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Characterization of Antisense Oligonucleotide Impurities by Ion-Pairing Reversed-Phase and Anion Exchange Chromatography Coupled to Hydrophilic Interaction Liquid Chromatography/Mass Spectrometry Using a Versatile Two-Dimensional Liquid Chromatography Setup.
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-03-31 , DOI: 10.1021/acs.analchem.0c00114
Alexandre Goyon 1 , Kelly Zhang 1
Affiliation  

Determination of phosphorothioate oligonucleotide purity and impurity profile is commonly performed by ion-pairing reversed-phase liquid chromatography (IPRP) with a mobile phase containing triethylamine (TEA) and hexafluoro-2-propanol (HFIP). However, ion-suppressing effects of TEA hamper mass spectrometry (MS) instrumentation sensitivity and HFIP can affect the robustness of the mass spectrometer due to its corrosive nature. Anion exchange chromatography (AEX) is an orthogonal separation mode to IPRP but typically cannot be directly coupled to MS. In this work, we developed a multiple heart-cutting IPRP-, AEX-hydrophilic interaction liquid chromatography(HILIC)/MS method for quantification and high sensitivity identification of antisense oligonucleotide (ASO) impurities using a Q-Exactive mass spectrometer. Notably, both AEX-HILIC and IPRP-HILIC modes could be operated on a versatile two-dimensional liquid chromatography (2D-LC) setup including several column selectors. The HILIC mobile phase contained 25 mM ammonium acetate and allowed identifying impurities at levels down to 0.3%. Careful selection of the sample loop volume and the 2D HILIC column dimension allowed straightforward coupling of HILIC for both IPRP and AEX without the need to use any solvent modulation. Overall, the 2D HILIC allowed online desalting of AEX and IPRP modes and further separation of additional impurities.

中文翻译:

使用多功能二维液相色谱装置通过离子对反相和阴离子交换色谱联用亲水相互作用液相色谱/质谱联用的离子对反相和阴离子交换色谱,对反义寡核苷酸杂质进行表征。

硫代磷酸酯寡核苷酸纯度和杂质分布的测定通常是通过离子对反相液相色谱(IPRP)和含有三乙胺(TEA)和六氟-2-丙醇(HFIP)的流动相进行的。但是,TEA妨碍质谱仪(MS)仪器灵敏度和HFIP的离子抑制作用由于其腐蚀性而会影响质谱仪的耐用性。阴离子交换色谱(AEX)是IPRP的正交分离模式,但通常无法直接与MS偶联。在这项工作中,我们开发了一种多重切割IPRP-,AEX亲水相互作用液相色谱(HILIC)/ MS的方法,用于使用Q-Exactive质谱仪对反义寡核苷酸(ASO)杂质进行定量和高灵敏度鉴定。值得注意的是 AEX-HILIC模式和IPRP-HILIC模式均可在包含多个色谱柱选择器的通用二维液相色谱(2D-LC)设置上进行操作。HILIC流动相含有25 mM乙酸铵,可以鉴定出含量低至0.3%的杂质。仔细选择样品定量环体积和2D HILIC色谱柱尺寸,可以直接将HILIC用于IPRP和AEX,而无需使用任何溶剂调节剂。总体而言,二维HILIC允许在线对AEX和IPRP模式进行脱盐,并进一步分离其他杂质。仔细选择样品定量环体积和2D HILIC色谱柱尺寸,可以直接将HILIC用于IPRP和AEX,而无需使用任何溶剂调节剂。总体而言,二维HILIC允许在线对AEX和IPRP模式进行脱盐,并进一步分离其他杂质。仔细选择样品定量环体积和2D HILIC色谱柱尺寸,可以直接将HILIC用于IPRP和AEX,而无需使用任何溶剂调节剂。总体而言,二维HILIC允许在线对AEX和IPRP模式进行脱盐,并进一步分离其他杂质。
更新日期:2020-04-23
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