Mobile Phase Conductivity Adjustment to Compensate
Publication Date: 2015-Aug-14
The IP.com Prior Art Database
for Ion Exchange Characteristic Changes
Anion exchange resins (e.g., quaternary ammonium ion exchange resins) have been used for the separation of synthetically produced oligonucleotides for many years. The typical oligonucleotide purification process involves loading a crude oligonucleotide synthesis mixture (containing oligonucleotide product, impurities, and synthesis and deprotection solutions) onto an anion exchange resin followed by isolation of the oligonucleotide product of interest with either an isocratic or gradient salt elution.
A purification method that is slowly gaining popularity for oligonucleotide separations is a multi-step process that includes (1) an isocratic mid-to-low conductivity flush of the purification resin to remove impurities after the material is loaded onto the column followed by (2) a separation step of the product via an increasing salt concentration (conductivity) gradient elution. The isocratic flush of step (1) is also called a Shortmer Isocratic Removal step (SIR).
The SIR technique has been used for years in other purification processes utilizing the broad adsorption characteristics between the desired product and impurities in the crude sample and the resin media. In the purification of a desired full-length product (FLP) from a synthetic oligonucleotide synthesis reaction sample, the SIR step exploits the adsorption difference between the FLP containing the protecting 5-dimethoxytrityl (DMT) group and non-FLP (from synthesis coupling failures) that do not have a DMT group bound to them. For example, after a crude synthesis mixture has been loaded onto a purification resin, an SIR step can be performed in which the resin is washed with an isocratic salt solution to remove impurities that do not have the DMT (the non-FLPs). Following the SIR step, FLP-DMT (FLP with a DMT group) can either be further purified via a salt gradient elution, or via a first detritylated (removing the DMT group from the FLP) and then further purified via a salt gradient elution.
The SIR purification technique uses either an isocratic or gradient elution to facilitate the removal of non-DMT containing impurities. This solution’s conductivity and the volume passed through the column are set prior to the start of the purification process. If the solution’s conductivity and/or volume are too low, an inefficient removal of non-DMT containing impurities will occur, resulting in a low FLP yield and/or FLP quality. If the solution’s conductivity and/or volume are too high, in addition to the non-DMT containing impurities, FLP-DMT will elute during the SIR step resulting in loss of the desired product.
Resins used for oligonucleotide purification processes are commonly re-used multiple times. The conductivity of SIR mobile phase required for efficient FLP-DMT separation from non-DMT containing impurities can change over time from these repeated us...