混合抗体的亲和色谱分离策略

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。

Separation strategy of affinity chromatography for mixed antibodies

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.