In the biopharmaceutical environment, controlling the Critical Quality Attributes (CQA) of a product is essential to prevent changes that affect its safety or efficacy. Physico-chemical techniques and bioassays are used to screen and monitor these CQAs. The higher order structure (HOS) is a CQA that is typically studied using techniques that are not commonly considered amenable to quality control laboratories. Here, we propose a peptide mapping-based method, named native peptide mapping, which could be considered as straightforward for HOS analysis and applicable for IgG4 and IgG1 antibodies. The method was demonstrated to be fit-for-purpose as a stability-indicating assay by showing differences at the peptide level between stressed and unstressed material. The unfolding pathway induced by a heat stress was also studied via native peptide mapping assay. Furthermore, we demonstrated the structure–activity relationship between HOS and biological activity by analyzing different types of stressed samples with a cell-based assay and the native peptide mapping. The correlation between both sets of results was highlighted by monitoring peptides located in the complementary-determining regions and the relative potency of the biotherapeutic product. This relationship represents a useful approach to interrogate the criticality of HOS as a CQA of a drug.

在生物制药环境中，控制产品的关键质量属性（CQA）对于防止影响其安全性或功效的更改至关重要。物理化学技术和生物测定法被用来筛选和监测这些CQA。高阶结构（HOS）是一种CQA，通常使用通常认为不适用于质量控制实验室的技术进行研究。在这里，我们提出了一种基于肽图分析的方法，称为天然肽图分析，该方法可被视为直接进行HOS分析并适用于IgG4和IgG1抗体。通过显示压力物质和非压力物质之间在肽水平上的差异，该方法被证明适合用作稳定性指示试验。还通过天然肽图分析研究了由热应激诱导的解折叠途径。此外，我们通过基于细胞的分析和天然肽图分析，分析了不同类型的应激样品，从而证明了居屋与生物活性之间的结构-活性关系。通过监测位于互补决定区的肽段和生物治疗产品的相对效价，可以突出显示两组结果之间的相关性。这种关系代表了一种有用的方法，可用于质疑居屋作为药物的CQA的重要性。通过监测位于互补决定区的肽段和生物治疗产品的相对效价，可以突出显示两组结果之间的相关性。这种关系代表了一种有用的方法，可用于质疑居屋作为药物的CQA的重要性。通过监测位于互补决定区的肽段和生物治疗产品的相对效价，可以突出显示两组结果之间的相关性。这种关系代表了一种有用的方法，可用于质疑居屋作为药物的CQA的重要性。