抗ErbB2嵌合抗体chA21聚集现象的鉴定和分析

Characterization and Analysis of the Aggregation of a Single-chain Chimeric Anti-ErbB2 Antibody

摘要:

聚集现象是目前基因重组抗体面临的挑战之一。采用高效排阻色谱(HPSEC)、动态光散射(DLS)、SDS-PAGE和间接ELISA鉴定抗ErbB2嵌合抗体chA21聚集的形式和性质, 并比较温度和添加剂对抗体聚集程度、抗原结合活性的影响, 用圆二色谱(CD)检测不同聚集条件下蛋白构象的变化, 最后将抗体酶解分离分析聚集的部位。结果表明: chA21在溶液中可形成二聚和更高形式的聚集, 聚集体是通过非共价键相互作用形成, 并仍保留抗原结合活性, 聚集程度和活性受温度和添加剂影响, 而蛋白质的构象相对稳定, 抗体可变区的相互作用可能是引起聚集的主要原因之一。对抗体聚集现象的分析有助于建立稳定的抗体保存条件, 并为今后抗体进一步改造提供依据。

Abstract:

We studied the aggregation of a recombinant engineering antibody (chA21). Anti-ErbB2 antibody chA21 was produced by fusing single-chain Fv (scFv) with human IgG1 Fc fragment, and it was proved to be a drug candidate for cancer therapy. We characterized the aggregation of chA21 by high performance sized-exclusive chromatography (HPSEC), dynamic light scattering (DLS), SDS-PAGE, indirect ELISA assay, and compared the influence of temperature and additive on the level of aggregation and binding activity. Conformation changes of different levels of aggregation were also analyzed via circular dichroism (CD). Finally, we analyzed which part of chA21 was involved in aggregation by cleaving it into scFv and Fc fragments. The results showed that chA21 could form aggregates in the storage solution. The aggregates interacted through non-covalent bonds and remained binding activity. Temperature and additive could slightly affect the level of aggregation and binding activity, while the conformations of chA21 were stable. Aggregation propensity of scFv fragment was almost same as chA21, indicating that scFv may be the major part to form the aggregates. The research on aggregation may be helpful to develop a suitable formulation for chA21 clinical application as well as provide direction for future antibody design and reconstruction.