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Stage III-IV non-small cell lung cancer (NSCLC) is a devastating disease characterized by a poor prognosis. NSCLC tumors carry genetic mutations, which can lead to the expression of altered protein sequences. Peptides originating from mutated proteins and bound to MHC molecules on the tumor cell surface are referred to as neoantigens, as they are tumor-specific and not expressed in normal cells. Due to their tumor specificity, neoantigens have a strong potential to induce an anti-tumor immune response and have been investigated for development of personalized therapeutic cancer vaccines. The current study describes the development of a clinical grade neoantigen vaccine formulation (FRAME-001) intended as immunotherapy in advanced NSCLC in combination with the immune checkpoint inhibitor pembrolizumab. The detection of aberrant tumor-specific transcripts as well as an algorithm to select immunogenic neoantigen peptides are described. Subsequently, selected neoantigen peptides were synthesized with a high throughput synthesis platform and aseptically formulated under good manufacturing practice (GMP) conditions into four aqueous peptides mixtures that each contained six neoantigen peptides. A validated stability-indicating analytical method was developed in which we considered the personalized nature of the formulation. An extensive stability study performed either at -25 °C or -80 °C showed that the formulation was stable for up to 32 weeks. The formulation was mixed with the vaccine adjuvant Montanide ISA 51 VG, which yielded the final vaccine emulsion. The stability of the vaccine emulsion was demonstrated using microscopic examination, differential light scattering, and the water-drop test. The presented data show that FRAME-001 is a feasible personalized vaccine formulation for the treatment of stage III-IV NSCLC. The presented data may give guidance in the development of novel personalized therapeutic vaccines since this formulation strategy could be used for any cancer indication., Competing Interests: K.F., M.V.M., and W.P.K are employees and stock option holders of CurveVac Netherlands B.V.; B.G., L.T.O., L.A.G., J.A.J., M.W.d.J., D.P.A., J.v.Z.: declare no conflict of interest. The present study was a multidisciplinary project between Frame Therapeutics B.V. (now CureVac Netherlands B.V.; genetic analyses of tumor material and neoantigen peptide selection), Almac Sciences Scotland Ltd. (manufacturing of the freeze-dried peptide mixtures), 3D-PharmXchange (CMC regulatory affairs and compliance), and the University Medical Center Groningen (development of the stability-indicating analytical method; manufacturing of the aqueous peptide mixtures; aqueous peptide mixture as well as vaccine emulsion stability study). The different individual authors combined their expertise to design a scientifically sound study protocol. The individual authors collected, analyzed, and interpreted the data independently from each other after which these data were discussed with the entire team. The analyses and interpretation of the data gathered by one group was not influenced by the other groups. The four independent groups together decided to write the manuscript and publish the results.