Process development and production of cGMP grade Melan-A for cancer vaccine clinical trials

• We developed a pilot scale process for cGMP production of Melan-A.
• We measured the necessary parameters for phase I clinical trial application.
• Several modifications for process resulted in increased yields as compared to previous cGMP production of CT antigens.
• Product is stable for excess of 1 year in final formulation buffer.
• Over 11 g of product was obtained for a single 20 L pilot scale fermentation.

Melan-A is a cancer testis antigen commonly found in melanoma, and has been shown to stimulate the body’s immune response against cancerous cells. We have developed and executed a process utilizing current good manufacturing practices (cGMP) to produce the 6 times-His tagged protein in C41DE3 Escherichia coli for use in Phase I clinical trials. Approximately 11 g of purified Melan-A were produced from a 20 L fed-batch fermentation. Purification was achieved through a three column process utilizing immobilized metal affinity, anion exchange, and cation exchange chromatography with a buffer system optimized for low-solubility, high LPS binding capacity proteins. The host cell proteins, residual DNA, and endotoxin concentration were well below limits for a prescribed dose with a final purity level of 91%.