Determination of Purge Factors for Use in Oligonucleotide Control Strategies

Abstract

The understanding, control, and removal of nonoligonucleotide process-related impurities (PRI) are of key importance for the manufacturing of therapeutic oligonucleotides as their presence in the final product is both a quality and safety concern. Regulatory agencies require manufacturers to demonstrate that PRI are under control or adequately purged during the manufacturing process. Purging depends on the physicochemical properties of the impurities and the unit operations of the manufacturing process but should be independent of oligonucleotide size or type. The purging power of unit operations relevant to oligonucleotide manufacturing (synthesis, cleavage and deprotection, chromatography, ultrafiltration/diafiltration) was measured using representative solvents and other small molecules typical for oligonucleotide synthesis. The results show that each unit operation has significant purging capability (synthesis >1000; cleavage and deprotection >100 (reactivity, when applicable); chromatography >1000; ultrafiltration/diafiltration >10) and that large overall purge factors can be obtained (≥1 × 107). Experimentally determined purge values are aligned with theoretical purge values; thus, the use of purge arguments in oligonucleotide control strategies is a sound scientific approach. Guidance on reasonable purge values for oligonucleotide unit operations is presented. Additionally, the data demonstrate that solvents and reagents typically used in oligonucleotide synthesis are robustly cleared by the process and should not require testing in the final product.

Trace analysis of dimethoxytrityl alcohol (DMT-OH) in oligonucleotide matrices using liquid chromatography coupled with tandem mass spectrometry

“…The secondary goal of this work was to enable quantification of other small molecule impurities, such as succinamide, aniline, isobutyramide, benzamide, 2-phenylacetamide, and uny-CTP 5 (structures shown in Fig. 6), that may be generated during cleavage and deprotection.…”

“…The quantification of oligonucleotide samples from crude to drug substance in twelve 163 mmol scale batches (data for one batch is shown) demonstrated that all seven potential small molecule impurities are removed by the downstream processes, thus supporting control via purge arguments. 5 With the exception of DMT-OH, the small molecule impurities were sufficiently removed by the first purification step (HIC). Since the DMT protecting group was intentionally removed after HIC purification, the presence of DMT-OH in solution prior to cleavage was expected.…”

“…2 The quality of oligonucleotide therapeutics, specically the impurities, must be well controlled and understood. [3][4][5] Most oligonucleotide impurities arise during synthesis 6,7 in which nucleotides are sequentially added to a solid support to yield the target oligonucleotide sequence. 4,4 ′ -Dimethoxytrityl (DMT) is widely used as a protecting group for hydroxy groups on the solid supports, such as Unylinker or controlled pore glass (CPG), as well as for nucleoside derivatives 8,9 (Scheme 1).…”

“…The DMT protecting group prevents polymerization of the nucleoside during synthesis and is cleaved in each reaction cycle to generate dimethoxytrityl alcohol (DMT-OH). 5,[10][11][12] The DMT protecting group remaining on the 5 ′ -end of the oligonucleotide aer synthesis may be cleaved before or aer chromatographic purication, 13 resulting in a DMT-OH impurity. While DMT cleavage is routinely monitored by UV during the reaction, the resulting impurity, DMT-OH, is not.…”

“…12 These impurities may be removed during the subsequent purication and isolation steps. 5 Quantication of DMT-OH and other small molecule impurities in the oligonucleotide intermediates and drug substance can help determine an appropriate control strategy. In a puri-ed solid drug substance, quantication is relatively straightforward via LC-UV due to minimal presence of other species that may interfere with separation and UV absorption of DMT-OH.…”