Standards Debate

We recommend that the fraction of plasmid in supercoiled conformation be included in the bulk release criteria, and that you establish a minimum specification for supercoiled plasmid content (preferably >80%).^[20]

Ambiguity

Is the FDA referring to over 80% of plasmid supercoiling qualitatively OR quantitatively? This question was raised early on by the academia, because they mean totally different things.

Quality: each individual pDNA possessing a level of negative supercoiling which exceeds 80%

Quantity: the amount of negative supercoiled pDNA in the vaccine, in other words, the purity of the vaccine being over 80% purity

We have reason to therefore postulate that the >80% standard is ambiguous, arbitrary, and needs revision because it lacks relevant insight and perspective in the field of DNA vaccines.

Reasoning

Whilst it is true that supercoiled pDNA will increase transfection efficiency^[21], thereby stimulating a stronger immune response (improved immunogenicity), it does not necessarily mean plasmid supercoiling quality and quantity needs to be over 80%. This is because delivery methods such as particle mediated delivery and electroporation already ensures transfection by bombardment and enabling dramatic uptake of biological material in local tissue respectively. Furthermore, it is not necessary to achieve maximal supercoiling; what truly matters is that compactness, robustness to shear stress and strong immunogenicity has been achieved.

A study^[22] has proven that standards should be tailored for different vaccines. In the case of rabies DNA vaccine, research has shown that a vaccination with a plasmid containing at least 70% of supercoiled molecules triggered statistically significant specific antibody titres and specific Th-1 oriented cell-based immunity as early as 2 and 3 weeks post vaccination. This comes to show how an arbitrarily set standard of >80% is irrelevant and will only add to time delays and incur greater costs^[23] in manufacture.

Another study has indicated why maximal supercoiling is not most ideal. Failure to relax negative supercoiling of DNA leads to unintended consequences at a cellular level^[24]. In healthcare, if such an incident occurs, the DNA vaccine would be doing more harm than good to the patient. Once again, this is why we strongly urge regulatory bodies to revise their standards and industry guidance in DNA vaccine production.

Our suggestion

We hope for scientists, engineers, industrialists and policymakers to collaborate in reforming current standards. Every opinion from every field carries weight and should be taken into account when setting a new standard. This eliminates ambiguity, arbitrariness, misinterpretations and misallocation of valuable resources.