Clinical antibodies are an important class of drugs for the treatment of both chronic and acute diseases. Their manufacturability is subject to evaluation to ensure product quality and efficacy. One critical quality attribute is deamidation, a non-enzymatic process that is observed to occur during thermal stress, at low or high pH, or a combination thereof. Deamidation may induce antibody instability and lead to aggregation, which may pose immunogenicity concerns. The introduction of a negative charge via deamidation may impact the desired therapeutic function (i) within the complementarity-determining region, potentially causing loss of efficacy; or (ii) within the fragment crystallizable region, limiting the effector function involving antibody-dependent cellular cytotoxicity. Here we describe a transformative solution that allows for a comparative assessment of deamidation and its impact on stability and aggregation. The innovative streamlined method evaluates the intact protein in its formulation conditions. This breakthrough platform technology is comprised of a quantum cascade laser microscope, a slide cell array that allows for flexibility in the design of experiments, and dedicated software. The enhanced spectral resolution is achieved using two-dimensional correlation, co-distribution, and two-trace two-dimensional correlation spectroscopies that reveal the molecular impact of deamidation. Eight re-engineered immunoglobulin G4 scaffold clinical antibodies under control and forced degradation conditions were evaluated for deamidation and aggregation. We determined the site of deamidation, the overall extent of deamidation, and where applicable, whether the deamidation event led to self-association or aggregation of the clinical antibody and the molecular events that led to the instability. The results were confirmed using orthogonal techniques for four of the samples.

中文翻译：

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一系列临床阶段抗体的简化多属性评估：降解与稳定性之间的关系

临床抗体是治疗慢性和急性疾病的一类重要药物。它们的可制造性需要接受评估，以确保产品质量和功效。一个关键的质量属性是脱酰胺作用，这是一种非酶促过程，观察到在热应激、低或高 pH 值或其组合下发生。脱酰胺可能会导致抗体不稳定并导致聚集，这可能会引起免疫原性问题。通过脱酰胺引入负电荷可能会影响互补性决定区域内所需的治疗功能（i），可能导致功效丧失；或（ii）在片段可结晶区域内，限制涉及抗体依赖性细胞毒性的效应子功能。在这里，我们描述了一种变革性的解决方案，可以对脱酰胺及其对稳定性和聚集的影响进行比较评估。创新的简化方法评估其配制条件下的完整蛋白质。这一突破性的平台技术由量子级联激光显微镜、允许灵活实验设计的载玻片阵列以及专用软件组成。增强的光谱分辨率是通过二维相关、共分布和两道二维相关光谱来实现的，这些光谱揭示了脱酰胺的分子影响。对八种重新设计的免疫球蛋白 G4 支架临床抗体在控制和强制降解条件下的脱酰胺和聚集进行了评估。我们确定了脱酰胺的位点、脱酰胺的总体程度，以及在适用的情况下，脱酰胺事件是否导致临床抗体的自缔合或聚集以及导致不稳定的分子事件。使用正交技术对四个样品证实了结果。