WO2017115652A1 - Procédé de séparation et analyse - Google Patents

Procédé de séparation et analyse Download PDF

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Publication number
WO2017115652A1
WO2017115652A1 PCT/JP2016/087202 JP2016087202W WO2017115652A1 WO 2017115652 A1 WO2017115652 A1 WO 2017115652A1 JP 2016087202 W JP2016087202 W JP 2016087202W WO 2017115652 A1 WO2017115652 A1 WO 2017115652A1
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WO
WIPO (PCT)
Prior art keywords
column
eluent
synthetic oligonucleotide
separation
ion
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PCT/JP2016/087202
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English (en)
Japanese (ja)
Inventor
謙 小木戸
青木　裕史
潤治 貴家
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昭和電工株式会社
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Priority to JP2017558924A priority Critical patent/JPWO2017115652A1/ja
Publication of WO2017115652A1 publication Critical patent/WO2017115652A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/34—Regenerating or reactivating
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a method for advanced separation analysis of synthetic oligonucleotides.
  • Synthetic oligonucleotides are used in various molecular biological experiments such as polymerase chain reaction primers, diagnostic probes, single nucleotide polymorphism (SNP) detection, and DNA sequencing. Synthetic oligonucleotides are synthesized primarily by a continuous reaction using phosphoramidite nucleic acid monomers on a solid support.
  • RNAi RNA interference
  • Various factors such as binding strength, target specificity, serum stability, nuclease resistance, and cell permeability need to be considered for stable transport of synthetic oligonucleotides in the body.
  • techniques for improving these include skeletal modifications such as modification of phosphorothioate and methylphosphonate, use of locked nucleic acid (LNA), and the like. While these skeletal modifications impart nuclease resistance, they can exhibit non-sequence-specific effects, reduced permeation performance, and even cytotoxicity.
  • Oligonucleotide synthesis methods are optimized to the level that does not require purification when used for PCR primers, but more sophisticated purification and analysis are required for application to pharmaceuticals.
  • long-chain DNA and modified / backbone variants are prone to unreacted coupling and side reactions during synthesis.
  • a synthetic oligonucleotide reaction mixture contains mainly incomplete oligonucleotides or incomplete oligonucleotides.
  • nx fragment a fragment in which a nucleic acid is deleted from a target oligonucleotide
  • n is the number of target nucleotides (nucleic acids)
  • x is a number deleted
  • n, x are positive integers
  • n> x For example, n-1, n-2, etc.
  • n + 1 fragments incorporated in duplicate are included as impurities.
  • incomplete or inaccurately deprotected fragments and isomers having partial chirality are also included (Patent Document 1).
  • gel electrophoresis, gel filtration chromatography, reverse phase chromatography, and ion exchange chromatography are selected as methods for separating highly synthetic oligonucleotides. Chromatography is excellent in operability, and in particular, reverse phase chromatography that does not require desalting is widely used.
  • reverse-phase chromatography of synthetic oligonucleotides an ion-pairing reagent having a positive dissociation group and a hydrophobic functional group in the molecule is generally used (Patent Document 1).
  • the ion-pairing reagent forms a neutral ion pair with the negatively dissociated oligonucleotide, increasing the retention in the reverse-phase filler, and the difference in hydrophobicity between long-chain oligonucleotides and modified / backbone variants In many cases, the separation analysis of oligonucleotides and impurities of interest is small.
  • Non-Patent Document 1 and Non-Patent Document 2 describe a method by reverse phase chromatography using Hydroshare C18 manufactured by YMC.
  • Hydrosphere C18 is a particulate filler in which octadecyl groups are bonded to a silica substrate having a diameter of 12 nm and a diameter of 3 ⁇ m.
  • an ion pair reagent such as triethylamine or dibutylamine
  • Non-Patent Document 3 describes a method by reverse phase chromatography using XBridge OST C18 manufactured by WATERS.
  • XBridge OST C18 is a particle type filler in which octadecyl groups are bonded to an ethylene-crosslinked hybrid silica base material having a pore diameter of 130 mm and a diameter of 2.5 ⁇ m.
  • triethylamine as an ion-pairing reagent to acetic acid or HFIP as an eluent, it is possible to highly separate 10 to 60-mer synthetic oligonucleotides.
  • An object of the present invention is to provide a technique for highly separating and analyzing impurities such as incomplete length oligonucleotides contained in an oligonucleotide reaction mixture in order to apply synthetic oligonucleotides to pharmaceuticals.
  • the present inventors have highly separated a 31-mer or more synthetic oligonucleotide by reverse phase chromatography using an ion-pairing reagent containing a dibutylammonium salt.
  • the inventors have found that analysis is possible and have arrived at the present invention.
  • the present invention is as follows.
  • the reverse phase chromatography is column chromatography, and the separation and analysis method is performed by equilibrating with a first eluent containing the ion pair reagent and water or the ion pair reagent, water and a water-soluble organic solvent in the column.
  • a method for separating and analyzing synthetic oligonucleotides which can be separated and analyzed to a high degree by reverse phase chromatography even for long-chain synthetic oligonucleotides of 31-mer or more.
  • FIG. 2 is a chromatogram of an ultraviolet detection method (wavelength 254 nm) measured using the eluent for reverse phase chromatography of Example 1.
  • FIG. 2 is a chromatogram of an ultraviolet detection method (wavelength 254 nm) measured using the eluent for reverse phase chromatography of Example 2.
  • FIG. 2 is a chromatogram of an ultraviolet detection method (wavelength 254 nm) measured using the eluent for reverse phase chromatography of Comparative Example 1.
  • 6 is a chromatogram of an ultraviolet detection method (wavelength 254 nm) measured using the eluent for reverse phase chromatography of Comparative Example 2.
  • 6 is a chromatogram of an ultraviolet detection method (wavelength 254 nm) measured using the eluent for reverse phase chromatography of Comparative Example 3.
  • the method for separating and analyzing a synthetic oligonucleotide according to the present invention is a method for highly separating and analyzing impurities contained in a synthetic process of a 31-mer or more synthetic oligonucleotide by reverse phase chromatography using an ion-pairing reagent containing a dibutylammonium salt. It is a technique to do.
  • the “synthetic oligonucleotide” is generally synthesized by continuously reacting a nucleic acid monomer on a solid phase carrier, and a nucleic acid oligomer synthesized by a phosphoramidite method is preferable.
  • the target chain length of the synthetic oligonucleotide is 10 to 110 mer, preferably 10 to 70 mer, more preferably 31 to 62 mer.
  • the “ion pair reagent” is a reagent having a hydrophobic functional group that forms an ion pair with an ionic target object, and increases the retention of the formed target object in the hydrophobic column and improves the separation.
  • An ion-pairing agent having a positive dissociation group and a hydrophobic functional group can be used for nucleotide (nucleic acid) separation.
  • Typical ion pair reagents include trialkylammonium salts of organic or inorganic acids, for example, triethylammonium acetate described in Non-Patent Document 2 above.
  • “Impurity contained in the synthesis process” means an impurity having a molecular weight different from that of the target synthetic oligonucleotide, for example, an incomplete length oligonucleotide (oligonucleic acid) often referred to as an nx fragment (n is the target nucleotide (nucleic acid ) Number, x is a missing number, n, x is a positive integer, n> x, for example n-1, n-2, etc.), n + 1 fragment incorporated in duplicate, acetyl group, isobutyryl group, cyanoethyl Insufficient backbone modifications such as oligonucleotides with insufficient deprotection of functional groups that protect nucleic acid monomers such as groups and methyl groups, incomplete oligonucleotides such as depurines and depyrimidines, and phosphodiester impurities in phosphorothioates These include oligonucleotides and isomers having
  • the method for separating and analyzing a synthetic oligonucleotide is as follows. That is, in column reverse phase chromatography, an eluent containing an ion-pairing reagent containing dibutylammonium salt is passed in advance through a column containing a packing with a hydrophobic functional group immobilized thereon, and equilibrated. The solution in which the synthetic oligonucleotide is dissolved is passed through to bind the synthetic oligonucleotide into the column.
  • the impurities contained in the synthetic oligonucleotide synthesis process and the target synthetic oligonucleotide are separated and eluted by a gradient that gradually increases the concentration of the organic solvent in the eluent to be passed, that is, a so-called gradient. Pass the eluent that was passed again during equilibration to regenerate the column.
  • a filler composed of silica or a polymer having a hydrophobic functional group fixed can be used.
  • Silica is particularly preferable as the filler.
  • the shape is preferably non-porous particles, and the particle size is preferably 3 ⁇ m or less, more preferably 2 ⁇ m or less.
  • the hydrophobic functional group to be fixed is preferably a butyl group, a phenyl group, an octyl group or an octadecyl group, and particularly preferably an octadecyl group.
  • the eluent of one embodiment of the present invention is not particularly limited except that an ion-pairing reagent containing a dibutylammonium salt is used, and an aqueous eluent containing a salt of dibutylamine and an acid and a C1 to 3 alcohol or nitrile. It is preferable to combine with an organic solvent of the system.
  • the acid is preferably carbonic acid, acetic acid, formic acid, trifluoroacetic acid or propionic acid. A combination of an aqueous eluent containing dibutylamine and acetic acid and acetonitrile is most preferred.
  • the salt concentration of the aqueous eluent is not particularly limited, but is preferably 1 to 100 mM, more preferably 5 to 50 mM, and particularly preferably 10 to 20 mM. Further, the pH of the aqueous eluent is not limited as long as it can be bound to and eluted from the column, preferably 6 to 8, and more preferably 6.5 to 7.5.
  • an ion pair reagent containing a dibutylammonium salt and an alkylammonium salt other than the dibutylammonium salt for example, a triethylammonium salt
  • the ratio of the dibutylammonium salt to the ion-pairing reagent is preferably 30% mol or more, more preferably 40%, and even more preferably 50% or more.
  • the ion pair reagent is a dibutylammonium salt.
  • a commercially available reverse phase chromatography apparatus can be used, and examples include analytical apparatuses such as Shimadzu Corporation Prominence and Waters Acquity.
  • a commercially available analytical or separation reversed phase column can be used.
  • MICRA registered trademark
  • NPS ODS-1 EPROGEN
  • Examples include a column for analysis or fractionation such as a particle size of 1.5 ⁇ m, 33 ⁇ 4.6 mm ID) Waters X Bridge OST C18 (particle size 1.7 ⁇ m, pore size 135 mm, 50 ⁇ 4.6 mm ID).
  • Examples of the detection method used in the method for separating and analyzing a synthetic oligonucleotide according to one embodiment of the present invention include a detector using ultraviolet / visible absorption such as a synthetic oligonucleotide, a detector using a mass spectrum of a synthetic oligonucleotide, and the like. Can be mentioned. In the case of an ultraviolet / visible absorption detector, the detection wavelength is, for example, 254 nm. By such a method, the synthetic oligonucleotide can be highly separated and analyzed.
  • Acetonitrile was added to 10 mM dibutylammonium acetate aqueous solution (pH 7.0), and 9.6 mM dibutylammonium acetate aqueous solution containing acetonitrile having a final concentration of 4.5 v / v% was prepared as the first eluent.
  • the column was equilibrated by passing 10 mL or more. The mobile phase flow rate was 1.0 mL / min.
  • NPS ODS-1 is a column containing a non-porous silica particle filler to which octadecyl groups are bonded.
  • Synthetic oligonucleotide binding step 10 uL of the prepared analytical sample was injected into the column equilibrated in (1), and the synthetic oligonucleotide was bound to the column using the first eluent.
  • the mobile phase flow rate was 1 mL / min.
  • the mobile phase is a first stage from the first eluent, and the linear gradient has a gradient that increases the final concentration of acetonitrile to 18 v / v% in the first 7.5 minutes.
  • the linear gradient has a gradient that increases the final concentration of acetonitrile to 18 v / v% in the first 7.5 minutes.
  • a two-stage gradient including a second-stage gradient with a gradient that increased the final concentration of acetonitrile to 31.5 v / v% in 32.5 minutes.
  • Synthetic oligonucleotides and impurities bound to the column were separated and eluted by a linear gradient gradient that gradually increased the acetonitrile concentration.
  • the detector is an ultraviolet / visible (uv / vis) detector, and the detection wavelength is 254 nm.
  • the detector was implemented in the same manner as in Example 1.
  • the peak of the synthetic oligonucleotide 40-mer at the retention time of 24 minutes the peak of the synthetic oligonucleotide 62-mer at the retention time of 29 minutes, and the synthetic oligonucleotide 80-mer at the retention time of 32 minutes.
  • a peak of the synthetic oligonucleotide 100-mer was observed at a retention time of 35 minutes, and a peak of the synthetic oligonucleotide 110-mer was observed at a retention time of 37 minutes.
  • Example 1 Separation and analysis of synthetic oligonucleotide using triethylammonium salt as ion-pairing reagent. Except that the eluent used in the column equilibration step (1) of Example 1 was 10 mM triethylammonium acetate (pH 7.0). Performed as in Example 1. A synthetic oligonucleotide peak was observed at a retention time of 8.5 minutes. However, the retention on the column is weak, and the impurity peak detected before the main peak as seen in Example 1 cannot be separated, and the separation of the synthetic oligonucleotide is insufficient. It was impossible. The results are shown in FIG.
  • Example 2 Separation and analysis of synthetic oligonucleotide using triethylammonium salt as ion-pairing reagent
  • the eluent used in the column equilibration step (1) of Example 1 was 100 mM triethylammonium acetate (pH 7.0).
  • the linear gradient of the binding step (3) was carried out in the same manner as in Example 1 except that the linear gradient was increased to 30% in 30 minutes to increase the final concentration of acetonitrile to 31.5 v / v%.
  • a synthetic oligonucleotide peak was observed at a retention time of 15 minutes. However, it could not be separated from the impurity peak detected before the main peak as seen in Example 1, the synthetic oligonucleotide was insufficiently separated, and high-level separation analysis was impossible. The results are shown in FIG.
  • Example 3 Separation and analysis of synthetic oligonucleotide using triethylammonium salt as ion-pairing reagent. Except that the eluent used in the column equilibration step (1) of Example 2 was 10 mM triethylammonium acetate (pH 7.0). Performed as in Example 2. A synthetic oligonucleotide peak was observed at a retention time of 8.5 minutes. However, the retention on the column was weak, and the five main peaks as seen in Example 2 could not be separated and eluted together. Therefore, the separation of the synthetic oligonucleotide was insufficient and advanced separation analysis was impossible. The results are shown in FIG.

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  • Chemical Kinetics & Catalysis (AREA)
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Abstract

L'invention fournit une technique destinée à séparer et analyser à un niveau élevé des impuretés telles qu'un oligonucléotide de longueur incomplète, ou similaire, contenu dans un mélange réactif d'oligonucléotide, dans le but d'une application d'un oligonucléotide synthétique à un médicament. Plus précisément, l'invention concerne un procédé selon lequel un oligonucléotide synthétique d'un 31 mère ou plus est soumis à une séparation et à une analyse par chromatographie en phases inversées mettant en œuvre un réactif d'appariement d'ions. Ce réactif d'appariement d'ions contient un sel de dialkylammonium.
PCT/JP2016/087202 2015-12-28 2016-12-14 Procédé de séparation et analyse WO2017115652A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7459814B2 (ja) 2021-02-03 2024-04-02 株式会社島津製作所 液体クロマトグラフ質量分析装置を用いた一次代謝物の分析方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003532088A (ja) * 2000-05-01 2003-10-28 トランスジエノミツク・インコーポレーテツド ポリマー分離媒体でのポリヌクレオチド分離

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003532088A (ja) * 2000-05-01 2003-10-28 トランスジエノミツク・インコーポレーテツド ポリマー分離媒体でのポリヌクレオチド分離

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GUAN, F. ET AL.: "Use of an ion-pairing reagent for high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry determination of anionic anticoagulant rodenticides in body fluids", JOURNAL OF CHROMATOGRAPHY B, vol. 731, 1999, pages 155 - 165, XP004177443, DOI: doi:10.1016/S0378-4347(99)00126-7 *
SASAKI, T. ET AL.: "High-performance ion-pair chromatographic behaviour of conjugated bile acids with di-n-butylamine acetate", JOURNAL OF CHROMATOGRAPHY A, vol. 888, 2000, pages 93 - 102, XP004209865, DOI: doi:10.1016/S0021-9673(00)00509-4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7459814B2 (ja) 2021-02-03 2024-04-02 株式会社島津製作所 液体クロマトグラフ質量分析装置を用いた一次代謝物の分析方法

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