Glycoengineering and biosimilarity are the key factors for growing, promising and progressive approaches in monoclonal antibodies development. In this study, the physicochemical stability of anti-CD20 rituximab (RTX); originator and biosimilar was compared to its glycoengineered humanized version; obinutuzumab (OBZ). An orthogonal stability-indicating protocol using a set of validated bioanalytical techniques; size exclusion high performance liquid chromatography (SE-HPLC), reversed phase liquid chromatography (RP-HPLC), quantitative gel electrophoresis by TapeStation, receptor binding assay and dynamic light scattering (DLS) was used to investigate the effect of different stress factors on the pattern and kinetics of degradation. SE-HPLC results supported with spectral purity showed similar degradation extent with a different pattern of degradation between RTX and OBZ. A lower tendency to form degraded fragments and a relatively higher favorability for degradation through aggregate formation has been revealed in case of OBZ. Results were in agreement with those of DLS and receptor binding assay which showed specificity to the intact antibodies in the presence of their degradation products. Furthermore, results were additionally confirmed through denaturing quantitative gel electrophoresis which suggested reducible covalent bonds as the mechanism for aggregates formation. RP-HPLC results showed two oxidized forms via excessive oxidation of RTX and OBZ with nearly the same degradation percent. Comparability data of RTX and OBZ using the applied methodologies showed that although glycoengineering; carried out to enhance the therapeutic and biological activity of OBZ altered the pattern of degradation but did not significantly affect the overall stability. Results showed also consistent stability profile between the biosimilar and its originator RTX products.

糖工程和生物相似性是单克隆抗体开发中增长，有前途和渐进方法的关键因素。在这项研究中，抗CD20利妥昔单抗（RTX）的理化稳定性；将鼻祖和生物仿制药与其糖工程化的人源化版本进行了比较；奥比妥单抗（OBZ）。使用一组经过验证的生物分析技术的正交稳定性指示方案；尺寸排阻高效液相色谱（SE-HPLC），反相液相色谱（RP-HPLC），TapeStation定量凝胶电泳，受体结合测定和动态光散射（DLS）用于研究不同应力因素对药物的影响模式和降解动力学。光谱纯度支持的SE-HPLC结果显示相似的降解程度，RTX和OBZ之间的降解方式不同。在OBZ的情况下，已经显示出形成降解片段的趋势较低，并且通过聚集体形成的降解相对较高的有利性。结果与DLS和受体结合测定的结果一致，后者在降解产物存在下对完整抗体表现出特异性。此外，通过变性定量凝胶电泳进一步证实了结果，该电泳表明可还原的共价键是聚集体形成的机理。RP-HPLC结果表明，通过RTX和OBZ的过度氧化，两种氧化形式具有几乎相同的降解率。使用所应用方法的RTX和OBZ的可比性数据表明，尽管糖工程化；进行增强OBZ的治疗和生物活性的实验改变了降解方式，但并未显着影响整体稳定性。结果还显示，生物仿制药与其原始RTX产品之间具有一致的稳定性。