{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,17]],"date-time":"2026-10-17T09:42:27Z","timestamp":1792230147764,"version":"4.4.0"},"reference-count":29,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2026,10,17]],"date-time":"2026-10-17T00:00:00Z","timestamp":1792195200000},"content-version":"vor","delay-in-days":16,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T00:00:00Z","timestamp":1790812800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["J of Separation Science"],"published-print":{"date-parts":[[2026,10]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>Affinity liquid chromatography (aLC) with immobilized oligonucleotides provides high selectivity for nucleic acid targets through on\u2010column hybridization. We describe the preparation and evaluation of prototype 2.1 mm \u00d7 20\u00a0mm affinity columns packed with a 3.5 \u00b5m nonporous polystyrene\/divinylbenzene sorbent covalently modified with streptavidin. Two different affinity columns were prepared by immobilizing biotinylated oligonucleotides on\u2010column via biotin\u2010streptavidin bond: (i) biotinylated 25\u2010nt oligodeoxythymidine, dT25 or (ii) biotinylated 21\u00a0nt TP53 oligonucleotide. dT25 column was used for poly(A) denylated messenger RNA (mRNA) and adenosine oligonucleotides variants analysis. The TP53 column was applied to single\u2010nucleotide polymorphism analysis. Nucleic acid binding was performed in 0.1\u00a0M sodium phosphate buffer (pH 7.4); retained species were eluted with water or 10% acetonitrile solution in water. We demonstrate that dT25 column selectively retains poly(A)\u2010tailed EPO, EGFP, and Cas9 mRNAs, while tailless mRNA is unretained. dT25 column resolved dA14\u2013dA25 oligoadenosine length variants and dA25 variants bearing cytidine substitutions. The TP53 column baseline\u2010separated a fully complementary 21\u2010nt sequence from single\u2010nucleotide mismatches. Direct coupling of the dT25 column with a 250 \u00c5, 1.7 \u00b5m size exclusion chromatography (SEC) column enabled improved poly(A) tail sizing after RNase T1 digestion of intact mRNA; mRNA recovery exceeded 98%, and carryover remained below 0.1% after cleanup.<\/jats:p>","DOI":"10.1002\/jssc.70555","type":"journal-article","created":{"date-parts":[[2026,10,17]],"date-time":"2026-10-17T09:05:36Z","timestamp":1792227936000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Affinity Chromatography With Selective Nucleic Acid Hybridization: Direct Coupling With Size Exclusion Chromatography"],"prefix":"10.1002","volume":"49","author":[{"given":"Martin","family":"Gilar","sequence":"first","affiliation":[{"name":"Waters Corporation  Milford Massachusetts USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brittney","family":"Racioppo","sequence":"additional","affiliation":[{"name":"Waters Corporation  Milford Massachusetts USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Beatrice","family":"Muriithi","sequence":"additional","affiliation":[{"name":"Waters Corporation  Milford Massachusetts USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kevin","family":"Wyndham","sequence":"additional","affiliation":[{"name":"Waters Corporation  Milford Massachusetts USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,10,17]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"crossref","DOI":"10.1016\/j.jchromb.2020.122332","article-title":"Affinity Chromatography: A Review of Trends and Developments Over the Past 50 Years","volume":"1157","author":"Rodriguez E. L.","year":"2020","journal-title":"Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences"},{"key":"e_1_2_9_3_1","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1016\/j.jpba.2012.01.004","article-title":"Pharmaceutical and Biomedical Applications of Affinity Chromatography: Recent Trends and Developments","volume":"69","author":"Hage D. S.","year":"2012","journal-title":"Journal of Pharmaceutical and Biomedical Analysis"},{"key":"e_1_2_9_4_1","doi-asserted-by":"crossref","DOI":"10.1016\/j.chroma.2020.461835","article-title":"G Protein\u2010Coupled Receptor\u2010In\u2010Paper, a Versatile Chromatographic Platform to Study Receptor\u2010Drug Interaction","volume":"1637","author":"Feng G.","year":"2021","journal-title":"Journal of Chromatography A"},{"key":"e_1_2_9_5_1","volume-title":"HPLC Columns: Theory, Technology, and Practice","author":"Neue U. D.","year":"1997"},{"key":"e_1_2_9_6_1","volume-title":"Modern HPLC Separations in Theory and Practice","author":"Nov\u00e1kov\u00e1 L.","year":"2024"},{"key":"e_1_2_9_7_1","doi-asserted-by":"crossref","first-page":"2208","DOI":"10.1093\/clinchem\/44.10.2208","article-title":"Oligo(dT)\u2010Immobilized Pipette Tip: Efficient New Methodology for mRNA Preparation and Direct Gene Amplification","volume":"44","author":"Eguchi K.","year":"1998","journal-title":"Clinical Chemistry"},{"key":"e_1_2_9_8_1","doi-asserted-by":"crossref","first-page":"2133","DOI":"10.1007\/s00216-012-6568-4","article-title":"Affinity Monolith Chromatography: A Review of Principles and Recent Analytical Applications","volume":"405","author":"Pfaunmiller E. L.","year":"2013","journal-title":"Analytical and Bioanalytical Chemistry"},{"key":"e_1_2_9_9_1","doi-asserted-by":"crossref","first-page":"2837","DOI":"10.1002\/elps.201700101","article-title":"Affinity Monolith Chromatography: A Review of General Principles and Applications","volume":"38","author":"Li Z.","year":"2017","journal-title":"Electrophoresis"},{"key":"e_1_2_9_10_1","doi-asserted-by":"crossref","DOI":"10.1016\/j.chroma.2024.464670","article-title":"Improved mRNA Affinity Chromatography Binding Capacity and Throughput Using an Oligo\u2010dT Immobilized Electrospun Polymer Nanofiber Adsorbent","volume":"1717","author":"Dewar E. A.","year":"2024","journal-title":"Journal of Chromatography A"},{"key":"e_1_2_9_11_1","doi-asserted-by":"crossref","DOI":"10.1016\/j.seppur.2022.122320","article-title":"Development and Scale\u2010Up of Oligo\u2010dT Monolithic Chromatographic Column for mRNA Capture Through Understanding of Base\u2010Pairing Interactions","volume":"304","author":"Mencin N.","year":"2023","journal-title":"Separation and Purification Technology"},{"key":"e_1_2_9_12_1","doi-asserted-by":"crossref","DOI":"10.1016\/j.chroma.2026.467212","article-title":"Advances and Perspectives in Oligo(dT) Affinity Chromatography for mRNA Capture: Resins, Ligands and Process Intensification","volume":"1784","author":"Tian X.","year":"2026","journal-title":"Journal of Chromatography A"},{"key":"e_1_2_9_13_1","doi-asserted-by":"crossref","DOI":"10.1016\/j.chroma.2025.466575","article-title":"Hybridization Liquid Chromatography\u2010Mass Spectrometry for Quantitative Bioanalysis of Oligonucleotides: The Upcoming Paradigm Shift","volume":"1766","author":"Yuan L.","year":"2026","journal-title":"Journal of Chromatography A"},{"key":"e_1_2_9_14_1","doi-asserted-by":"crossref","first-page":"2700","DOI":"10.3390\/ijms24032700","article-title":"A Comprehensive Review of mRNA Vaccines","volume":"24","author":"Gote V.","year":"2023","journal-title":"International Journal of Molecular Sciences"},{"key":"e_1_2_9_15_1","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1016\/S0021-9673(00)91270-6","article-title":"High\u2010Performance Affinity Chromatography of DNA","volume":"508","author":"Goss T. A.","year":"1990","journal-title":"Journal of Chromatography"},{"key":"e_1_2_9_16_1","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1016\/0021-9673(91)85017-A","article-title":"High\u2010Performance Affinity Chromatography of Messenger RNA","volume":"588","author":"Goss T. A.","year":"1991","journal-title":"Journal of Chromatography"},{"key":"e_1_2_9_17_1","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1016\/j.chroma.2018.11.029","article-title":"Direct Identification of Microribonucleic Acid miR\u2010451 From Plasma Using Liquid Chromatography Mass Spectrometry","volume":"1584","author":"Basiri B.","year":"2019","journal-title":"Journal of Chromatography A"},{"key":"e_1_2_9_18_1","doi-asserted-by":"crossref","first-page":"22.1.1","DOI":"10.1002\/0471142727.mb2201s101","article-title":"Overview of DNA Microarrays: Types, Applications, and Their Future","volume":"101","author":"Bumgarner R.","year":"2013","journal-title":"Current Protocols in Molecular Biology"},{"key":"e_1_2_9_19_1","doi-asserted-by":"crossref","DOI":"10.1016\/j.jpba.2024.116556","article-title":"Analyte and Probe Melting Temperature Guided Method Development Strategy for Hybridization LC\u2010MS\/MS Quantification of siRNAs","volume":"253","author":"Song Z.","year":"2025","journal-title":"Journal of Pharmaceutical and Biomedical Analysis"},{"key":"e_1_2_9_20_1","doi-asserted-by":"crossref","first-page":"12331","DOI":"10.1021\/bi9808722","article-title":"Hybridization of Peptide Nucleic Acid","volume":"37","author":"Ratilainen T.","year":"1998","journal-title":"Biochemistry"},{"key":"e_1_2_9_21_1","doi-asserted-by":"crossref","first-page":"24117","DOI":"10.1021\/acs.analchem.6c01884","article-title":"On\u2010Column Preparation of High\u2010Performance Affinity Chromatography Columns","volume":"98","author":"Gilar M.","year":"2026","journal-title":"Analytical Chemistry"},{"key":"e_1_2_9_22_1","doi-asserted-by":"crossref","DOI":"10.1016\/j.chroma.2024.465285","article-title":"Impact of Mobile and Stationary Phases on siRNA Duplex Stability in Liquid Chromatography","volume":"1733","author":"Gilar M.","year":"2024","journal-title":"Journal of Chromatography A"},{"key":"e_1_2_9_23_1","doi-asserted-by":"crossref","first-page":"335","DOI":"10.18609\/cgti.2022.049","article-title":"Purification of Therapeutic & Prophylactic mRNA by Affinity Chromatography","volume":"8","author":"Grinsted J.","year":"2022","journal-title":"Cell and Gene Therapy Insights"},{"key":"e_1_2_9_24_1","doi-asserted-by":"crossref","DOI":"10.1016\/j.chroma.2025.466382","article-title":"Structure\u2010Performance Analysis of Oligo(dT) Resin Affinity Chromatography for RNA Separations","volume":"1762","author":"Grinsted J.","year":"2025","journal-title":"Journal of Chromatography A"},{"key":"e_1_2_9_25_1","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1016\/j.chroma.2007.09.005","article-title":"Peak Capacity in Gradient Reversed\u2010Phase Liquid Chromatography of Biopolymers. Theoretical and Practical Implications for the Separation of Oligonucleotides","volume":"1169","author":"Gilar M.","year":"2007","journal-title":"Journal of Chromatography A"},{"key":"e_1_2_9_26_1","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1016\/j.chroma.2005.03.008","article-title":"Theory of Peak Capacity in Gradient Elution","volume":"1079","author":"Neue U. D.","year":"2005","journal-title":"Journal of Chromatography A"},{"key":"e_1_2_9_27_1","doi-asserted-by":"crossref","first-page":"14308","DOI":"10.1021\/acs.analchem.3c02552","article-title":"Liquid Chromatography Methods for Analysis of mRNA Poly(A) Tail Length and Heterogeneity","volume":"95","author":"Gilar M.","year":"2023","journal-title":"Analytical Chemistry"},{"key":"e_1_2_9_28_1","doi-asserted-by":"crossref","first-page":"40","DOI":"10.3390\/vaccines11010040","article-title":"mRNA COVID\u201019 Vaccines\u2010Facts and Hypotheses on Fragmentation and Encapsulation","volume":"11","author":"Demongeot J.","year":"2022","journal-title":"Vaccines"},{"key":"e_1_2_9_29_1","unstructured":"World Health Organization Messenger RNA Encoding the Full\u2010Length SARS\u2010CoV\u20102 Spike Glycoprotein(World Health Organization 2020)."},{"key":"e_1_2_9_30_1","unstructured":"\u201cNew NIST Research Grade Test Material to Support mRNA Therapeutics \u201dNIST last modified September 24 2026 https:\/\/www.nist.gov\/news\u2010events\/news\/2025\/07\/new\u2010nist\u2010research\u2010grade\u2010test\u2010material\u2010support\u2010mrna\u2010therapeutics."}],"container-title":["Journal of Separation Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/jssc.70555","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/jssc.70555","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/jssc.70555","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,17]],"date-time":"2026-10-17T09:05:42Z","timestamp":1792227942000},"score":1,"resource":{"primary":{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/10.1002\/jssc.70555"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,10]]},"references-count":29,"journal-issue":{"issue":"10","published-print":{"date-parts":[[2026,10]]}},"alternative-id":["10.1002\/jssc.70555"],"URL":"https:\/\/doi.org\/10.1002\/jssc.70555","archive":["Portico"],"relation":{},"ISSN":["1615-9306","1615-9314"],"issn-type":[{"value":"1615-9306","type":"print"},{"value":"1615-9314","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,10]]},"assertion":[{"value":"2026-07-27","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-10-05","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-10-17","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e70555"}}