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https://www.arca.fiocruz.br/handle/icict/63902
ASSESSING MRNA INTEGRITY USING CAPILLARY ELECTROPHORESIS: INSIGHTS INTO SCIENTIFIC PARAMETERS
Author
Affilliation
Fundação Oswaldo Cruz. Instituto de Tecnologia.em imunobiológicos (Bio-manguinhos). Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia.em imunobiológicos (Bio-manguinhos). Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia.em imunobiológicos (Bio-manguinhos). Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia.em imunobiológicos (Bio-manguinhos). Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia.em imunobiológicos (Bio-manguinhos). Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia.em imunobiológicos (Bio-manguinhos). Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia.em imunobiológicos (Bio-manguinhos). Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia.em imunobiológicos (Bio-manguinhos). Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia.em imunobiológicos (Bio-manguinhos). Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia.em imunobiológicos (Bio-manguinhos). Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia.em imunobiológicos (Bio-manguinhos). Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia.em imunobiológicos (Bio-manguinhos). Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia.em imunobiológicos (Bio-manguinhos). Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia.em imunobiológicos (Bio-manguinhos). Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia.em imunobiológicos (Bio-manguinhos). Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia.em imunobiológicos (Bio-manguinhos). Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia.em imunobiológicos (Bio-manguinhos). Rio de Janeiro, RJ, Brasil.
Abstract in Portuguese
Introduction: The rise of mRNA-based vaccines through via in vitro transcription (IVT) has seen rapid
development and widespread acceptance due to their potential in the combating of diseases. Ensuring
mRNA integrity in these therapies is critical for efficacy and safety. Evaluating mRNA purity using
capillary gel electrophoresis is crucial, involving a meticulous analysis of mRNA degradation and abortive
mRNA species, directly impacting vaccine effectiveness and safety.
Objectives: Due to insufficient parameters in scientific literature for mRNA integrity analyses via IVT
in biopharmaceutical development, our study aimed to explore two critical parameters. We investigated
the impact of user-controlled variables: adjusting the minimum RFU (Relative Fluorescence Units)
threshold for signal processing and selecting the ‘smear analysis’ function (specific areas) in capillary
electrophoresis.
Methodology: We evaluated three IVT mRNAs, each about 4,000 nucleotides long, using capillary
electrophoresis on the Agilent 5200 Fragment Analyzer with DNF-471 RNA kit, following the
manufacturer’s guidelines. Sample preparation and analysis assessed IVT mRNA size and integrity.
Results: Our analysis involved three IVT mRNA samples, with ‘smear analysis’ set at 10% on both sides.
We tested minimum RFU values of 5, 10, 20, and 100. At 5 RFU, peak percentages were 91.1%, 88.3%,
and 67.1%. With 10 RFU, the values were 91.9%, 88.7%, and 68.8%, while at 20 RFU they increased
to 93.7%, 89.6%, and 71.9%. Employing 100 RFU yielded 100%, 96.8%, and 92.9%. Higher RFU and
broader ‘smear analysis’ windows led to less stringent quality control for IVT mRNA samples.
Conclusion: The gap in standardized parameters in the current literature highlights the potential for
diverse estimations of mRNA integrity in IVT samples. Addressing this is crucial for the rigorous analysis
of IVT-based immunotherapeutic products. Maintaining a standardized minimum RFU value enhances
the reliability and comparability of the results. Selecting a fixed peak window percentage, like 10-20%
on both sides of RNA size, enables a comprehensive evaluation of mRNA integrity. Implementing these
Abstract
Introduction: The rise of mRNA-based vaccines through via in vitro transcription (IVT) has seen rapid
development and widespread acceptance due to their potential in the combating of diseases. Ensuring
mRNA integrity in these therapies is critical for efficacy and safety. Evaluating mRNA purity using
capillary gel electrophoresis is crucial, involving a meticulous analysis of mRNA degradation and abortive
mRNA species, directly impacting vaccine effectiveness and safety.
Objectives: Due to insufficient parameters in scientific literature for mRNA integrity analyses via IVT
in biopharmaceutical development, our study aimed to explore two critical parameters. We investigated
the impact of user-controlled variables: adjusting the minimum RFU (Relative Fluorescence Units)
threshold for signal processing and selecting the ‘smear analysis’ function (specific areas) in capillary
electrophoresis.
Methodology: We evaluated three IVT mRNAs, each about 4,000 nucleotides long, using capillary
electrophoresis on the Agilent 5200 Fragment Analyzer with DNF-471 RNA kit, following the
manufacturer’s guidelines. Sample preparation and analysis assessed IVT mRNA size and integrity.
Results: Our analysis involved three IVT mRNA samples, with ‘smear analysis’ set at 10% on both sides.
We tested minimum RFU values of 5, 10, 20, and 100. At 5 RFU, peak percentages were 91.1%, 88.3%,
and 67.1%. With 10 RFU, the values were 91.9%, 88.7%, and 68.8%, while at 20 RFU they increased
to 93.7%, 89.6%, and 71.9%. Employing 100 RFU yielded 100%, 96.8%, and 92.9%. Higher RFU and
broader ‘smear analysis’ windows led to less stringent quality control for IVT mRNA samples.
Conclusion: The gap in standardized parameters in the current literature highlights the potential for
diverse estimations of mRNA integrity in IVT samples. Addressing this is crucial for the rigorous analysis
of IVT-based immunotherapeutic products. Maintaining a standardized minimum RFU value enhances
the reliability and comparability of the results. Selecting a fixed peak window percentage, like 10-20%
on both sides of RNA size, enables a comprehensive evaluation of mRNA integrity. Implementing these
factors bolsters scientific integrity, instilling confidence in findings’ accuracy and reproducibility.
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