Multi-Attribute Monitoring Method for Process Development of Engineered Antibody for Site-Specific Conjugation.
Multi-Attribute Monitoring Method for Process Development of Engineered Antibody for Site-Specific Conjugation.
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DOI：
10.1021/jasms.3c00037
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发表时间：
2023-07-05
影响因子：
3.2
通讯作者：
Bond, Nicholas J.
中科院分区：
文献类型：
--
作者：
Hines, Alistair R.;Edgeworth, Matthew;Devine, Paul W. A.;Shepherd, Samuel;Chatterton, Nicholas;Turner, Claire;Lilley, Kathryn S.;Chen, Xiaoyu;Bond, Nicholas J.
关键词：
抗体药物偶联物是一类生物活性蛋白，近年来得到了广泛的发展，导致了新的批准和癌症患者护理标准的提高。在将细胞毒性有效载荷缀合至单克隆抗体的众多策略中，半胱氨酸残基的插入实现了严格控制的位点特异性药物与抗体的比率。需要定制的分析工具来指导能够制造具有所需产品质量的新型抗体支架的工艺的开发。在这里，我们描述了一个12分钟，质谱法为基础的方法，能够同时监测四个不同的质量属性的发展：插入的半胱氨酸，N-连接的糖基化，减少链间二硫键，和多肽片段的巯基状态的变化。该方法为抗体中间体的性质和相关生产工艺提供了新的见解。在生物反应器内形成氧化巯基状态，其中产生含有额外二硫键的变体，并在整个补料分批工艺中保持相对恒定;在收获时引入还原巯基变体。近20%的N-连接聚糖含有唾液酸，大大高于野生型IgG 1的预期。最后，在C239 i恒定结构域中鉴定了先前未报道的多肽片段化位点，并探索了片段化与糖型之间的关系。这项工作说明了应用高通量液相色谱-质谱多属性监测方法来支持工程抗体支架的开发的实用性。
Antibody drug conjugates, a class of biotherapeutic proteins, have been extensively developed in recent years, resulting in new approvals and improved standard of care for cancer patients. Among the numerous strategies of conjugating cytotoxic payloads to monoclonal antibodies, insertion of a cysteine residue achieves a tightly controlled, site-specific drug to antibody ratio. Tailored analytical tools are required to direct the development of processes capable of manufacturing novel antibody scaffolds with the desired product quality. Here, we describe the development of a 12 min, mass-spectrometry-based method capable of monitoring four distinct quality attributes simultaneously: variations in the thiol state of the inserted cysteines, N-linked glycosylation, reduction of interchain disulfide bonds, and polypeptide fragmentation. This method provides new insight into the properties of the antibody intermediate and associated manufacturing processes. Oxidized thiol states are formed within the bioreactor, of which a variant containing an additional disulfide bond was produced and remained relatively constant throughout the fed-batch process; reduced thiol variants were introduced upon harvest. Nearly 20 percent of N-linked glycans contained sialic acid, substantially higher than anticipated for wildtype IgG1. Lastly, previously unreported polypeptide fragmentation sites were identified in the C239i constant domain, and the relationship between fragmentation and glycoform were explored. This work illustrates the utility of applying a high-throughput liquid chromatography–mass spectrometry multi-attribute monitoring method to support the development of engineered antibody scaffolds.
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影响因子：
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