However, as the global demand for these vaccines surged, questions arose about the presence of residual DNA within the vaccines, raising concerns about their safety and quality. In this article, we explore the manufacturing processes, the detection of residual DNA, and the potential implications for vaccine recipients.
Manufacturing Processes: From RCT to Large-Scale Production
Both Pfizer-BioNTech and Moderna used similar processes to manufacture their mRNA COVID-19 vaccines. Initially, for randomized clinical trials (RCT), Pfizer employed a process (Process 1) to produce the mRNA material. Subsequently, they transitioned to a more efficient process (Process 2) similar to Moderna’s. The core of this manufacturing process involves cloning the SARS-CoV-2 spike sequence into a plasmid, containing a bacterial origin of replication (ori) that is active in Escherichia coli (E. coli).
The E. coli cells are harvested, lysed, and the DNA is extracted and linearized with a restriction enzyme. This linear DNA then acts as the template for in vitro transcription (IVT) using T7 RNA Polymerase in the presence of N1-methyl-pseudouridine. Post-IVT, DNA is hydrolyzed, thus reducing its prevalence in the final drug product.
Residual DNA Concerns
The presence of residual DNA in mRNA vaccines is a matter of concern, as it may potentially affect the safety and efficacy of the vaccines. Leaked documents from the European Medicines Agency (EMA) highlighted variations in the levels of residual DNA in mRNA products made using the manufacturing processes described above.
Generalizability of Findings
To assess the generalizability of these findings, a study obtained and analyzed 24 unopened, expired vials of Pfizer and Moderna vaccines, along with remnants of Moderna XBB.1.5 COVID-19 vaccines distributed in Ontario, Canada. Using Qubit® fluorometry and qPCR, the researchers targeted specific sequences such as the spike, plasmid ori, and the SV40 promoter-enhancer-ori to detect residual DNA.
Safety and Adverse Event Reporting
In addition to investigating the presence of residual DNA, this study aimed to evaluate any adverse events (AEs), including serious AEs (SAEs), associated with the lots of vaccines examined.
Understanding DNA Packaging in Lipid Nanoparticles (LNPs)
Furthermore, the study delved into the size distribution of DNA fragments and the DNase I sensitivity of the vaccine. These analyses aimed to determine whether the residual DNA is packaged within the lipid nanoparticles (LNPs) used to encapsulate the mRNA.
Discussion
The presence of residual DNA in mRNA COVID-19 vaccines is a subject of significant concern, and the findings from our study shed light on the extent of this issue. In our examination of 27 vaccine vials, we observed that all of them contained residual DNA, with varying concentrations. These results underscore the need for a comprehensive understanding of the implications of DNA contamination in mRNA vaccines.
Consistency and Reliability of Assays
Moderna vs. Pfizer: Variations in DNA Concentration
Moderna’s vaccine exhibited the lowest DNA concentration when assessed using qPCR, but the highest concentration when analyzed using Qubit. This discrepancy between the two quantification methods may be attributed to the differences in the size and nature of the DNA fragments present.
Moderna’s vials displayed a remarkably uniform distribution of DNA levels, suggesting a more standardized and robust manufacturing process. Notably, in Moderna vaccines, the ori sequence displayed lower DNA loads than the spike sequence in all vials except lot AS0467D. This may indicate that homologous modified RNA in the vaccines hinders the digestion of template DNA by hybridization.
Pfizer’s Purple Top Vials and Formulation Change
Two lots of Pfizer monovalent purple top vials, which require dilution before administration, exhibited the highest DNA concentrations. These lots were associated with the highest number of adverse events (AEs) and serious adverse events (SAEs) reported in the Vaccine Adverse Event Reporting System (VAERS).
The change in formulation to a Tris/sucrose buffer for other Pfizer vaccines was authorized by the US FDA on October 29, 2021. This formulation change aimed to enhance stability and simplify storage requirements, offering a ready-to-use formulation. It is essential to note that the purple-topped Pfizer lots were not included in this formulation change and were linked to increased adverse events.
Exploratory Analysis of DNA and SAEs
Our preliminary analysis suggested a positive dose-response relationship between the quantity of residual DNA estimated by qPCR and SAEs for the Pfizer lots. In contrast, different relationships were observed for Moderna lots, both in qPCR data and in estimates based on fluorometry. These variations may result from differences in the DNA content, the size distribution of DNA fragments, plasmid vector composition, and lipid nanoparticle composition between the two products.
Potential Impurities and Impacts on Vaccine Safety
The presence of impurities and contaminants, such as fragmented mRNA, double-stranded RNA (dsRNA), and lipopolysaccharides, can influence vaccine safety. These impurities may trigger immune-inflammatory reactions and contribute to adverse events, including conditions like myocarditis. Wider sampling and further research are necessary to understand the relationships between DNA contamination and adverse events in greater detail, including severe outcomes like death.
SV40 Enhancer and Potential Impacts
While genomic integration of DNA fragments in the COVID-19 mRNA vaccines has not been demonstrated, the presence of DNA could lead to unwarranted innate immune responses, prothrombotic effects, and potentially contribute to ischemic diseases. The presence of DNA sequences, such as the SV40 enhancer, with nuclear targeting potential may introduce new considerations for vaccine safety.
Quantification Challenges
Quantifying residual DNA in vaccines is not without challenges. qPCR is limited in its ability to detect molecules smaller than the amplicon size (105-114 bp), potentially leading to underestimations of the total DNA content. The large discrepancies observed between qPCR and Qubit quantifications highlight the need for consistent and standardized methods when assessing residual DNA. The use of specific fluorometric dyes in samples with high concentrations of N1-methyl-pseudouridine modRNA may also introduce uncertainties in quantification.
Guidelines and Methodological Clarity
The guidelines established by the US FDA and the World Health Organization (WHO) recommend limiting residual DNA to below 10 ng/dose for parenteral inoculations and restricting DNA fragment sizes to below the size of a functional gene (~200 base pairs). These guidelines were conceived before the era of efficient transfection using lipid nanoparticles (LNPs). The presence of DNA in LNPs may lead to longer DNA persistence and increased transfection efficiency, which the guidelines did not consider.
Cumulative Dosing and Future Considerations
The cumulative dosing of vaccines using the same plasmid and LNP-based modRNA platform across different infections presents a novel concern. The guidelines in place may not adequately address the risks associated with administering multiple doses, sometimes with short intervals, and the potential for accumulating DNA over time. A comprehensive reevaluation of the guidelines is necessary to ensure the safety of such practices.
Implications and Transparency
The implications of our findings are far-reaching. Ensuring the safety and efficacy of vaccines, especially those developed through innovative technologies like mRNA, is of paramount importance. The presence of residual DNA in these vaccines, in such high quantities, calls into question the existing guidelines set by regulatory agencies.
Transparency and open disclosure of vaccine composition and manufacturing processes are critical to public trust. The precautionary principle should guide decision-making, particularly when dealing with emerging technologies that may have unforeseen consequences.
Further Research and Replication
Our study is preliminary and limited in sample size, and therefore, its findings should be seen as a starting point for further research. Replication under controlled, forensic conditions is essential to validate our results and delve deeper into the implications of residual DNA in mRNA vaccines.
In Conclusion
The presence of residual DNA in mRNA COVID-19 vaccines is a matter of concern that warrants thorough investigation and reevaluation of existing guidelines. The vast quantities of DNA found in these vaccines, the potential implications for vaccine safety, and the cumulative dosing of vaccines using the same platform all require careful consideration.
Public health agencies, regulatory bodies, and the pharmaceutical industry must work together to ensure the continued safety and effectiveness of vaccines. As the world grapples with the ongoing challenges of the COVID-19 pandemic and future health threats, a transparent and evidence-based approach to vaccine development and safety is imperative. This will not only enhance public trust but also promote the responsible use of emerging vaccine technologies.
REFERENCE LINK : https://osf.io/mjc97/
