Effective vaccines that mediate clinical responses in cancer patients may require generation of broadly specific cytotoxic T lymphocytes (CTL) directed against multiple epitopes and tumor-associated antigens (TAA). Pursuant to this goal we developed a synthetic peptide vaccine, EP-2101, composed of 10 synthetic peptide epitopes and formulated in Montanide ISA 51 adjuvant. Nine of the HLA-A*0201-restricted CTL epitopes were derived from five well-characterized TAA. The universal HLA-DR binding epitope PADRE was also included for T-cell help. Herein we describe studies on the formulation and characterization of the EP-2101 vaccine which supports generation of a sterile single-vial emulsion using standardized processes. The physicochemical properties of the peptides were highly disparate and as such, solubilization studies were required to identify a process which supported sterile filtration of the EP-2101 peptide mix. A homogenization-based formulation process with Montanide ISA 51 and 0.5 mg/ml of each peptide was developed to generate a water-in-oil emulsion. Physical studies indicated the vaccine emulsion to be stable, with little change in visual appearance, viscosity and water droplet size for at least three months. The physical stability of individual peptides in the vaccine emulsion was demonstrated using HPLC and immunogenicity of the vaccine formulation was confirmed in HLA-A*0201/K(b) transgenic mice where T-cell responses could be induced to all epitopes in EP-2101 following vaccination. Our study process is scalable for production of approximately 1.5 liters of potent experimental vaccine for preclinical animal toxicity and phase 1 clinical testing in patients with breast, colon or lung cancer.