On-Column Oligonucleotide Deprotection for Faster High-Purity Purification
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Solution Overview
Problem
Existing purification processes for oligonucleotides require multiple steps and substantial operation time due to the presence of acid labile 5′hydroxy protecting groups, leading to low yields and impurities.
Innovation Solution
An on-column deprotection process using anion-exchange chromatography with specific buffer and acid solutions to remove the acid labile 5′hydroxy protecting group directly on the chromatography column, reducing the number of purification steps and time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If the crude oligonucleotide is purified using conventional multi-step processes (reversed phase chromatography, concentration and desalting, removal of protecting group in solution, further concentration and desalting), then the purity of oligonucleotides can be achieved, but the operation time is substantial and the overall yield is reduced
Solution Approach 1:
The patent combines multiple separate purification steps (chromatography, concentration, desalting, and deprotection) into a single integrated anion-exchange chromatography process. The oligonucleotide is loaded onto the column in ammonia form, deprotected while bound to the column, and eluted in a single continuous operation, eliminating the need for separate handling steps between purification and deprotection.
Solution Approach 2:
The anion-exchange column serves multiple functions simultaneously: it performs chromatographic separation to purify the oligonucleotide, retains the oligonucleotide in ammonia form for subsequent deprotection, and enables direct elution of the deprotected product. This multi-functional approach replaces several specialized steps with a single versatile process.
2Manufacturing precision
If the crude oligonucleotide is purified using conventional multi-step processes, then the purity of oligonucleotides can be achieved, but the number of operation steps is high which reduces overall yield
Solution Approach 1:
The patent combines multiple separate purification steps (chromatography, concentration, desalting, and deprotection) into a single integrated anion-exchange chromatography process. The oligonucleotide is loaded onto the column in ammonia form, deprotected while bound to the column, and eluted in a single continuous operation, eliminating the need for separate handling steps between purification and deprotection.
Solution Approach 2:
The process maintains continuous useful action by keeping the oligonucleotide bound to the column throughout the deprotection process, eliminating interruptions and transfers. The deprotection occurs in-situ on the column while the oligonucleotide is retained, and the deprotected product is immediately eluted, creating a seamless continuous process that maximizes yield.
3Manufacturing precision
If the acid labile 5′hydroxy protecting group is removed in solution after chromatography, then the protecting group can be removed effectively, but additional concentration and desalting steps are required increasing operation time
Solution Approach 1:
The anion-exchange column acts as an intermediary that enables deprotection to occur in a fixed-phase environment rather than solution. The column matrix provides a controlled environment for acid treatment while the oligonucleotide remains bound, and the deprotected product is then eluted directly, eliminating the need for separate solution-based deprotection, concentration, and desalting steps.
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
This method significantly reduces operation time and increases yield by directly removing the protecting group on the column, resulting in higher purity oligonucleotides suitable for therapeutic applications.
Implementation Method 1
The oligonucleotide, after its cleavage from the solid support, is loaded onto an anion-exchange chromatography column
Implementation Method 2
anion-exchange chromatography column
Implementation Method 3
removal of an acid labile 5′hydroxy protecting group at the 5′-O-oligonucleotide terminus of the oligonucleotide by way of an on-column de-protection with an acid
Data Source
AI summary
The invention relates to a new process for the purification of oligonucleotides which comprises the removal of an acid labile 5′hydroxy protecting group at the 5′-O-oligonucleotide terminus of the oligonucleotide by means of an on-column de-protection with an acid.