Analysis and Purification of Synthetic Nucleic Acids Using HPLC

Nanda D. Sinha1, Kyeong Eun Jung2

1 Independent Consultant, Boxborough, Massachusetts, 2 ST Pharm Co. Ltd, Gyeonggi‐do
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 10.5
DOI:  10.1002/0471142700.nc1005s61
Online Posting Date:  June, 2015
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Abstract

Chromatographic methods have been essential tools for analysis and purification of synthetic oligonucleotides since the 1970s. Significant developments in terms of instruments and stationary phases (media) have been made during the past several decades; among the latest are sub‐micron to micron particles for the media, as well as ultra performance liquid chromatography (UPLC). Micron and sub‐micron particles have increased product resolution. Applications of recently developed methods such as IP‐RP‐HPLC and LC‐MS have been discussed for analysis, along with use of various methods for purification. Utilization of UPLC has decreased analysis time, increasing the throughput for analysis. Commonly used methods for analysis and purification of synthetic oligonucleotides have been described in this unit. © 2015 by John Wiley & Sons, Inc.

Keywords: synthetic oligonucleotides; aptamers; siRNA; antisense; HPLC; anion‐exchange; reversed‐phase; ion‐paired HPLC; UPLC; LC‐MS chromatography

     
 
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Table of Contents

  • Introduction
  • Chromatographic Methods
  • Chromatographic Analyses
  • Basic Protocol 1: Considerations for Sample Preparation
  • Anion‐Exchange Chromatography
  • Basic Protocol 2: Analysis of DMTr‐on or DMTr‐off Oligonucleotides by Anion‐Exchange HPLC
  • Basic Protocol 3: Purification of DMTr‐on Oligonucleotides Using Anion‐Exchange Chromatography (from Hundreds of Milligrams to Hundreds of Grams)
  • Alternate Protocol 1: Purification of DMTr‐off Crude Oligonucleotides Using Anion‐Exchange HPLC
  • Support Protocol 1: Post–Anion‐Exchange Purification
  • Reversed‐Phase Chromatography
  • Basic Protocol 4: Analysis by Reversed‐Phase HPLC
  • Alternate Protocol 2: Analysis by Ion‐Paired Reversed‐Phase HPLC (IP‐RP‐HPLC)
  • Alternate Protocol 3: Analysis by UPLC
  • Basic Protocol 5: RP‐HPLC for Purification
  • Basic Protocol 6: LC‐MS Chromatography
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Considerations for Sample Preparation

  Materials
  • Crude oligonucleotide solution with DMTr‐on or DMTr‐off and appropriate elution buffer or solvent
  • Elution buffer or solvent:
    • For oligodeoxynucleotides:
    • Mobile phase A = 25 mM sodium hydroxide solution
    • Mobile phase B = 2 M sodium chloride solution containing 25 mM sodium hydroxide
    • For oligoribonucleotides and chimeric oligonucleotides (also see recipefor mobile phase solutions A and B for reversed‐phase and ion‐paired RP columns):
    • Mobile phase A = 10 mM sodium phosphate/10% (v/v) acetonitrile or 10 mM sodium perchlorate/10% acetonitrile
    • Mobile phase B = 10 mM sodium phosphate/10% acetonitrile containing 1 M sodium chloride, 1 M sodium bromide, or 0.4 M sodium perchlorate
  • HPLC‐grade H 2O
  • 80% (v/v) acetic acid or 5% to 10% (w/v) dichloroacetic acid
  • Analytical instruments:
    • Any one of these instruments from Waters HPLC (Alliance, Acquity, UPLC), Agilent (1290 Infinity LC Systems), Thermo Fisher (Dionex UltiMate 3000 series), Shimadzu (i‐Series, Prominence‐1, and Nexera‐I series) can be utilized for analysis of oligonucleotides; additional information regarding analytical HPLCs can be obtained using the following URLs:http://www.waters.com/waters/en_US/Alliance‐HPLC‐System/nav.htm?cid=534293http://www.dionex.com/en‐us/products/liquid‐chromatography/lc‐systems/lp‐72448.htmlhttp://www.chem.agilent.com/en‐US/products‐services/Instruments‐Systems/Liquid‐Chromatography/1260‐Infinity‐Quaternary‐LC‐System/Pages/default.aspxhttp://shimadzu.com/an/hplc/index.html
  • Analytical columns:
    • Two types of anion‐exchange media—weak anion exchange (functionalized with tertiary amine such as DEAE, diethylaminoethyl‐linked) and strong anion exchange (functionalized with alkyl quaternary ammonium, mostly trimethylammonium)—are used; weak anion exchange columns include Aquapore (Perkin Elmer) and Gen Pak Fax (Waters Corporation); strong anion‐exchange columns include:
    • DNAPAC Pa‐100/DNAPAC Pa‐200 (Thermo Fisher Dionex), PL‐SAX columns (Agilent), Source‐Q (GE Life Sciences), TSK‐gel SuperQ‐5PW (Tosoh Biosciences), which can be used for analysis of oligonucleotides. The most commonly used anion‐exchange columns are from Dionex's DNA‐PAC‐100/200 and Waters’ Gen‐Pac series; additional information regarding analytical HPLCs can be obtained using the following URLs:http://www.chem.agilent.com/Library/applications/5990‐8297EN.pdfhttp://www.dionex.com/en‐us/products/columns/bio/nucleic‐acid/dnapac‐pa100/lp‐73370.htmlhttp://www.dionex.com/en‐us/products/columns/bio/nucleic‐acid/dnapac‐pa200/lp‐73371.htmlhttps://www.gelifesciences.com/gehcls_images/GELS/RelatedContent/Files/1314807262343/litdoc18106043AF_20110831215748.pdfhttp://www.perkinelmer.com/Catalog/Product/ID/07120040http://www.separations.us.tosohbioscience.com/Products/ProcessMedia/ByMode/IEC/TSKgelSuperQ5PW.htmhttp://www.waters.com/webassets/cms/support/docs/WAT015493.pdf

Basic Protocol 2: Analysis of DMTr‐on or DMTr‐off Oligonucleotides by Anion‐Exchange HPLC

  Materials
  • Crude oligonucleotide solution with DMTr‐on or DMTr‐off and appropriate elution buffer or solvent
  • Elution buffer or solvent:
    • For oligodeoxynucleotides:
    • Mobile phase A = 25 mM sodium hydroxide solution
    • Mobile phase B = 2 M sodium chloride solution containing 25 mM sodium hydroxide
    • For oligoribonucleotides and chimeric oligonucleotides:
    • Mobile phase A = 10 mM sodium phosphate/10% (v/v) acetonitrile,
    • Mobile phase B = 10 mM sodium phosphate/10% acetonitrile containing 1 M sodium chloride, 1 M sodium bromide, or 0.4 M sodium perchlorate
  • Chromatography instruments:
    • HPLCs/FPLCs are widely used for method development and purification of oligonucleotides, based on analytical results. The common HPLC instruments for large‐scale purifications are ÄKTA Pilot/Bio Process (GE Life Sciences), Bio Process Chromatography (AsahiKasei/Technikrom), and Kilo Prep (Biotage, a Division of Dyax); more information on these instruments can be obtained using the following URLs:http://www.bioprocessonline.com/doc/chromatography‐systems‐0003https://www.ak‐bio.com/products/chromatography‐equipment/bioprocess‐chromatography‐systems/key‐features/http://www.biotage.com/product‐page/biotage‐flash‐400
  • Anion‐exchange media for large‐scale purification:
    • Q‐Sepharose FF/HP (loading capacity 700 to 1000 OD 260 or 30 to 35 mg/mL)
    • Source‐Q or TSK‐gel Super‐Q PW5 (loading capacity 375 to 500 OD 260 or 15 mg to 20 mg/mL)
    • Column: Pre‐packed (1, 5, or 10 mL from GE Life Sciences for method development) or custom‐packed (47 × 300 mm, 50.4 × 600 mm, or other sizes) column can be used depending on the amount to be purified. Additional information is available through the following URLs:https://www.gelifesciences.com/gehcls_images/GELS/RelatedContent/Files/1314729545976/litdoc18116179_20130721222811.pdfhttp://www.separations.asia.tosohbioscience.com/NR/rdonlyres/C0A5135E‐C1FC‐48ED‐8490‐71A22048183E/0/TP138.pdf

Basic Protocol 3: Purification of DMTr‐on Oligonucleotides Using Anion‐Exchange Chromatography (from Hundreds of Milligrams to Hundreds of Grams)

  Materials
  • Crude oligonucleotide samples (with or without DMTr‐group at 5′‐end)
  • Triethylamine
  • Eluent buffers for analytical applications (also see reciperecipes for mobile phase solutions A and B for reversed‐phase and ion‐paired RP columns):
    • Mobile phase A: 0.1 M triethylammonium acetate (TEAA), pH 7 (see recipe)
    • Mobile phase B: 100% acetonitrile
  • Eluent buffers for purification:
    • Mobile phase A: 100 mM ammonium acetate/200 mM sodium acetate
    • Mobile phase B: 100 mM ammonium acetate/200 mM sodium acetate in methanol
  • HPLC instrumentation (instruments same as described in anion exchange analysis section), capable of analyzing oligonucleotide with following features and specifications:
    • Injector: autosampler (preferred) or manual syringe
    • Pumping system: ternary (preferred) or binary, 0.1 to 5 mL/min
    • Detector: UV/fluorescence (preferred) or UV/VIS variable between 190 and 600 nm
    • Data: integrating data system (preferred) or chart recorder
    • Gradient system: displays and stores for redisplay and reformatting (preferred) or programmable
  • Reversed‐phase columns:
    • Aquapore RP‐300 (4.6 × 220 mm; http://www.perkinelmer.com/catalog/family/id/brownlee; aquapore octyl c8)
    • Spheri‐5RP‐18 (http://www.perkinelmer.com/catalog/product/id/07110013)
    • Nova‐Pak C‐18 (http://www.waters.com/waters/partDetail.htm?partNumber=WAT086344)
    • Xterra (RP 18, 5 μm, 4.6 × 150 mm; http://www.waters.com/waters/en_US/XTerra‐Columns/nav.htm?cid=513769&semCBUXterraColumnsNonBrandUS&gclid=CjwKEAiAxsymBRCegqiLzI7Q1S8SJADOgQrzhGx‐GqDjWlsR8GWjY‐ob_hj4IQxtHI6OOXxQXR‐RNB°CXHXw_wcB)
    • XBridge OST C‐18 (http://www.waters.com/waters/en_US/XBridge‐Columns/nav.htm?cid=513767)
    • PRP‐1/PRP‐3 (polymeric reversed‐phase HPLC columns; http://www.teknokroma.es/en/Productos/hplc‐chromatography/5/reverse‐phase‐polymeric‐hplc‐columns/43/674/hamilton‐prp‐1.aspx)
    • Resource RPC (6.4 × 30 mm/Source RPC 4.6 × 100 mm; https://www.gelifesciences.com/gehcls_images/GELS/Related Content/Files/1314807262343/litdoc71717400AH_20110831204915.pdf
    • Custom packed columns (47 × 300 mm, 520 mL; 50.4 × 600 mm, ∼1200 mL; GE Life Sciences)
    • Radial compressed column of various dimensions (Waters Corporation)

Alternate Protocol 1: Purification of DMTr‐off Crude Oligonucleotides Using Anion‐Exchange HPLC

  Materials
  • Crude 5′‐DMTr‐on oligonucleotide samples
  • Triethylamine
  • Sodium acetate or sodium chloride
  • Ethanol
  • Elution buffers (also see recipe for mobile phase solutions A and B for reversed‐phase and ion‐paired RP columns):
    • Mobile phase A: 0.1 M TEAA solution pH 7
    • Mobile phase B: Acetonitrile containing 5% solution A
    • Mobile phase A: 0.2 M sodium acetate or 0.2 M NaCl solution pH 7.2
    • Mobile phase B: Methanol
  • Ethanol, cold
  • 10 mM sodium acetate, pH 3.0 (adjust pH with glacial acetic acid)
  • 3 M sodium acetate, pH 7.2 ( appendix 2A)
  • Rotary evaporator
  • Chromatography instrument: ÄKTA Pilot/AKTA Process, Bi°Cad (Thermo Fisher/formerly PE Biosystems) Technikrom, or Kilo Prep KP100 (Biotage, division of Dyax): these Bio‐Process instruments should have a detector system, pH, and conductivity meters and pressure gauge, with fraction collector (see protocol 3 for URLs related to these instruments)
  • Media (Sephasil and BondaPac/NovaPac are silica‐based purification media and only suitable for pH ∼7, whereas PS‐DV‐based media can be used at pH 1 to14; no sample preparation is necessary for columns filled with polystyrene (PS/DV)–based materials such as Oligo R3, PRP‐1/PRP‐3, or Source 15/30 RPC; sample preparation is essential for silica‐based column such as Bonda‐Pac C‐18 or Sephasil columns, as silica tends to dissolve at high pH (ammonia solution):
    • Source 15 RPC (PS‐DV, 15 μm), Source 30 RPC (PS‐DV, 30 μm), Sephasil Protein C18 silica, 12 μm) from GE Life Sciences (http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences‐US/products/AlternativeProductStructure_17488/)
    • Oligo R3 from Thermo Fisher Life Technologies or formerly PE Biosystems (http://www.lifetechnologies.com/order/catalog/product/1133903)
    • PRP‐1 and PRP3 from Hamilton (http://www.teknokroma.es/en/Productos/hplc‐chromatography/5/reverse‐phase‐polymeric‐hplc‐columns/43/674/hamilton‐prp‐1.aspx)
    • NovaPac or BondaPac HC18 HA from Waters (http://www.waters.com/webassets/cms/library/docs/lcPrep.pdf)
  • Lyophilizer
  • Additional reagents and equipment for analytical anion‐exchange ( protocol 2) and reversed‐phase HPLC ( protocol 6)

Support Protocol 1: Post–Anion‐Exchange Purification

  Materials
  • Crude 5′‐DMTr‐on or DMT‐off oligonucleotide samples
  • Appropriate mobile phase solutions for anion‐exchange or reversed‐phase chromatography (see reciperecipes)
  • Methanol
  • LC‐MC instruments:
    • Agilent: HPLC 1200 Series with mass spectrometer MSD6130
    • Shimadzu: LC‐MS System 2020
    • Thermo Fisher: Dionex HPLC Acclaim with mass spectrometer MSQ
    • Waters Corporation: Acquity UPLC with Premier mass spectrometer Q‐TOF
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Figures

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Literature Cited

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