Fei Mengdan, Zhang Qiang, Zhang Lei, Zhu Xiaoqi, Du Chaofu, Zhang Zhongli
Department of Analytical Science and Development, Shanghai Henlius Biologics Co., Ltd., Shanghai 201600, China.
Department of Analytical Science and Development, Shanghai Henlius Biologics Co., Ltd., Shanghai 201600, China.
J Pharm Biomed Anal. 2023 Jan 20;223:115121. doi: 10.1016/j.jpba.2022.115121. Epub 2022 Oct 22.
Aggregate of therapeutic antibodies is usually considered as one of the most important critical quality attributes (CQA). The propensity of aggregates formation for bevacizumab is higher than other monoclonal antibody (mAb) drugs due to its tendency of self-association via the non-covalent interaction between the Fab arm of one bevacizumab molecule and the K445 residue on the heavy chain of another bevacizumab molecule. HLX04 has been developed as a biosimilar to bevacizumab (Avastin®) by Shanghai Henlius Biotech. To perform a head-to-head similarity evaluation with respect to aggregates or higher molecular weight species (HMWS) between HLX04 and Avastin®, we developed a robust high performance liquid chromatography (SEC-HPLC) method for aggregates analysis. Our characterization data indicated that HMWS of bevacizumab were mainly composed of dimers, and the dimer formation-dissociation equilibrium was influenced by protein concentration and storage temperature. Based on the characterization data of aggregates, we optimized the key parameters for SEC-HPLC based aggregates analysis method including mobile phase components and pH, autosampler temperature, as well as incubation conditions for sample pretreatment. The developed method was applied in HLX04 and Avastin® aggregates assessment and the similarity were confirmed among HLX04, China-sourced, and Europe-sourced Avastin® using both the pharmaceutical dosage forms and forced degradation samples. The method was also validated per ICH Q2 (R1) guidelines by challenging the parameters including specificity, accuracy, precision, linearity, range, limit of quantitation, and robustness. The validated method was applied in release test and stability study of HLX04 samples generated from commercial manufacturing process.
治疗性抗体的聚集体通常被认为是最重要的关键质量属性(CQA)之一。贝伐单抗形成聚集体的倾向高于其他单克隆抗体(mAb)药物,这是由于其一个贝伐单抗分子的Fab臂与另一个贝伐单抗分子重链上的K445残基之间通过非共价相互作用发生自缔合的趋势。HLX04是上海复宏汉霖生物技术股份有限公司开发的贝伐单抗(安维汀®)生物类似药。为了对HLX04和安维汀®之间的聚集体或更高分子量物质(HMWS)进行直接的相似性评估,我们开发了一种用于聚集体分析的稳健高效液相色谱法(SEC-HPLC)。我们的表征数据表明,贝伐单抗的HMWS主要由二聚体组成,并且二聚体形成-解离平衡受蛋白质浓度和储存温度的影响。基于聚集体的表征数据,我们优化了基于SEC-HPLC的聚集体分析方法的关键参数,包括流动相成分和pH值、自动进样器温度以及样品预处理的孵育条件。所开发的方法应用于HLX04和安维汀®聚集体评估,并使用药物剂型和强制降解样品确认了HLX04、中国来源和欧洲来源的安维汀®之间的相似性。该方法还按照ICH Q2(R1)指南进行了验证,对特异性、准确性、精密度、线性、范围、定量限和稳健性等参数进行了考察。经过验证的方法应用于商业化生产过程中产生的HLX04样品的放行测试和稳定性研究。