Multivalent mRNA vaccines show suppressed antigen expression in cell-based potency testing
Interference with antigen expression in multivalent mRNA vaccines
In potency testing using human cells, multivalent mRNA formulations showed lower antigen expression than dose-matched single-antigen formulations. Messenger RNA (mRNA) vaccines are used to prevent infectious diseases, but multivalent and combination vaccines containing several antigens also need to be considered to address viral diversity. Whether different mRNAs interfere with translation of their target proteins when combined is also a question for quality assessment. Here, potency refers to biological activity measured in a cell assay and is distinct from protection against infection in people.
Expression suppression confirmed by mass spectrometry and antibody-based analyses
The researchers introduced mRNA into human cells and analyzed antigen proteins using liquid chromatography–tandem mass spectrometry (LC-MS/MS). In multivalent formulations, antigen protein translation was suppressed compared with dose-matched single-antigen formulations. Western blotting and intracellular immunostaining with type-specific antibodies also confirmed suppressed expression.
In the same assay, LC-MS/MS proteomics data obtained without additional sample preparation also revealed host cell responses. Cells given a high mRNA dose showed proteomic changes suggestive of reduced overall translation capacity. However, these results alone do not establish the specific molecular mechanism behind the suppressed expression. The provided abstract does not report the cell line, antigen types, mRNA doses, percentage reduction in expression, or statistical values, making the magnitude and scope of the suppression difficult to assess.
Potency assessment and dose evaluation for multivalent formulations
The study showed that LC-MS/MS could serve as an antibody-independent alternative for potency testing. This does not mean that a vaccine’s clinical protective efficacy was quantified; the findings concern a method for assessing antigen proteins produced in cells.
The researchers proposed carefully designing cell-based assays when evaluating multivalent mRNA formulations and considering reference materials suited to these formulations, along with mRNA dose optimization. Checking whether expression results obtained with a single-antigen formulation also hold for a multivalent formulation thus emerges as a task for quality assessment. These cell assay results alone cannot establish changes in vaccine recipients’ immune responses or in vaccine safety or protective efficacy.
Source: Journal of Pharmaceutical and Biomedical Analysis article abstract
Messenger RNA (mRNA) vaccines are highly effective against infectious diseases, but viral diversity has underscored the necessity for multivalent and combination vaccines. We hypothesize there could be interference of target protein translation in multivalent mRNA vaccines in cell-based assays used for potency testing. Following transfection into human cells, we observed that antigen protein translation is inhibited in multivalent formulations compared to their dose-matched monovalent counterparts using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Inhibited expression was further validated using Western blot and intra-cellular immunostaining with type-specific antibodies. Beyond direct antigen-specific analysis, LC-MS/MS proteomics data from the same assay without additional sample preparation provided a broad view of host cell responses, revealing proteome-level changes indicative of impaired global translation capacity in cells transfected with high dose mRNA(s). To the best of our knowledge, we report for the first time in literature that antagonistic interactions among mRNA constructs can reduce protein expression in multivalent drug products. Our study also demonstrates that LC-MS/MS is a viable alternative for potency testing without dependence on antibodies. These findings highlight the need for careful cell-based assay design, multivalent drug product reference standards for potency assessment, and deliberate mRNA dose optimization in multivalent formulations.
Developers of multivalent mRNA vaccines can compare results from testing each mRNA separately with antigen expression in the final mixed formulation to review quality assessment conditions. For example, if antigen expression differs between dose-matched single-antigen and multivalent formulations, they could consider reference materials and assay conditions suited to the multivalent formulation, adjust mRNA doses, and compare expression changes. LC-MS/MS was proposed as an antibody-independent alternative for potency assessment. However, the provided abstract alone cannot establish optimal doses or assay criteria for individual products, and such assessments do not demonstrate improvements in clinical safety or protective efficacy.