单克隆抗体(mAb)等基于蛋白质的药物的临床疗效和安全性依赖于产品开发过程中蛋白质高级结构(HOS)的完整性,制造,storage,和病人管理。随着基于mAb的药物在许多疾病的治疗中变得越来越普遍,通过高分辨率技术建立mAb疗法质量属性指标的需求也变得越来越明显。为此,在这里我们使用了一种强制降解方法,过氧化氢的时间依赖性氧化,模型生物治疗NISTmAb,并通过正交分析方法和功能测定评估对HOS的影响。为了监测氧化过程,实验工作流程涉及NISTmAb与过氧化氢在台式核磁共振波谱仪(NMR)中的孵育,遵循反应动力学,实时通过水质子横向弛豫率R2(1H2O)。在确定的时间点采集的等分试样通过高场2D1H-13C甲基相关指纹图谱与其他分析技术并行进行进一步分析。包括热展开,尺寸排阻色谱法,和表面等离子体共振,为了评估稳定性的变化,异质性,和有约束力的亲和力。来自不同技术的互补测量输出证明了将NMR与其他分析工具相结合以监测氧化动力学并提取功能相关的mAb的结构变化的实用性。允许严格评估与基于mAb的药物产品的功效和安全性相关的HOS属性。
The clinical efficacy and safety of protein-based drugs such as monoclonal antibodies (mAbs) rely on the integrity of the protein higher order structure (HOS) during product development, manufacturing, storage, and patient administration. As mAb-based drugs are becoming more prevalent in the treatment of many illnesses, the need to establish metrics for quality attributes of mAb therapeutics through high-resolution techniques is also becoming evident. To this end, here we used a forced degradation method, time-dependent oxidation by hydrogen peroxide, on the model biotherapeutic NISTmAb and evaluated the effects on HOS with orthogonal analytical methods and a functional assay. To monitor the oxidation process, the experimental workflow involved incubation of NISTmAb with hydrogen peroxide in a benchtop nuclear magnetic resonance spectrometer (NMR) that followed the reaction kinetics, in real-time through the water proton transverse relaxation rate R2(1H2O). Aliquots taken at defined time points were further analyzed by high-field 2D 1H-13C methyl correlation fingerprint spectra in parallel with other analytical techniques, including thermal unfolding, size-exclusion chromatography, and surface plasmon resonance, to assess changes in stability, heterogeneity, and binding affinities. The complementary measurement outputs from the different techniques demonstrate the utility of combining NMR with other analytical tools to monitor oxidation kinetics and extract the resulting structural changes in mAbs that are functionally relevant, allowing rigorous assessment of HOS attributes relevant to the efficacy and safety of mAb-based drug products.