Since cells prohibit entry to large molecules like DNA, unique delivery and formulation technologies must be fine-tuned to optimize the transfection and delivery of DNA vaccines.
“Technologies for delivering DNA vaccines include liposomes, polymers, or electroporation,” noted Magda Marquet, Ph.D., co-founder and co-chairperson of Althea Technologies. “However, these methods are often cumbersome for patients to receive, and present difficulties in manufacturing and scale up.”
Althea is pursuing complex formulations, such as polymers and liposomes, to overcome the obstacle of DNA vaccine delivery by delivering DNA directly to cells. Some formulations have challenging requirements.
Dr. Marquet cited one case in which aseptic processing was required throughout the formulation since product could not be sterile filtered prior to filling. “Althea successfully scaled up the process and brought in GMP formulation equipment that allowed the liposomal complex to be processed aseptically and filled into vials,” she explained.
Martin Schleef, Ph.D., CEO of the PlasmidFactory, echoed Chambers’ concerns over the high standard of purity required of DNA for delivery in humans, and cited a recent study (Woodell et al., J. Gene Med), which demonstrated that chromosomal bacterial DNA, which is typically present in kit-grade plasmid DNA preparations, is a significant contaminant leading to low efficacy and severe toxic effects.
“We have developed a manufacturing technology to avoid such contamination,” said Dr. Schleef. In addition, he highlighted plasmid topology as an important parameter in determining transfection efficiency. Covalently closed circular (ccc), or supercoiled, DNA adopts a compact form due to internal tensions in the DNA molecule that is optimal for transfection efficiency.
“Ideally the proportion of ccc-DNA in the preparation should be 95% or higher,” said Dr. Schleef, “and we use a manufacturing technology to obtain pure preparations of this form.”
PlasmidFactory has also developed a capillary gel electrophoresis-based analytical tool to quantify the major plasmid topologies in a given plasmid preparation, namely ccc-DNA, oc (open circular)-DNA, and linear DNA. In addition to fine-tuning the conformation of DNA for transfection, stripping plasmids of genes encoding resistance to antibiotics or other selection markers is an important step for preparing DNA for transfection.
“We have designed strategies to remove such genes from the bacterial backbone of the plasmids used, in addition to the origin of replication, resulting in a simply circular and extremely small DNA for vaccination, the minicircle,” said Dr. Schleef.
According to Dr. Schleef, PlasmidFactory has obtained all relevant patents for this technology and supplies researchers in gene therapy and DNA vaccination worldwide with this safe, nonviral vector. PlasmidFactory is also tackling the issue of scaling of plasmid preparations to the amounts required for more demanding applications, e.g., large clinical trials.
“We are developing technologies for ultra-large scale production of plasmid DNA (e.g., kg scale) by use of fed-batch and large scale lysis to obtain pure ccc-plasmid-DNA,” he said.
As both Chambers and Dr. Schleef have pointed out, the purity of a plasmid preparation is a critical determinant of successful vaccine transfection or delivery.
“If you don’t start with pure DNA, you can forget about reproducible and effective transfection,” said Bill Kuhlman, vp North America for BIA Separations. “Removal of endotoxins and genomic DNA is commonly achievable at small scale, but producing DNA vaccines at commercial scale requires process steps that can efficiently produce tens of grams of highly pure supercoiled plasmid DNA per run.”
BIA Separations specializes in separations, with particular emphasis on monolithic HPLC columns. Rather than traditional bead-based separation columns, monolithic columns are composed of an organic or inorganic substrate and multiple highly permeable and porous channels that afford a large surface area to the stationary phase.
“We’ve achieved homogeneity of greater than 97% supercoiled DNA with greater than 99% removal of host DNA, protein, and RNA at development through industrial scales,” Kuhlman pointed out. BIA Separations has recently introduced large-scale disposable Monolith columns allowing 48 grams of super coiled DNA to be purified in a single run, while maintaining the purity and recovery seen at small scale.
In addition to manufacturing and delivery, another important consideration for DNA vaccines, like any other candidate therapeutic, is safety. The potential integration of DNA vaccines into the host cell genome is of concern due to the possibility of insertional mutagenesis resulting in the inactivation of tumor suppressor genes or the activation of oncogenes in the host genome.
According to Dr. Marquet, “DNA vaccines have been proven to be safe in multiple studies, with no integration of the DNA into the host chromosomes ever being observed.”
Her assessment is reinforced by Dr. Kim from Inovio Pharmaceuticals. “We have vaccinated or treated over 600 patients with good safety and tolerability profiles,” he said. “Adverse events are typically mild to moderate, and any injection sites resolve without sequelae.”