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Simultaneous separation of small interfering RNA and lipids using ion-pair reversed-phase liquid chromatography.

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Title: Simultaneous separation of small interfering RNA and lipids using ion-pair reversed-phase liquid chromatography.
Authors: Li, Li1,2 (AUTHOR), Foley, Joe P.1,2 (AUTHOR) jfoley@drexel.edu, Helmy, Roy1 (AUTHOR)
Source: Journal of Chromatography A. Sep2019, Vol. 1601, p145-154. 10p.
Subjects: Small interfering RNA, Liquid chromatography, Lipids, Cationic lipids, Gradient elution (Chromatography), Lipid analysis
Abstract: • Simultaneous gradient elution separation of siRNA duplexes and lipids via IP-RPLC. • Investigated 5 UHPLC columns and 4 ion-pairing agents. • Best retention conditions achieved with phenyl column and DBAA. • Optimal temperature depends on melting characteristics of siRNA duplex. • Forced-stress testing and analysis of lipid nanoparticle formulation of siRNA. RNA interference offers a novel approach for the development of new therapeutics for targets that are otherwise "undruggable" using traditional modalities. The safety and efficacy of siRNA-based therapy mainly rely on lipid or polymer-based nanocarriers to overcome inherent barriers to a systemic delivery of siRNA. A multicomponent lipid nanoparticle (LNP) system is a promising delivery platform, typically consisting of a cationic lipid, phospholipid, PEG-containing short-chain lipid, and cholesterol. Characterization and chemical analysis of the LNP formulation is important to assure drug product stability, a key consideration for chemistry, manufacturing and control strategy. Here we report an ion-pair reversed phase UHPLC method capable of simultaneously separating both siRNA and functional lipids in LNPs with a minimal retention gap for two classes of biologically essential yet chemically distinct molecules. Key chromatographic parameters critical to the separation are discussed, including the structure of the ion-pair agent, stationary phase chemistry, column temperature and an organic additive. The results showed that the retention time of siRNA is tunable by using various ion-pair reagents. The retention factor of the siRNA exhibited a first order relationship with the number of carbons in the alkyl chain of the ion-pair reagents. In contrast, the type of ion-pair reagent has no significant impact on the separation of phospholipids. Separations using a BEH phenyl column and dibutylammonium acetate as the ion-pair reagent showed satisfactory selectivity for a range of double-stranded siRNAs and phospholipids, key components for lipid nanoparticle formulations. Furthermore, the method was applied to the separation of an experimental LNP formulation, demonstrating good selectivity for siRNA, functional lipids and their potential degradation products. [ABSTRACT FROM AUTHOR]
Copyright of Journal of Chromatography A is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
Database: Engineering Source
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Header DbId: egs
DbLabel: Engineering Source
An: 137432767
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PubType: Academic Journal
PubTypeId: academicJournal
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Items – Name: Title
  Label: Title
  Group: Ti
  Data: Simultaneous separation of small interfering RNA and lipids using ion-pair reversed-phase liquid chromatography.
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  Label: Authors
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  Data: <searchLink fieldCode="AR" term="%22Li%2C+Li%22">Li, Li</searchLink><relatesTo>1,2</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Foley%2C+Joe+P%2E%22">Foley, Joe P.</searchLink><relatesTo>1,2</relatesTo> (AUTHOR)<i> jfoley@drexel.edu</i><br /><searchLink fieldCode="AR" term="%22Helmy%2C+Roy%22">Helmy, Roy</searchLink><relatesTo>1</relatesTo> (AUTHOR)
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  Data: <searchLink fieldCode="JN" term="%22Journal+of+Chromatography+A%22">Journal of Chromatography A</searchLink>. Sep2019, Vol. 1601, p145-154. 10p.
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  Data: <searchLink fieldCode="DE" term="%22Small+interfering+RNA%22">Small interfering RNA</searchLink><br /><searchLink fieldCode="DE" term="%22Liquid+chromatography%22">Liquid chromatography</searchLink><br /><searchLink fieldCode="DE" term="%22Lipids%22">Lipids</searchLink><br /><searchLink fieldCode="DE" term="%22Cationic+lipids%22">Cationic lipids</searchLink><br /><searchLink fieldCode="DE" term="%22Gradient+elution+%28Chromatography%29%22">Gradient elution (Chromatography)</searchLink><br /><searchLink fieldCode="DE" term="%22Lipid+analysis%22">Lipid analysis</searchLink>
– Name: Abstract
  Label: Abstract
  Group: Ab
  Data: • Simultaneous gradient elution separation of siRNA duplexes and lipids via IP-RPLC. • Investigated 5 UHPLC columns and 4 ion-pairing agents. • Best retention conditions achieved with phenyl column and DBAA. • Optimal temperature depends on melting characteristics of siRNA duplex. • Forced-stress testing and analysis of lipid nanoparticle formulation of siRNA. RNA interference offers a novel approach for the development of new therapeutics for targets that are otherwise "undruggable" using traditional modalities. The safety and efficacy of siRNA-based therapy mainly rely on lipid or polymer-based nanocarriers to overcome inherent barriers to a systemic delivery of siRNA. A multicomponent lipid nanoparticle (LNP) system is a promising delivery platform, typically consisting of a cationic lipid, phospholipid, PEG-containing short-chain lipid, and cholesterol. Characterization and chemical analysis of the LNP formulation is important to assure drug product stability, a key consideration for chemistry, manufacturing and control strategy. Here we report an ion-pair reversed phase UHPLC method capable of simultaneously separating both siRNA and functional lipids in LNPs with a minimal retention gap for two classes of biologically essential yet chemically distinct molecules. Key chromatographic parameters critical to the separation are discussed, including the structure of the ion-pair agent, stationary phase chemistry, column temperature and an organic additive. The results showed that the retention time of siRNA is tunable by using various ion-pair reagents. The retention factor of the siRNA exhibited a first order relationship with the number of carbons in the alkyl chain of the ion-pair reagents. In contrast, the type of ion-pair reagent has no significant impact on the separation of phospholipids. Separations using a BEH phenyl column and dibutylammonium acetate as the ion-pair reagent showed satisfactory selectivity for a range of double-stranded siRNAs and phospholipids, key components for lipid nanoparticle formulations. Furthermore, the method was applied to the separation of an experimental LNP formulation, demonstrating good selectivity for siRNA, functional lipids and their potential degradation products. [ABSTRACT FROM AUTHOR]
– Name: AbstractSuppliedCopyright
  Label:
  Group: Ab
  Data: <i>Copyright of Journal of Chromatography A is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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RecordInfo BibRecord:
  BibEntity:
    Identifiers:
      – Type: doi
        Value: 10.1016/j.chroma.2019.04.061
    Languages:
      – Code: eng
        Text: English
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      Pagination:
        PageCount: 10
        StartPage: 145
    Subjects:
      – SubjectFull: Small interfering RNA
        Type: general
      – SubjectFull: Liquid chromatography
        Type: general
      – SubjectFull: Lipids
        Type: general
      – SubjectFull: Cationic lipids
        Type: general
      – SubjectFull: Gradient elution (Chromatography)
        Type: general
      – SubjectFull: Lipid analysis
        Type: general
    Titles:
      – TitleFull: Simultaneous separation of small interfering RNA and lipids using ion-pair reversed-phase liquid chromatography.
        Type: main
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            NameFull: Li, Li
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            NameFull: Foley, Joe P.
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            NameFull: Helmy, Roy
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          Dates:
            – D: 13
              M: 09
              Text: Sep2019
              Type: published
              Y: 2019
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              Value: 1601
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            – TitleFull: Journal of Chromatography A
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