ABSTRACT

Therapeutic monoclonal antibodies (mAbs) are highly complex proteins that must be exhaustively characterized according to the regulatory authorities' recommendations. MAbs display micro-heterogeneity mainly due to their post-translational modifications, but also to their susceptibility to chemical and physical degradations. Among these degradations, aggregation is quite frequent, initiated by protein denaturation and then dimer formation. Here, we investigated the nature and structure of the high molecular weight species (HMW) present at less than 1% in an unstressed formulated roledumab biopharmaceutical, as a model of high purity mAb. HMW species were first purified through preparative size-exclusion chromatography (SEC) and then analyzed by a combination of chromatographic methods (ion-exchange chromatography (IEX), SEC) coupled to native mass spectrometry (MS), as well as sodium dodecyl sulfate–polyacrylamide gel electrophoresis and capillary gel electrophoresis under non-reducing conditions. Both covalently and non-covalently bound dimers were identified at a proportion of 50/50. In-depth characterization of the HMW fraction by SEC and IEX hyphenated to native MS revealed the presence of three mAb dimer forms having the same mass, but differing by their charge and size. They were attributed to different compact and elongated dimers. Finally, high-resolution middle-up approaches using different enzymes (IdeS and IgdE) were performed to determine the mAb domains implicated in the dimerization. Our results revealed that the roledumab dimers were associated mainly by a single Fab-to-Fab arm-bound association.

摘要

治疗性单克隆抗体（mAb）是高度复杂的蛋白质，必须根据监管机构的建议进行详尽鉴定。MAb表现出微异质性，这主要是由于它们的翻译后修饰，还在于它们对化学和物理降解的敏感性。在这些降解中，聚集是非常频繁的，由蛋白质变性引发，然后形成二聚体。在这里，我们调查了高分子量mAb的模型，该分子在无应力的Roledumab生物药物中的不足1％的高分子量物质（HMW）的性质和结构。HMW物种首先通过制备型体积排阻色谱（SEC）进行纯化，然后通过色谱方法（离子交换色谱（IEX），SEC）与天然质谱法（MS）以及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和毛细管凝胶电泳在非还原条件下结合使用。共价和非共价结合的二聚体均以50/50的比例鉴定。通过SEC和IEX与天然MS联用对HMW馏分进行的深入表征，揭示了存在三种质量相同，但电荷和大小不同的mAb二聚体形式。它们归因于不同的紧密和细长的二聚体。最后，使用不同的酶（IdeS和IgdE）进行高分辨率的中上方法来确定与二聚化有关的mAb结构域。我们的结果表明，Roledumab二聚体主要通过单个Fab与Fab臂结合的关联而关联。以及在非还原条件下的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和毛细管凝胶电泳。共价和非共价结合的二聚体均以50/50的比例鉴定。通过SEC和IEX与天然MS联用对HMW馏分进行的深入表征，揭示了存在三种质量相同，但电荷和大小不同的mAb二聚体形式。它们归因于不同的紧密和细长的二聚体。最后，使用不同的酶（IdeS和IgdE）进行高分辨率的中上方法来确定与二聚化有关的mAb结构域。我们的研究结果表明，Roledumab二聚体主要通过单个Fab与Fab臂结合缔合。以及在非还原条件下的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和毛细管凝胶电泳。共价和非共价结合的二聚体均以50/50的比例鉴定。通过SEC和IEX与天然MS联用对HMW馏分进行的深入表征，揭示了存在三种质量相同，但电荷和大小不同的mAb二聚体形式。它们归因于不同的紧密和细长的二聚体。最后，使用不同的酶（IdeS和IgdE）进行高分辨率的中上方法来确定与二聚化有关的mAb结构域。我们的研究结果表明，Roledumab二聚体主要通过单个Fab与Fab臂结合缔合。