Establishing Preferred Product Characterization for the Evaluation of RNA Vaccine Antigens

Abstract

The preferred product characteristics (for chemistry, control, and manufacture), in addition to safety and efficacy, are quintessential requirements for any successful therapeutic. Messenger RNA vaccines constitute a relatively new alternative to traditional vaccine development platforms, and thus there is less clarity regarding the criteria needed to ensure regulatory compliance and acceptance. Generally, to identify the ideal product characteristics, a series of assays needs to be developed, qualified and ultimately validated to determine the integrity, purity, stability, and reproducibility of a vaccine target. Here, using the available literature, we provide a summary of the array of biophysical and biochemical assays currently used in the field to characterize mRNA vaccine antigen candidates. Moreover, we review various in vitro functional cell-based assays that have been employed to facilitate the early assessment of the biological activity of these molecules, including the predictive immune response triggered in the host cell. Messenger RNA vaccines can be produced rapidly and at large scale, and thus will particularly benefit from well-defined and well-characterized assays ultimately to be used for in-process, release and stability-indications, which will allow equally rapid screening of immunogenicity, efficacy, and safety without the need to conduct often lengthy and costly in vivo experiments.

Addressing the Cold Reality of mRNA Vaccine Stability

“…reviewed key techniques currently available to monitor the critical quality attributes of mRNA vaccine candidates ( Table 3 ). 32 We expanded the list by including additional assays to characterize attributes related to formulated (i.e., lipid- and/or protein-containing) mRNA drug products including general quality attributes for parenterally administered vaccines. 19 , 33 Unfortunately, a critical review on analytical techniques to characterize mRNA-(cationic) lipid complexes, as has been published previously for plasmid DNA-lipid complexes, 34 is clearly missing.…”

Enlisting the mRNA Vaccine Platform to Combat Parasitic Infections

“…Moreover, we will go over some recently published studies on mRNA vaccines for parasitic diseases and briefly introduce our development plan for an mRNA vaccine to protect against Chagas disease. An accompanying manuscript in this special issue (Poveda et al) [25] reviews the necessary product characterizations for the initial evaluation of mRNA vaccine antigen candidates, and for more detailed information on the mRNA technology beyond the application for parasitic diseases, we refer the reader to a few excellent recent reviews on the topic [26][27][28][29][30][31].…”

“…Finally, the mRNA product is purified to remove any remaining DNA template, double-stranded RNA, and other contaminations by HPLC [32,33] (Figure 1). The quality of the mRNA transcript is a critical factor, requiring testing for stability, integrity, identity, purity, and homogeneity, as is testing for the desired innate immune response prior to animal testing [25]. Typically, IVT mRNA is comprised of a protein-encoding open reading frame (ORF) flanked by two untranslated regions (UTRs), to support translation ( Figure 2).…”

“…It is therefore suggested that the interferon Type I response should be controlled, in order to improve translation of the mRNA vaccine candidate and consequently increase vaccine potency [32,44]. The innate immune response against exogenous mRNA can be minimized by avoiding the activation of TLR receptors, by using highly purified transcripts (complete removal of production byproducts, such as dsRNA and DNA template) and modified nucleosides (replacing uridine for pseudouridine) [25,62]. Another strategy is to suppress the receptors by co-expressing mRNA encoding immune evasion proteins, such as vaccinia virus proteins E3, K3, and B18 [64][65][66].…”

“…The production and purification process of IVT mRNA can be standardized, therefore avoiding the need for costly product-specific production and purification steps. While it faces the same regulatory requirements and needs for quality control as a recombinant protein [25], the mRNA purification process is less complicated [26]. Due to the standardization of the production and purification process, the development time for new vaccine candidates can be dramatically reduced, which allows for the rapid testing of more vaccine candidates by high-throughput screening [28,31].…”