Presentation<\/h2>\n\n\n\nTitle<\/strong><\/td>Development of Chromatographic Separation Methods for the Purification of Single-Guide RNA Therapeutics<\/td><\/tr> Affiliations<\/strong><\/strong><\/td>R&D DEPT., YMC CO., LTD.,
Health & Medical Business Division, NIPPON SHOKUBAI CO., LTD.<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<\/div>\n\n\n\nBackground<\/h2>\n\n\n\n
Demand for highly pure single-guide RNA (sgRNA) is increasing as its use in clinical applications grows. Ion-pair reversed-phase liquid chromatography (IP-RP-LC) using alkylamine ion-pair reagents and hexafluoroisopropanol (HFIP) has been widely used for high-resolution separation of long (approximately 100-mer) oligonucleotides, enabling the removal of closely related impurities from synthetic sgRNA. This method is, however, hardly applicable to industry-scale purification of long oligonucleotides due to the high cost and environmental burden of HFIP; thus, an alternative method is required.<\/p>\n\n\n\n
<\/div>\n\n\n\nResearch Contents<\/h2>\n\n\n\n
This study aimed to develop an IP-RP-LC method for purifying phosphorothioate (PS)-modified sgRNA without using HFIP or other expensive additives. Previous research indicates that the addition of sodium phosphate buffer to alkylamine mobile phase solutions enhances the separation of long oligonucleotides. Here, we hypothesized that the following two factors are involved in this enhancement: 1) phosphate ions, which may prevent the adsorption of oligonucleotides in the LC system, including the column; 2) sodium ions in the mobile phase, which may stabilize the secondary structure of oligonucleotides. Our data show that the enhanced separation of oligonucleotides by the addition of phosphate buffer is due to sodium ions, rather than to phosphate ions. We observed that a similar enhancement was also caused by another potassium ion. By applying these findings, we established a novel method for separating 100-mer sgRNA from 5\u2019 truncated products and phosphodiester (PO) impurities without using HFIP. This method offers a cost-effective and environmentally favorable solution for the industrial manufacturing of sgRNA drug substances.<\/p>\n\n\n\n
<\/div>\n\n\n\n![\"\"](\"https:\/\/www.lifescience.shokubai.co.jp\/home\/wp-content\/uploads\/2026\/08\/\u99ac\u5834\u3055\u30932_kz-1-748x1024.jpg\")
Takeshi Baba, Health & Medical Business Division, NIPPON SHOKUBAI CO., LTD.<\/figcaption><\/figure>\n<\/div>\n\n\n<\/div>\n\n\n\nMeeting Information<\/h2>\n\n\n\nName<\/td> \u00a0The 11th Annual Meeting of the Nucleic Acids Therapeutics Society of Japan<\/td><\/tr> Meeting<\/td> July 7th<\/sup> (Tue) \u2013 July 9th<\/sup> (Thu), 2026<\/td><\/tr>Venue<\/td> Dejima Messe Nagasaki<\/td><\/tr> Official website<\/td> \u00a0https:\/\/www.natsj.jp\/2026\/natsj11<\/a><\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<\/p>\n","protected":false},"featured_media":0,"template":"","news_category":[16],"class_list":["post-2384","news","type-news","status-publish","hentry","news_category-technical-information-en"],"acf":{"blank_url":"","pdf_url":null,"select_link":"single_link"},"aioseo_notices":[],"aioseo_head":"\n\t\t\n\t\n\t\n\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t
Title<\/strong><\/td>| Development of Chromatographic Separation Methods for the Purification of Single-Guide RNA Therapeutics<\/td><\/tr> | Affiliations<\/strong><\/strong><\/td> | R&D DEPT., YMC CO., LTD., | Health & Medical Business Division, NIPPON SHOKUBAI CO., LTD.<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n <\/div>\n\n\n\n Background<\/h2>\n\n\n\nDemand for highly pure single-guide RNA (sgRNA) is increasing as its use in clinical applications grows. Ion-pair reversed-phase liquid chromatography (IP-RP-LC) using alkylamine ion-pair reagents and hexafluoroisopropanol (HFIP) has been widely used for high-resolution separation of long (approximately 100-mer) oligonucleotides, enabling the removal of closely related impurities from synthetic sgRNA. This method is, however, hardly applicable to industry-scale purification of long oligonucleotides due to the high cost and environmental burden of HFIP; thus, an alternative method is required.<\/p>\n\n\n\n <\/div>\n\n\n\n Research Contents<\/h2>\n\n\n\nThis study aimed to develop an IP-RP-LC method for purifying phosphorothioate (PS)-modified sgRNA without using HFIP or other expensive additives. Previous research indicates that the addition of sodium phosphate buffer to alkylamine mobile phase solutions enhances the separation of long oligonucleotides. Here, we hypothesized that the following two factors are involved in this enhancement: 1) phosphate ions, which may prevent the adsorption of oligonucleotides in the LC system, including the column; 2) sodium ions in the mobile phase, which may stabilize the secondary structure of oligonucleotides. Our data show that the enhanced separation of oligonucleotides by the addition of phosphate buffer is due to sodium ions, rather than to phosphate ions. We observed that a similar enhancement was also caused by another potassium ion. By applying these findings, we established a novel method for separating 100-mer sgRNA from 5\u2019 truncated products and phosphodiester (PO) impurities without using HFIP. This method offers a cost-effective and environmentally favorable solution for the industrial manufacturing of sgRNA drug substances.<\/p>\n\n\n\n <\/div>\n\n\n \n <\/div>\n\n\n\n Meeting Information<\/h2>\n\n\n\n
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