P4: Process Development and Technology Transfer of a High Yield Fermentation Process for cGMP Production of Plasmid DNA Vaccines

Plasmid DNA (pDNA) is a vector of choice for many new experimental vaccines and therapeutic products. The Vaccine Clinical Materials Program (VCMP), at the Vaccine Research Center (VRC/NIAID) operated by SAIC-Frederick, Inc., manufactures DNA vaccine products for use in preclinical safety studies and clinical trials.  Various pDNA products have been successfully produced at the 15L and 100L fermentation scales using the HyperGRO^TM pDNA technology under a license with Nature Technology Corporation (NTC).  This technology incorporates exponential nutrient feeding with a temperature shifting strategy for induction of plasmid replication, shown to achieve high plasmid yields in E. coli DH5α.  Previously, the exponential feed method at VCMP included user interventions to minimize glycerol accumulation during exponential growth.  Recently, poor culture performance with high acetate levels was encountered with two pDNA vaccine products, leading to low plasmid yield and higher levels of host cell contaminants.

Based on experiments by NTC and VCMP, changing transformation temperature and cell banking conditions led to acceptable fermentation of one of these plasmids encoding influenza hemagglutinin.  However, the same strategy when applied to an HIV glycoprotein construct still resulted in poor culture performance. In addition, process differences were encountered between the 15L and 100L scales for this same construct.  As a result, studies were performed to address this production problem including feed regimes and k_La studies at these fermenter scales.  Finally, by the design and implementation of a simplified feeding strategy, robust pDNA production for several vector constructs was achieved, meeting all purity requirements upon plasmid purification.