Antibody aggregation can lead to reduced Protein A binding capacity and low step yield

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2023-10-01 DOI:10.1016/j.pep.2023.106315

Gaoya Yuan , Meng Qu , Qi Geng , Wanyuan Dong , Xudong Zhang , Yifeng Li

引用次数: 2

Abstract

Protein A affinity chromatography has been widely used for antibody capture and initial purification. In general, the yield of this step is relatively high (i.e., >90%). However, recently while purifying a bispecific antibody (bsAb) in appended IgG format, the Protein A capture step experienced low yield (i.e., ∼80%). It was found that the target bsAb started appearing in flow-through at a relatively low load density, suggesting that a portion of the expressed bsAb has compromised Protein A binding capability. Further studies indicated that the bsAb in flow-through was mainly in aggregate form. In addition, normal Protein A step yield was restored when the column was loaded with bsAb monomer. Thus, all the evidence pointed to the fact that aggregate with compromised binding capacity was the cause of low Protein A step yield in this case.

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抗体聚集可导致蛋白A结合能力降低，步产率降低

蛋白A亲和层析已被广泛用于抗体捕获和初始纯化。通常，该步骤的产率相对较高（即，>；90%）。然而，最近在纯化附加IgG形式的双特异性抗体（bsAb）时，蛋白质a捕获步骤的产率较低（即～80%）。发现靶bsAb在相对低的负载密度下开始在流通中出现，这表明部分表达的bsAb已经损害了蛋白a的结合能力。进一步的研究表明，流动通道中的bsAb主要以聚集体形式存在。此外，当柱负载bsAb单体时，蛋白质A的阶跃产率恢复正常。因此，所有的证据都表明，在这种情况下，具有受损结合能力的聚集体是低蛋白A步骤产率的原因。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.