Tumor neoantigen peptides, characterized by tumor-specific mutations and aberrant gene expression profiles, demonstrate superior immunogenicity and minimal off-target effects. Neoantigen peptide vaccines are able to activate the immune system, break immune tolerance, and induce tumor cell cytotoxicity through enhanced antigen-specific T-cell responses, thereby emerging as promising candidates for personalized cancer immunotherapy. However, due to their personalized characteristics, the current commercial production of tumor neoantigen peptide vaccines face challenges in difficult synthesis, long preparation cycles and complex purification processes, which limit their widespread clinical applications. To address these issues, this study proposes a novel method utilizing fragmented peptide resin storage, in which the shared Link-NitraTh peptide sequence is first synthesized, and then it is used as the starting material for the subsequent preparation of full-length neoantigen peptides. This approach reduces the overall synthesis cycle from two months to one month, significantly shortening the production time for synthetic peptides and increasing the accessibility of neoantigen peptide vaccines for clinical patients. Additionally, optimization of the shared Link-NitraTh synthesis method greatly improves the purity of crude neoantigen peptides and simplifies the purification process, enabling robust production of neoantigen peptide vaccines with stringent quality control and acceptable manufacturing time. Using this method, twelve tumor neoantigen peptides, each comprising thirty-eight amino acids, can be efficiently produced within twenty-three working days to meet clinical demands. The quality specifications for neoantigen peptides include: purity>95% by high-performance liquid chromatography (HPLC), conversion to the acetate salt form and trifluoroacetate residue<1%, mass exceeding 20 mg, and endotoxin level below 10 EU/mg. This work has achieved significant progress in the design and optimization of the tumor neoantigen peptide preparation process, and established a standardized operational framework including parameter optimization for peptide solid-phase synthesis and liquid-phase purification protocols. Our optimized methodologies not only improve the synthesis efficiency and enhance batch-to-batch consistency of tumor neoantigen peptides, but also reduce production timelines. These advancements represent a pivotal step toward realizing the therapeutic potential of patient-specific neoantigen peptide vaccine platforms and accelerating their clinical translation within precision medicine frameworks for oncological and immunological indications.

Key words: tumor vaccine, neoantigen, peptide synthesis, peptide purification, process optimization