Challenges in the Process Development of a Novel Zero CpG CFTR Plasmid for Human Clinical Use.

Hebel HL, Cai Y, Davies LA, Hyde SC, Pringle IA, Gill DR

Molecular Therapy, 16 S1 S110

The American Society of Gene Therapy Annual Conference, Boston, 2008

Demand for high purity plasmid DNA for gene therapy and DNA vaccination is escalating. A greater variety of products will be presented to manufacturers, each with specific obstacles to overcome.

The UK Cystic Fibrosis Gene Therapy Consortium has previously shown that a plasmid containing a single CpG motif can cause significant inflammation when delivered to the murine lung. Based on these findings, a novel zero-CpG plasmid expressing the therapeutic cystic fibrosis transmembrane conductance regulator gene product was constructed and provided to VGX Pharmaceuticals for process development and manufacture for subsequent use in human clinical trials.

The therapeutic plasmid has several features resulting in significant challenges to its large-scale production for human clinical use. Employment of a zero-CpG R6K origin of replication (Cayla-Invivogen, Toulouse, France) constrained the choice of the E. Coli host strain for plasmid replication and production. The high recombination tendency of this plasmid vector required extensive studies in transformation conditions to maintain plasmid monomer, as plasmid form was affected by colony growth rate.

Unusual E. Coli growth characteristics required optimization of fermentation conditions and harvest at high cell density. The plasmid also showed unexpected characteristics compared with a typical 6.5 kb plasmid in terms of its ionic and hydrophobic characteristics.

First, development and verification of processes with increased throughput were necessary for production of this low yield product.

Second, lysis scale-up without prolonging hold time was critical to avoid adverse conditions leading to plasmid denaturation. Third, a more stringent purification process was required to achieve a very low endotoxin standard (5 mg/ml) for patient delivery presented several challenges in the ultrafiltration and diafiltration steps to obtain highly concentrated product while limiting shear damage. During process development, samples were tested for physical/chemical properties and biologic responses in animal models. The optimized manufacturing protocol resulted in plasmid DNA able to form a homogenous population of stable DNA/liposome complexes suitable for nebulization to patients. This is in contrast to earlier DNA batches, using alternative manufacturing techniques, which demonstrated significant adverse physicochemical interactions when complexed with cationic liposomes.

Ethical permission has now been granted for a clinical study in the UK. The DNA/liposomes will be nebulised to the lungs of patients with cystic fibrosis to evaluate the safety of this gene therapy formulation.