生物药物在癌症治疗中至关重要，因为它们能够刺激人体的自然免疫反应来对抗癌症，但它们价格昂贵。生物类似物相对于专利生物药物来说更加经济实惠，但必须验证它们与创新药物一样有效。生物类似物的特征和可替代性评估需要大量的数据点来进行验证。本研究提出了通过开发和绿色评估正交测试方案和纯度指示法来对两种新的贝伐单抗（BVZ）生物类似物进行质量评估，其中包括凝胶过滤（SE-HPLC）、反相（RP-HPLC）和阳离子交换色谱（CEX-HPLC），以及动态光散射（DLS）和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）。采用SE-HPLC方法对BVZ单体及其聚集物和/或碎片进行筛选和验证。计算峰纯度和系统适用性参数。结果表明，正交方案是评估单克隆抗体稳定性的有用工具，它是生物类似性评估的关键指标。DLS和SDS-PAGE的结果相互比较，揭示了BVZ创新型药物和其生物类似物之间的保持时间和条带模式的相似性。这些结果显示Avastin®和所研究的生物类似物在相关化合物峰面积和视觉分子量方面具有相同的特征，并且SDS-PAGE技术被认为是最环保的技术之一。所获得的结果突出了评估相似性和差异对于确保所研究产品的生物相似性和可替代性的重要性。© 2023作者。由牛津大学出版社代表AOAC INTERNATIONAL出版。保留所有权利。如需许可，请发送电子邮件至journals.permissions@oup.com。

Biologics are essential in cancer treatment because they stimulate the body's natural response to fight cancer, but they are expensive. Biosimilars are more affordable they have to be compared to patent biologicals, but it must be verified that they are as effective as their innovators. Characterization of biosimilars and assessment of interchangeability requires many data points for verification.The proposed study provides a quality assessment of two new bevacizumab (BVZ) biosimilars, produced by Amgen and Biocad Inc, through the development and greenness assessment of an orthogonal testing protocol and purity indicating assay, including size-exclusion (SE-HPLC), reversed-phase (RP-HPLC) and cation exchange chromatography (CEX-HPLC) in addition to dynamic light scattering (DLS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).SE-HPLC method was performed and validated to screen the BVZ monomer and its aggregates and/or fragments. Peak purity and system suitability parameters were calculated. Results indicate that the orthogonal protocol is a useful tool for assessing monoclonal antibody stability. It is a key criterion for biosimilarity assessment. DLS and SDS-PAGE results were compared to each other to reveal close retention times and banding patterns between BVZ innovator and its biosimilars. These results showed that Avastin® and the investigated biosimilars have the same profile in terms of peak area of related compounds within the acceptance limit and apparent molecular weight, and the SDS-PAGE technique was found to be the most eco-friendly technique among others.The results obtained highlighted the importance of assessing similarities and differences in ensuring the biosimilarity and interchangeability of the studied products.© The Author(s) 2023. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.