DNA-kontaminering i mRNA vaccin

Abstract
The primary safety concern for DNA vaccines is their potential to integrate into the host cell genome. We describe an integration assay based on purification of high-molecular-weight genomic DNA away from free plasmid using gel electrophoresis, such that the genomic DNA can then be assayed for integrated plasmid using a sensitive PCR method. The assay sensitivity was approximately 1 plasmid copy/µg DNA (representing ∼150,000 diploid cells). Using this assay, we carried out integration studies of three different plasmid DNA vaccines, containing either the influenza hemagglutinin, influenza matrix or HIV gag gene. Six weeks after intramuscular injection, free plasmid was detected in treated muscle at levels ranging from approximately 1,000 to 4,000 copies/µg DNA. At 6 months, the plasmid levels ranged between 200 and 800 copies/µg DNA. Gel purification of genomic DNA revealed that essentially all of the detectable plasmid in treated quadriceps was extrachromosomal. If integration had occurred, the frequency was ≤1–8 integrations per 150,000 diploid cells, which would be at least three orders of magnitude below the spontaneous mutation rate. Our results suggest that the risk of mutation due to integration of plasmid DNA vaccines following intramuscular injection is negligible.

This DNA can and likely will integrate into the genomic DNA of cells that got transfected with the vaccine mix

DNA plasmid pieces smaller than 250 base pairs can diffuse right into the nucleus.
The average base pair length of the pieces of DNA plasmid contamination is 120.

2.2.6. Recommendations for future quality development
In the context of the obligation of the Marketing Authorisation Holder (MAH) to take due account of
technical and scientific progress, the CHMP recommends the following points for investigation:
Active substance
1. The MAH should implement relevant testing strategies to ensure an adequate microbiological
control for the starting materials.
2. The MAH should implement a relevant testing strategy to ensure that HEPES (Pfizer) raw
material, included in the formulation buffer of FP, is free from contaminating RNases.
3. The MAH should implement in-house functional activity analytical methods for release testing
of enzymes used in the manufacturing process at all relevant manufacturing sites, by Q1 2021.
4. The MAH should reassess the specification for the linear DNA template purity and impurities.
The Applicant has already agreed to supply these by Q2 2021.
5. The MAH should perform and document a gap analysis to identify any supplemental
qualification needed to align the methods used for the DNA template control with ICH
requirements. The gaps identified should be addressed either prior to transferring the methods
to relevant sites or during the transfer activities.
6. The MAH should provide active substance process validation data regarding the finalised
Assessment report
EMA/707383/2020 Page 40/140
indirect filter qualification assessment and the shipping validation between sites.
7. The MAH should provide the results of the studies performed to enhance the robustness of the
DNase digestion step.
8. The MAH should tighten the low limits of the proven acceptable ranges for the target volumes
for ATP and CTP, to the levels needed to ensure a sufficiently high mRNA integrity in the active
substance manufacturing process.
9. The MAH should comprehensively describe the capability of the next generation sequencing
technology platform to detect lower amounts of RNA species of alternative sequence in the
presence of the correct, more abundant RNA for the active substance.
10. The MAH should discuss the results and the assay suitability for the cell-based flow cytometry
and the western blot method used for biological characterization of protein expression for the
active substance.
11. The MAH should provide a summary of the validation/verification status of the immunoblot
analytical procedure used to detect double stranded RNA (dsRNA) in BNT162b2 active
substance.
12. In order to improve the control strategy, the MAH should provide the protocol on preparation
and qualification of future primary and working reference standards for the active substance.

Furthermore, the poly(A)tail of the 3’ end was characterised by LC-UV/MS. While the results were
demonstrated to be comparable between Process 1 and Process 2 batches, significant differences were
identified. As expected, poly(A) tail heterogeneity was observed both for Process 1 and Process 2
batches. Thus, slight differences in the poly(A)tail pattern were observed when comparing Process 1
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and Process 2 AS batches. The Applicant explains that the redistribution is probably due to the use of a
linearised DNA plasmid template in Process 2 instead of a PCR-derived DNA template in Process 1

The sole product-related impurity addressed is double-stranded RNA, derived from the in-vitro
transcription reaction. Results from the active substance batches demonstrate that the level of double
stranded RNA is low, acceptable and consistent

For those making the argument that the DNA plasmids are dealt with in the nucleus when cell division occurs (asymmetric partitioning), this primarily occurs when the plasmid DNA or exogenous DNA is in circular form.
With the current DNA contamination, this has been cut up and linearized. It is no longer in its formerly closed, circular form.