Development of Biolayer Interferometry (BLI)-Based Double-Stranded RNA Detection Method with Application in mRNA-Based Therapeutics and Vaccines.
Pharmaceutics
; 16(9)2024 Sep 19.
Article
in En
| MEDLINE
| ID: mdl-39339263
ABSTRACT
Background:
In vitro-transcribed (IVT) mRNA has been established as a promising platform for therapeutics and vaccine development. Double-stranded RNA (dsRNA) is a major impurity of IVT mRNA and can trigger unfavored immune responses, potentially causing adverse events in patients. Existing dsRNA detection and quantitation methods, such as gel electrophoresis, ELISA, or homogeneous time-resolved fluorescence (HTRF), have low sensitivity or are time-consuming. A recently published lateral flow immunoassay (LFSA) was shown to be fast, but it lacks the sensitivity for dsRNA with uridine modifications.Methods:
In this study, we provided a possible explanation for the reduced sensitivity of existing quantitation methods for dsRNA with modified uridines by characterizing the binding affinities of commonly used anti-dsRNA antibodies. Then, a rapid and sensitive biolayer interferometry (BLI) dsRNA detection assay utilizing Flock House Virus (FHV) B2 protein was developed to overcome the challenges in dsRNA detection and the reduced sensitivity.Results:
This assay allows the detection of dsRNA with different uridine modifications (ψ, m1ψ, 5 moU) with similar sensitivity as dsRNA without modification. Furthermore, we demonstrated this method can be used to quantify both short and long dsRNA, as well as hairpin-structured dsRNA, providing a more comprehensive detection for dsRNA impurities. Moreover, we applied this assay to monitor dsRNA removal through a purification process.Conclusions:
Taken together, this BLI method could enable real-time monitoring of impurities in IVT mRNA production to prevent immunogenicity stemming from dsRNA.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Language:
En
Journal:
Pharmaceutics
Year:
2024
Document type:
Article
Affiliation country:
United States
Country of publication:
Switzerland