatographic process, including ammonium sulfate precipitation, IEC and GFC, was developed for the purification of PT and FHA through the screening of chromatographic adsorbents, the addition of additives and the design of chromatographic procedures. The PT recovery and its purification-fold were 26.8% and 37.8, respectively. The FHA recovery and its purification-fold were 37.5% and 23.3, respectively. The chromatographic efficiency by CM Sepharose FF was very effective, and most of the impurities were removed after IEC. PT, its subunits and FHA can be separated effectively in GFC process. |
| 语种 | 中文 |
| 公开日期 | 2013-09-13 |
| 页码 | 205 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1123]  |
| 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 | 黄永东. 多聚亚基疫苗的稳定性及层析过程优化[D]. 过程工程研究所. 中国科学院过程工程研究所. 2008. |
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