Feasibility of Rapid Regulatory Differentiation of TNF Receptor 2-Fc Fusion Protein Products from Various Manufacturers in the Chinese Market Using a Novel Mass Spectrometry-Based Multi-attribute Method (MAM)

作者：Mengjiao Xu, Mingming Xu, Tao Liu, Dan Mao, Chunguang Zheng, Wei Yu, Qingcheng Guo, Zhixin Li, Tianyu Gao, Yule Ren, Weifan Zhu, Huangzhen Zhuang, Zhiyuan Pan, Fugui Wang, Xinxin Fang, Shanshan Dong, Lankun Song, Xi Chen, Aiying Nie, Lusha Ji, Weizhu Qian, Sheng Hou, Jun Li, Yajun Guo, Dapeng Zhang, Jin Xu, Hong Shao, Huaizu Guo · 发表于：Journal of the American Society for Mass Spectrometry · 年份：2025 · DOI：10.1021/jasms.5c00085 · 被引用次数：3 · 研究领域：Biosimilars and Bioanalytical Methods、Monoclonal and Polyclonal Antibodies Research、Protein purification and stability

Ensuring the quality of pharmaceutical products, particularly for complex recombinant protein drugs such as TNF receptor 2-Fc fusion proteins (TNFR2-Fc, Etanercept), poses significant public health challenges. These products, including biosimilars and follow-on versions, exhibit intricate glycosylation patterns and heterogeneous post-translational modifications, complicating their analytical assessment. The Chinese market, hosting four different TNFR2-Fc products, presents a unique regulatory challenge for rapid differentiation and quality control. This study developed a novel mass spectrometry-based multiattribute method (MAM) to address this challenge, enabling simultaneous monitoring of multiple quality attributes and effective differentiation among products from various manufacturers. Conventional techniques initially indicated high purity across all products, but these methods provided limited capabilities for differentiation. The improved MAM approach, involving desialylation, partial deglycosylation, and digestion steps, minimizes heterogeneity and simplifies analysis. This method successfully indicates differences in primary amino acid sequences and specific quality attributes, allowing for a clear differentiation among manufacturers. Notably, products from manufacturers A and B, as well as C and D, despite their high similarity, could be differentiated by their O-glycan profiles. Further activity evaluations revealed that the products from manufacturers C and D exhib...