The use of intact antibodies, such as trastuzumab, in radioimmunotherapy often causes off-target radiation due to prolonged circulation and increased non-specific binding. This study developed a refined pepsin-based method to generate F(abʹ)₂ fragments of trastuzumab and evaluated the radiochemical stability of [131I]I-F(abʹ)2-trastuzumab. A pepsin-to-antibody ratio of 1:20 yielded highly pure (> 95%) F(abʹ)₂ fragments, confirming effective enzymatic cleavage. The radiolabeled fragment maintained radiochemical purity above 95% under all storage conditions over 24h, demonstrating high stability. These findings establish a reliable strategy for producing radioiodinated antibody fragments with improved pharmacokinetic properties for targeted HER2-positive breast cancer therapy. © Akadémiai Kiadó Zrt 2026.