人源化抗人TNFα单克隆抗体纯化工艺的优化Development and optimization of purification process for humanized anti-human TNFα monoclonal antibody
魏建玲,张波,刘颖,夏焕章
WEI Jian-ling,ZHANG Bo,LIU Ying,XIA Huan-zhang
摘要(Abstract)：
目的考察中国仓鼠卵巢细胞(Chinese hamster ovary,CHO细胞)表达的人源化抗人TNFα单克隆抗体的亲和色谱过程中的杂质洗脱工艺,优化目的蛋白洗脱工艺条件。方法以目的蛋白回收率和宿主细胞蛋白残留量为评价指标,筛选最佳的杂质洗脱缓冲液;利用实验设计方法,以纯度、洗脱峰pH值、宿主细胞蛋白残留量为评价指标,考察目的蛋白洗脱缓冲液的pH值和浓度对工艺的影响,优化目的蛋白洗脱工艺条件。结果优化后的亲和色谱工艺,在保持目的蛋白高回收率(优化前为86.5%,优化后为86.7%)的同时,可显著降低目的蛋白洗脱峰中宿主细胞蛋白残留量(由1 218×10-6降低至78×10-6),提高目的蛋白纯度(优化前为95.5%,优化后为96.6%),减轻后续纯化压力;提高了目的蛋白洗脱峰的pH值(由3.59提高到4.25),有利于目的蛋白的稳定。结论优化了人源化抗人TNFα单克隆抗体的亲和色谱工艺,本工艺有效、可行。
Objective To optimize the step of impurity removal and targeted protein elution in the process purification of humanized TNFα antibody that expressed in CHO cell by affinity chromatography.Methods In order to screen the optimal impurity elution buffer,the recovery and residual HCP of elution peek collected were investigated.The factors influencing target protein elusion were optimized by design of experiment method.The effects of p H and concentration of target protein elution buffer on HPLC purity,p H,residual HCP of elution peek collected were investigated.Results The results indicated that residual HCP in targeted protein peak collection was reduced dramatically from 1 218 × 10- 6to 78 × 10- 6,without compromising of recovery(86.5% before optimization,86.7% afer optimization).The purity of target protein was improved from 95.5% to 96.6%,and p H was increased from 3.59 to 4.25 meanwhile after the process optimization,which was beneficial for the stability of the target protein and subsequent operation.Conclusions The process of affinity chromatography was optimized and it was proved to be effective and feasible for monoclonal antibody purification.
关键词(KeyWords)：
单克隆抗体;TNFα;亲和色谱;工艺优化;纯化
monoclonal antibody;TNFα;affinity chromatography;process optimization;purification
基金项目(Foundation):
作者(Author):
魏建玲,张波,刘颖,夏焕章
WEI Jian-ling,ZHANG Bo,LIU Ying,XIA Huan-zhang
DOI: 10.14066/j.cnki.cn21-1349/r.2017.01.014
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