Determination of linearized pDNA template in mRNA production process using HPLC

Area of Science:

• Biotechnology
• Analytical Chemistry
• Pharmaceutical Science

Background:

• Messenger RNA (mRNA) therapeutics have shown significant clinical success, particularly during the COVID-19 pandemic.
• Production of mRNA involves in vitro transcription from plasmid DNA (pDNA), necessitating the removal of residual pDNA as a process-related impurity.
• Current regulatory limits require pDNA levels below 10 ng per human dose, emphasizing the need for sensitive detection methods.

Purpose of the Study:

• To develop and validate a generic, cost-efficient, and sequence-agnostic analytical procedure for determining residual pDNA in mRNA samples.
• To establish a reliable method for in-process monitoring during mRNA production.
• To offer an alternative to quantitative polymerase chain reaction (qPCR) for mRNA quality control.

Main Methods:

• Development of a procedure involving enzymatic degradation of mRNA followed by anion-exchange high-performance liquid chromatography (HPLC) separation.
• Quantification of residual pDNA using established chromatographic methods for pDNA quality analytics.
• Validation of the method with a limit of quantification (LOQ) of 2.3 ng and a limit of detection (LOD) below 0.1 ng.

Main Results:

• The developed method successfully quantified residual pDNA in mRNA samples with high sensitivity (LOQ: 2.3 ng, LOD: <0.1 ng).
• The procedure was effectively applied for in-process monitoring of three model mRNAs and a self-amplifying RNA (saRNA).
• The method demonstrated cost-efficiency, speed, and sequence agnosticism compared to traditional qPCR methods.

Conclusions:

• The enzymatic digestion and chromatographic separation method provides a robust and sensitive approach for residual pDNA determination in mRNA products.
• This generic procedure can serve as a valuable substitute for qPCR in mRNA manufacturing, enhancing analytical strategies.
• The method's efficiency and broad applicability support the quality control and regulatory compliance of mRNA-based therapeutics.