Abstract:
Biopharmaceuticals, such as monoclonal antibodies, are considered as life-saving drugs for autoimmune diseases, cancer and infectious diseases. However, biotherapeutics tend to undergo chemical degradation during various stages of manufacturing. The conditions of chemical degradation, along with the physical degradation pathways, have a direct influence on the overall stability, safety and efficacy of these therapeutics. While site-specific chemical changes have been well-explored and investigated using various analytical approaches, the resulting conformational and structural changes have not been much studied. Thus, we explored various biophysical techniques for assessing the influence of three representatives forced degradation conditions viz. oxidation, deamidation, and glycation, in a model therapeutic trastuzumab biosimilar. The site-specific modifications caused by these stress conditions were analysed using high resolution mass spectrometry. While their thermodynamic and conformational consequences were investigated by using differential scanning colorimetry (Nano-DSC), circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AUC), and dynamic light scattering (DLS). The investigated stress conditions resulted in reduced thermodynamic stability of mAb, as confirmed using Nano-DSC. Secondary structure analysis performed with CD spectroscopy indicated detectable structural alterations in the beta sheets of stressed samples. DLS and SV-AUC studies demonstrated an enhanced level of aggregation and fragmentation in presence of all stress conditions. Thus, the biophysical analytical toolkits, when used simultaneously, could offer deeper insights into the subtle conformational changes that result from site-specific chemical modifications in mAbs. Hence, these analytical approaches may serve as significant additions to the battery of techniques used for forced degradation analysis of biopharmaceuticals.
摘要:
生物制药,如单克隆抗体,被认为是治疗自身免疫性疾病的救命药物,癌症和传染病。然而,生物治疗剂倾向于在制造的各个阶段经历化学降解。化学降解的条件,以及物理降解途径,对整体稳定性有直接影响,这些疗法的安全性和有效性。尽管已使用各种分析方法对特定地点的化学变化进行了充分的探索和研究,由此产生的构象和结构变化还没有得到太多的研究。因此,我们探索了各种生物物理技术来评估三个代表强制降解条件的影响。氧化,脱酰胺,和糖化,在模型治疗曲妥珠单抗生物仿制药。使用高分辨率质谱法分析由这些应激条件引起的位点特异性修饰。虽然他们的热力学和构象后果是通过使用差示扫描比色法(纳米DSC)研究,圆二色性(CD)光谱,分析超速离心(AUC),和动态光散射(DLS)。研究的应力条件导致mAb的热力学稳定性降低,使用Nano-DSC确认。用CD光谱学进行的二级结构分析表明在受应力样品的β折叠中可检测的结构改变。DLS和SV-AUC研究表明在所有胁迫条件存在下聚集和片段化水平提高。因此,生物物理分析工具包,当同时使用时,可以提供对mAbs中位点特异性化学修饰导致的微妙构象变化的更深入的见解。因此,这些分析方法可以作为用于生物制药强制降解分析的一系列技术的重要补充。