色谱

色谱 ›› 2013, Vol. 31 ›› Issue (10): 974-979.DOI: 10.3724/SP.J.1123.2013.03026

• 研究论文 • 上一篇    下一篇

混合抗体的亲和色谱分离策略

周月芳1,2, 张焱2, 罗坚2, 康丽梅2,3, 陈毅2, 石红2, 孟庆雄1, 苏志国2   

  1. 1. 昆明理工大学生命科学与技术学院, 云南 昆明 650224;
    2. 中国科学院过程工程研究所 生化工程国家重点实验室, 北京 100190;
    3. 四川大学华西药学院, 四川 成都 610041
  • 收稿日期:2013-03-13 修回日期:2013-04-25 出版日期:2013-10-28 发布日期:2013-10-08
  • 通讯作者: 张焱, 孟庆雄, 苏志国
  • 基金资助:

    国家重点基础研究发展计划("973"计划)项目(2013CB733600,2013CB733604);国家高技术研究发展计划("863"计划)项目(2012AA021202);国家自然科学基金项目(21306207,21275134,3100960);中国计量科学研究院基础科学研究项目(21-JB1127).

Separation strategy of affinity chromatography for mixed antibodies

ZHOU Yuefang1,2, ZHANG Yan2, LUO Jian2, KANG Limei2,3, CHEN Yi2, SHI Hong2, MENG Qingxiong1, SU Zhiguo2   

  1. 1. Institute of Life Science and Technology, Kunming University of Science and Technology, Kunming 650224, China;
    2. National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China;
    3. West China School of Pharmacy, Sichuan University, Chengdu 610041, China
  • Received:2013-03-13 Revised:2013-04-25 Online:2013-10-28 Published:2013-10-08

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摘要：

乳腺生物反应器可以高效表达重组人单克隆抗体,但是目标产品与乳液原料中的牛抗体性质、结构非常类似,分离难度很大。本文对牛抗体和重组人抗体的种属差异进行了分析,并在此基础上制定了新型分离策略,采取Protein A亲和色谱和免疫亲和色谱来解决混合抗体的分离问题,并讨论了色谱洗脱模式对分离效果的影响。结果表明,Protein A亲和色谱结合梯度洗脱可以有效地纯化得到混合抗体,但是难以彻底分离重组人抗体和牛抗体;相比之下,使用Protein A亲和色谱结合置换色谱模式可以更加高效地分离混合抗体,最终可以得到纯度高达95%以上的重组人抗体,回收率可达95%以上。免疫亲和色谱同样可以有效地分离纯化重组单克隆抗体,且其通用性更强,可以应用于任何动物乳腺表达重组人抗体的分离纯化中。

关键词: Protein A亲和色谱, 免疫亲和色谱, 牛抗体, 乳腺生物反应器, 重组人抗体

Abstract:

A mammary gland bioreactor can efficiently express human recombinant monoclonal antibody. However, the target products are similar to the bovine antibody in the raw emulsion material in properties and structures. Thus it is difficult to achieve effective separation of the target products. In this work, the species differences between bovine antibody and recombinant human antibody were analyzed and a new separation strategy was raised based on it. We employed two kinds of affinity chromatography to separate these two antibodies from each other and studied the effect of elution mode upon separation. The results demonstrated that Protein A affinity chromatography could get hybrid antibodies using gradient elution mode, but hardly separate the recombinant human antibody and bovine antibody from each other. In contrast, the combination of Protein A affinity chromatography and displacement chromatography could separate the hybrid antibodies effectively and finally give recombinant human IgG (rHGG) product with the purity of 95%and the yield of more than 95%. Immuno-affinity chromatography could also effectively purify recombinant monoclonal antibodies and owned better generality, which could be used in purification of recombinant antibody expressed by any animal mammary gland.

Key words: bovine antibody, immuno-affinity chromatography, mammary gland bioreactor, Protein A affinity chromatography, recombinant human antibody

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