mRNA Sequence Mapping With Flow-Through Limited Digestion
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Solution Overview
Problem
Existing methods for sequencing mRNA therapeutics face limitations in achieving comprehensive sequence coverage due to the generation of short and non-unique oligos from common nuclease digestion, which hinders the identification and characterization of process-related and product-related impurities.
Innovation Solution
A flow-through (FT)-based limited digestion method using immobilized RNase T1 enzyme on a solid support, combined with ion-pairing reversed phase liquid chromatography-tandem mass spectrometry (IP RP-LC-MS/MS), allows for precise control of digestion conditions to produce longer, unique oligonucleotides, enhancing sequence coverage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If common nuclease digestion is used to sequence mRNA therapeutics, then the process is simple and fast, but the sequence coverage is limited due to generation of short and non-unique oligos
Solution Approach 1:
The patent changes the digestion parameters by using limited digestion conditions instead of complete digestion, controlling the amount of nuclease and digestion time to produce longer, unique oligonucleotides that enable comprehensive sequence coverage while maintaining practicality
Solution Approach 2:
The patent applies partial digestion rather than complete digestion, using controlled amounts of nuclease that are insufficient for complete degradation, thereby generating a mixture of full-length and partially digested fragments with unique sequences for comprehensive mapping
2Measurement precision
If limited digestion with controlled conditions is used to produce longer unique oligos, then sequence coverage is improved, but the process complexity and control requirements increase
Solution Approach 1:
The patent optimizes digestion parameters including nuclease amount, incubation time, and temperature to achieve controlled limited digestion, producing sufficient unique oligonucleotides for accurate sequence mapping while managing process complexity
Solution Approach 2:
The patent employs monitoring and adjustment mechanisms during the digestion process to ensure optimal conditions are maintained, allowing real-time control over the digestion extent to balance sequence coverage with process manageability
3Productivity
If flow-through based limited digestion is implemented, then high-throughput sequence coverage mapping is achieved, but the system complexity and automation requirements increase
Solution Approach 1:
The patent utilizes flow-through chromatography technology where the sample flows through a column containing immobilized nuclease, enabling automated, high-throughput digestion with controlled conditions and subsequent separation of digestion products
Solution Approach 2:
The patent introduces a solid support column as an intermediary medium that contains the nuclease enzyme, allowing the digestion process to be automated and scaled up while maintaining controlled conditions for producing unique oligonucleotides
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
The method achieves ultra-high coverage and reproducible sequence mapping of mRNA therapeutics, ensuring product quality and characterization by generating uniquely mappable oligonucleotides.
Implementation Method 1
contacting the sample comprising the polynucleotide of interest with a solid support having a digestive enzyme immobilized thereto to thereby form a digested sample
Implementation Method 2
analyzing the eluate by liquid chromatography-mass spectrometry
Implementation Method 3
analyzing the eluate by liquid chromatography-mass spectrometry, to identify and/or characterize the sequence of the polynucleotide of interest
Data Source
AI summary
The present disclosure provides methods for mapping the sequence of a polynucleotide of interest in a sample using flow-through based limited digestion. A digestive enzyme is immobilized to a solid surface, and a sample including the polynucleotide of interest is contacted to the solid surface in flow-through mode to digest the polynucleotide of interest. The flow-through fraction comprising the digested polynucleotide of interest is collected and then subjected to liquid chromatography-mass spectrometry (LC-MS) analysis to map the sequence of the polynucleotide of interest.