［医薬品医療機器レギュラトリーサイエンス，PMDRS, 54（5），439〜454（2023）］

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複数種の液体クロマトグラフ質量分析計を用いたモデル核酸
医薬品の分析データの比較
廣瀬 賢治＊1,†，吉田 徳幸＊2,†，寺崎 真樹＊1，瀬崎 浩史＊3，唐澤 薫＊4，岩崎 了教＊4，高原 健太郎＊5，
滝口 直美＊6 ，関口 光明＊7，南海 浩一＊8，斎藤 恵美＊8，佐藤 秀昭＊9，大澤 昂志＊10，山口 卓男＊10，
伊藤 浩介＊11，川上 純司＊12，小比賀 聡＊10, ＃，井上 貴雄＊2, ＃（受付：令和 5 年 2 月 27 日，受理：令和 5 年 7 月 3 日）

Comparison of Analysis Data of Model Oligonucleotide Therapeutics Obtained with Different
Types of Liquid Chromatograph-Mass Spectrometers
Kenji HIROSE ＊1, †, Tokuyuki YOSHIDA ＊2, †, Maki TERASAKI ＊1, Hiroshi SEZAKI ＊3,Kaoru KARASAWA ＊4, Noriyuki IWASAKI ＊4, Kentaro TAKAHARA ＊5, Naomi TAKIGUCHI ＊6,Mitsuaki SEKIGUCHI ＊7, Hirokazu NANKAI ＊8, Emi SAITO ＊8, Hideaki SATO ＊9,Takashi OSAWA ＊10, Takao YAMAGUCHI ＊10, Kosuke ITO ＊11, Junji KAWAKAMI ＊12,
Satoshi OBIKA ＊10, # and Takao INOUE ＊2, #Summary
In evaluating the quality of oligonucleotide therapeutics, it is necessary to pay close attention to the presence ofoligonucleotide impurities. As an analysis method, LC / MS, combining liquid chromatography (LC) and mass
spectrometry (MS), is generally used for quality assessment. However, since the physicochemical properties of active
pharmaceutical ingredient (API)-derived impurities are likely to be similar to those of the API, there are technicallimitations on their separation and purification. Therefore, it is important to be aware of such limitations and tounderstand the capabilities of the analytical techniques used in evaluating the quality of oligonucleotide therapeutics withdue consideration of the characteristics of the oligonucleotides and their manufacturing processes.
In order to achieve this, we analyzed model oligonucleotides using several different types of commercial liquid
chromatograph-mass spectrometers (LC-MS). We investigated the LC separation of impurities from the parent
oligonucleotide, the relative quantification of impurities, the characterization of the parent oligonucleotide, and theidentification of impurities. In the LC separation of the impurities from the parent oligonucleotide, none of the LC-MS
analyses achieved complete separation (Rs > 1.5) of all the major impurities. However, almost complete separations wereobserved between the parent oligonucleotide and three or more nucleotide-deleted oligonucleotide impurities. The relative
quantification of impurities was not consistent among the LC-MS instruments tested here, and inconsistent ion
suppressions or enhancements were observed. For the characterization of the parent oligonucleotide, the accuracy of
deconvoluted mass was demonstrated to be within 3 ppm for all LC-MS analyses, and MS/MS sequence analysis showed
100% coverage in more than half of the cases. For the structural estimation of impurities, the mass accuracy was within 2
ppm for all LC-MS analyses when impurities were spiked at 0.1% or more, and the MS/MS sequence coverage was 76%
or more when the spiked amounts of impurities were 1% or more. These results indicate that generally used LC/MS
methods could provide reliable information for estimating the composition of impurities present at a level of 1% or more.

Key words
Oligonucleotide therapeutics, Quality evaluation, Impurities, Liquid chromatography, Mass spectrometry
Pharmaceutical and Medical Device Regulatory ScienceVol. 54 No. 5（2023）

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