“DNA vaccines offer a host of advantages over conventional vaccine technology,” noted Henry Hebel, Ph.D., COO of VGXI, a CMO. “Because they can be produced rapidly, they are ideal for combating emerging diseases. Moreover, they are safe to produce and administer, and do not constitute a threat to the production team.”
But as other participants in the conference noted, plasmid manufacturing poses a number of critical challenges. The E. coli host is renowned for the large quantities of endotoxins that it produces, and plasmids are notoriously hard to work with.
“Ease of manufacture” is a relative term, and what may be easy on the benchtop may be devilishly difficult when scale-up time arrives. The field is also subject to the “intellectual property thicket syndrome,” in which patents crowd out one another, raising expenses and confusing legal issues. “Plan early and build twice as much product as you think you’ll need,” Dr. Hebel advised.
The VGXI team searches for the most straightforward solution, weighing relative risks and benefits, while taking into account intellectual property. The purification procedure that it selected consists of anion exchange membranes, followed by hydrophilic interaction chromatography, which is then followed by thiophilic interaction chromatography for polishing.
“We consider cell lysis the critical step in the purification process,” said Dr. Hebel, “At this stage we avoid any procedure that could shear the plasmids. We also look for alternatives to traditional purification chromatography during the subsequent steps.”
The plasmid concentration step is another difficult issue, as mishandling can cause shearing and other destructive changes to the preparation. “As we optimize our protocol, we look to remove roadblocks,” he continued. “We need to justify the designation of contamination levels, degree of acceptable supercoiling, limits of cleaning, and the amount of plasmid DNA required.”
Althea Technologies offers services in plasmid design and production, and possesses core capabilities in biological process development, according to Magda Marquet, Ph.D., co-founder. This includes formulation and analytical development, as well as all phases of upstream and downstream processing and the final testing and IND filing from preclinical to commercial scale.
The company’s initial goal was to implement a scalable process adapted to clinical applications. Manufacturing of plasmid DNA is now well defined, and scale-up is achievable. “We have developed a high-cell density fermentation and purification process for plasmid-based DNA vaccines that yields 200–500 mg/L initially, and up to 2 grams/L following optimization,” Dr. Marquet stated. “This process has been used successfully on our multiple cGMP campaigns to produce lot sizes as large as 30 grams of plasmid DNA.”
The purification process development must take into account the problem of endotoxin removal. In conjunction with clients moving into Phase II trials, Althea has reportedly advanced its handling of the material, resulting in an additional log reduction in both endotoxin (<0.1 EU/mg) and RNA levels.