mRNA vaccines—What’s in a name?
The sudden and mysterious emergence of COVID-19 in 2020 represented a Black Swan event that drew the world's attention to the mRNA platform and its potential to usher in a new golden age of vaccinology. Much debate has since ensued regarding this new modality being treated as a vaccine or a gene therapy product. It is not unreasonable to say that the mRNA technology stands at the confluence of both product families. As per FDA guidance, ‘Content and Format of Chemistry, Manufacturing and Controls Information and Establishment Description Information for a Vaccine or Related Product,’ 1999, “A vaccine is an immunogen, the administration of which is intended to stimulate the immune system to result in the prevention, amelioration or therapy of any disease or infection.” However, such a definition does not entirely suffice as the mRNA proper is not intended to be immunogenic but rather encodes for a protein/immunogen that arises after the mRNA is ferried across the cell membrane and is translated.
With regard to gene therapy, elements of the FDA Guidance ‘Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs),’ 2020, seem applicable as per the wording “FDA generally considers human gene therapy products to include all products that mediate their effects by transcription or translation of transferred genetic material or by specifically altering host (human) genetic sequences.”
The mRNA technology has advanced at warp speed and has eclipsed the commensurate growth of regulatory oversight; thus, semantics are important to dictate the proper quality control oversight and guidance necessary to ensure that at each stage the mRNA vaccine product is safe, pure, and reproducible. This article, which is restricted in scope to cationic lipid encapsulated mRNAs that are not self-amplifying, reviews the vaccine manufacture and testing processes, as well as the tools and technologies used to ensure purity and integrity of the intermediates and final products.
Overview of mRNA vaccine manufacturing
The attractiveness of the mRNA platform lies in its inherent simplicity and amenability to scale-up and scale-out. Animal cell lines do not need to be developed and grown in sophisticated bioreactors. As such, the technology is predicated upon a cell-free system that will not introduce adventitious eukaryotic viral agents. The structure of the mRNA component of an mRNA vaccine comprises a single-stranded mRNA molecule with a cap at the 5′ end, a poly(A) tail at the 3′ end, and an open reading frame (ORF) flanked by untranslated regions (UTR). The naked mRNA, which is negatively charged, is then sheathed in a cationic-lipid based formulation which serves to form what is termed as a lipid nanoparticle (LNP).
Currently, a well-established manufacturing platform is still somewhat embryonic and is very much in evolution. Several combinations of steps are possible, which, at the most general level, can be grouped into upstream processing (plasmid isolation, enzymatic generation of mRNA, post-transcriptional mRNA modification) and downstream processing, which includes the unit operations required to purify the mRNA product and then properly combine it with cationic lipids to achieve a predefined particle size, lipid:mRNA ratio, and encapsulation efficiency.
The manufacture of an mRNA vaccine consists of the following basic steps or variations thereof:
- Plasmid/template creation and maintenance
- Plasmid/template isolation, purification, and preparation for transcription into mRNA
- mRNA transcription
- mRNA post-transcriptional modification, i.e., 5’ capping and 3’ poly-A tail addition
- mRNA purification
- Formulation with cationic lipids
Manufacturing quality controls, critical quality attributes, and acceptance criteria
Plasmid DNA—It all starts here
mRNA vaccines typically start with a plasmid cDNA as the template for mRNA transcription. During the purification process, little, if any, source plasmid should remain as the DNA is digested with DNase post-transcription; hence, plasmid persistence studies will not be necessary for mRNA vaccines and most likely will not be covered by a future mRNA vaccine-dedicated guidance. In contrast, plasmid DNA vaccines are covered in great detail in FDA Guidance ‘Considerations for Plasmid DNA Vaccines for Infectious Disease Indications,’ 2007. Such detailed regulatory oversight for the case of plasmid vaccines ostensibly originated from the theoretical risk of unintended plasmid integration into the host genome, which potentially could result in tumorigenesis if insertion compromises the activity of a tumor suppressor or increases the activity of an oncogene. However, intramuscular studies in mice or guinea pigs using four different DNA vaccine plasmids demonstrated that there was no evidence of integration using an assay with a sensitivity of about one plasmid copy/microgram of DNA, which would at least three orders of magnitude below the spontaneous mutation frequency (Ledwith BJ et al, 2000).
Nevertheless, although residual plasmid contamination is not inherently risky, there must be appropriate mechanisms in place to ensure the quality of the plasmid template and the master cell bank from which it is derived. A summary of suggested quality control mechanisms and acceptance values are presented in Table 1. Concerning the master cell bank, antibiotics and other components used in the culture but neither required nor specifically intended to be in the final vaccine product should be removed at the working cell bank level, and no animal-derived products should be used at any point in the process.
Table 1: Suggested plasmid and master cell bank quality attributes
Abbreviations: EU = endotoxin units; LAL = limulus amoebocyte lysate; MCB = master cell bank; Ph. Eur. = European Pharmacopoeia; QT-PCR = quantitate PCR; USP = United States Pharmacopeia; UV = ultraviolet; WCB = working cell bank. aAs per FDA Guidance for Industry, ‘Considerations for Plasmid DNA Vaccines for Infectious Disease Indications,’ 2007.
mRNA
As an mRNA-based vaccine, the quality of the mRNA is critical to the intended performance of the vaccine. RNA is an inherently fragile molecule and is prone to hydrolysis, oxidation, and depurination. To further complicate matters, reaction kinetics not only depend on the levels of oxygen and pH but are also influenced by the RNA secondary structure and the presence of cationic lipids (Mikkola S et al, 2001; Wayment-Steele HK et al, 2021). In addition to the mRNA needing to be integrous, the presence of residual amounts of DNA, enzymes, and solvents, as well as dsRNA and truncated RNA fragments, must either be determined or mitigated against. The Center for Biologics Evaluation and Research (CBER) decided that the LNP should be evaluated as part of drug product as opposed to separately (Liu MA et al, 2022).
The main contaminant in the in vitro transcription process is double-stranded RNA (dsRNA). The production of dsRNA byproducts occurs through two distinct mechanisms. In the first mechanism, the RNA transcript synthesized by the T7 RNA polymerase (RNAP) serves as a template for the RNA-dependent RNA polymerase activity of the T7 RNAP in subsequent rounds of transcription. If the 3’-end of the runoff transcript has sufficient complementarity (in cis), it will fold back and result in extension of the runoff transcript. In the second mechanism, the formation of dsRNA byproducts results from the RNAP using the nontemple strand, resulting in an RNA molecule that is complementary to the runoff product but synthesized in a promoter-independent manner. Because the size of the antisense molecule will be similar to the size of the runoff product, it cannot be distinguished by denaturing gel electrophoresis; rather, analysis of the dsRNA byproducts formed because of the presence of an antisense RNA molecule will require native conditions.
The mRNA vaccine can be properly analyzed by a battery of compendial and non-compendial methods as illustrated in Table 2, which lists suggested and potential tests that can be used to ensure product efficacy and safety.
Table 2: Suggested mRNA vaccine drug product quality attributes
Abbreviations: ds = double stranded; EM = electron microscopy; EU = endotoxin units; HPLC = high-performance liquid chromatography; LAL = limulus amoebocyte lysate; LC-MS = liquid chromatography mass spectrometry; LNP = lipid nanoparticle; Ph. Eur. = European Pharmacopoeia; QT-PCR = quantitate PCR; USP = United States Pharmacopeia; UV = ultraviolet. aMay only need to be done during clinical development. bIdentified as being optimal by Hassett KJ et al, 2021. cTechnique was described by Malburet C et al, 2022 as being more appropriate than dynamic light scattering.
References
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