DNA Removal from mRNA via DNase Digestion and Solid Support Separation
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Solution Overview
Problem
Current methods for mRNA production do not effectively remove DNA templates, which can induce an innate immune response and are oncogenic, and regulatory guidelines require the quantification and removal of residual DNA in RNA products.
Innovation Solution
The methods involve DNase treatment or removal of labeled DNA using solid supports to separate DNA templates from RNA transcripts, with techniques such as centrifugation, ultrafiltration, or liquid chromatography-tandem mass spectrometry to ensure complete removal of DNA, achieving residual deoxynucleotide levels below specific ppm thresholds.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNase treatment is applied to remove DNA templates, then DNA removal efficiency is improved, but process complexity and cost increase
Solution Approach 1:
The patent extracts the harmful DNA template from the reaction mixture using DNase I enzyme treatment. The enzyme specifically degrades DNA while leaving RNA intact, thereby removing the harmful component without affecting the desired product. This is followed by separation steps to remove the digested DNA fragments.
Solution Approach 2:
The patent uses DNase I enzyme as an intermediary agent to facilitate DNA removal. The enzyme acts as a mediator that selectively targets and degrades DNA templates, enabling effective separation between DNA and RNA components through enzymatic digestion rather than direct physical separation methods.
2Reliability
If multiple separation steps are used to remove DNA, then DNA removal efficiency is improved, but manufacturing time and cost increase
Solution Approach 1:
The patent combines multiple functions into integrated steps. For example, DNase treatment is performed in the same reaction vessel without complete separation, and purification steps are merged to simultaneously remove enzymes, buffers, and digested DNA fragments in a single or limited number of operations, reducing overall processing time while maintaining effectiveness.
Solution Approach 2:
The patent performs DNase treatment during or immediately after the transcription reaction, rather than as a separate subsequent step. This preliminary action allows the enzyme to work on the DNA template while the RNA product is still present in the reaction mixture, streamlining the overall process and reducing total manufacturing time.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
These methods effectively remove DNA templates from mRNA products, ensuring the safety and efficacy of therapeutics by minimizing residual DNA, aligning with regulatory requirements and reducing immune responses.
Implementation Method 1
DNase I can be added to the sample. In some embodiments, 10 units, 2 units, 1 unit, 0.5 units, or 0.05 units of DNase I are added for each μg of DNA template in the sample.
Implementation Method 2
the DNA removal procedure includes removal of labeled DNA from a sample by contacting the sample with a solid support configured to bind the label
Implementation Method 3
the separating step may involve centrifugation, ultrafiltration, precipitation, liquid chromatography, gel electrophoresis, or use of a vacuum column
Data Source
AI summary
The present invention describes methods of removing DNA from an RNA transcript during the mRNA production process. The method embodies procedures for obtaining an in vitro transcription product, and removing any DNA from the product. The DNA can be removed by adding either free DNase or a resin containing immobilized DNase to the product, and recovering the RNA transcript. Alternatively, the DNA template used in the in vitro transcription reaction is labeled. After transcription, the product is applied to a resin that is configured to bind the label, and the RNA transcript is recovered. To detect whether any residual impurities are left in the RNA transcript product, the product is subjected to nuclease digestion and subsequently to liquid chromatography-tandem mass spectrometry analysis to quantitate any residual DNA. The present invention demonstrates efficient and effective methods of isolating an RNA transcript from an in vitro transcription product.