Abstract 5676: TFC Quick ‘n’ Clean, a next-gen platform for high-throughput production of true four-chain bispecific antibodies

Introduction: Bispecific antibodies (BsAbs) are a promising class of biologics with substantial therapeutic potential. However, the production of bsAbs, particularly four-chain bsAbs containing two distinct LCs, presents significant challenges. Issues such as LC mispairing, fragmentation, and aggregation complicate purification processes and impact overall yield, purity and throughput. To address these challenges, WuXi Biologics has developed the True Four Chain (TFC) Quick ‘n’ Clean platform. By leveraging the WuXiBody™ backbone, this platform eliminates LC mispairing and enables high-throughput, high-purity production of four-chain bsAbs. This innovative approach accelerates the identification of optimal four-chain bsAb pairings during early-stage drug discovery. Methods: All experiments were conducted at WuXi Biologics’ Protein Sciences Department. Briefly, the codons of DNA sequence were optimized, and the plasmids were constructed into WuXi Biologics’ proprietary plasmid vector. Transient transfection was performed in CHO cells, with the culture medium harvested after 7 days. The harvested culture medium was clarified and subsequently processed using an AKTA Pure system with a tandem purification strategy using Nickel, anti-DYKDDDDK, and SEC columns. Final products were analyzed using SDS-PAGE, SEC-HPLC, Intact Mass, and LAL assays. Results: Using the TFC Quick ‘n’ Clean platform, we successfully produced 15 four-chain bsAbs in 20 mL CHO cell cultures within 3-4 weeks. The platform achieved an average monomer purity of 98.23%, an average yield of 3.34 mg, and an average endotoxin level of 0.06 EU/mg. Intact Mass analysis confirmed the absence of impurities such as homodimers and LC mispairing, with heterodimer purity exceeding 99%. Conclusion: The TFC Quick ‘n’ Clean platform overcomes key challenges associated with four-chain bsAb production by harnessing the WuXiBody™ backbone, high-titer CHO expression systems and advanced tandem purification strategies. This platform provides high-throughput, high-purity production of four-chain bsAbs, enabling the rapid, cost-effective identification of optimal parings to accelerate early-stage drug discovery and development. Citation Format: Julia Su, Gaozhan Zhang, Xiayan Li, Dawei Zhang, Mengjie Lu, Zhumei Feng, Jiansheng Wu. TFC Quick ‘n’ Clean, a next-gen platform for high-throughput production of true four-chain bispecific antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5676.