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2000
Volume 21, Issue 7
  • ISSN: 1573-4110
  • E-ISSN: 1875-6727

Abstract

Ion Exchange Chromatography (IEX) is an efficient and widely employed technique for the comprehensive characterization of therapeutic proteins and peptides. IEX separates biomolecules based on differences in their surface charge arising from amino acid composition and Post-Translational Modifications (PTMs). As a non-denaturing method, IEX is particularly valuable for resolving and quantifying charge variants like deamidation, oxidation, glycosylation, and terminal truncations in monoclonal antibodies, antibody-drug conjugates, and other biotherapeutics. The review provides insights into the principles, advantages, applications, recent advances, and limitations of using IEX (cation and anion exchange modes) for detailed analysis of proteins and peptides.

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2024-10-01
2025-10-12

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References

  1. CoskunO. Separation tecniques: Chromatography.North. Clin. Istanb.20163215616010.14744/nci.2016.3275728058406
     [Google Scholar]
  2. SarkerS.D. NaharL. Applications of high performance liquid chromatography in the analysis of herbal products.Evidence-Based Validation of Herbal MedicineElsevier eBooks201540542510.1016/B978‑0‑12‑800874‑4.00019‑2
     [Google Scholar]
  3. FliegerJ. ALKALOIDS.Thin-Layer (Planar) Chromatography.Elsevier eBooks20001956196310.1016/B0‑12‑226770‑2/02591‑6
     [Google Scholar]
  4. LeventA. ŞentürkZ. Development of an ion-pair HPLC method for determination of acebutolol in pharmaceuticals.Anal. Lett.20104391448145610.1080/00032710903502116
     [Google Scholar]
  5. YardimY. LeventA. EkinS. KeskinE. OtoG. SenturkZ. Determination of 7,12-dimethylbenz[a]anthracene in orally treated rats by high-performance liquid chromatography and transfer stripping voltammetry.Comb. Chem. High Throughput Screen.201215541842610.2174/13862071280019444022263862
     [Google Scholar]
  6. LeventA. OtoG. EkinS. BerberI. Method validation and simultaneous determination of retinol, retinyl palmitate, β-carotene, α-tocopherol and vitamin C in rat serum treated with 7,12 dimethylbenz[a]anthracene and Plantago major L. by high- performance liquid chromatography using diode-array detection.Comb. Chem. High Throughput Screen.201316214214910.2174/13862071380480628323176060
     [Google Scholar]
  7. SorciM. BelfortG. Insulin Oligomers.Elsevier eBooks201410.1016/B978‑0‑12‑394431‑3.00021‑3
     [Google Scholar]
  8. JoshiS. UpadhyayK. S RathoreA. Ion exchange chromatography hyphenated with fluorescence detector as a sensitive alternative to UV detector: Applications in biopharmaceutical analysis.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.2022121212351110.1016/j.jchromb.2022.12351136334329
     [Google Scholar]
  9. KurilA.K. SaravananK. SubbappaP.K. Analytical considerations for characterization of generic peptide product: A regulatory insight.Anal. Biochem.202469411563310.1016/j.ab.2024.11563339089363
     [Google Scholar]
  10. Al MusaimiO. FDA’s stamp of approval: Unveiling peptide breakthroughs in cardiovascular diseases, ACE, HIV, CNS, and beyond.J. Pept. Sci.2024e3627e362710.1002/psc.362738885943
     [Google Scholar]
  11. WangL. WangN. ZhangW. ChengX. YanZ. ShaoG. WangX. WangR. FuC. Therapeutic peptides: Current applications and future directions.Signal Transduct. Target. Ther.2022714810.1038/s41392‑022‑00904‑435165272
     [Google Scholar]
  12. de la TorreB.G. AlbericioF. The pharmaceutical industry in 2023: An analysis of fda drug approvals from the perspective of molecules.Molecules202429358510.3390/molecules2903058538338330
     [Google Scholar]
  13. JaagS. ShirokikhM. LämmerhoferM. Charge variant analysis of protein-based biopharmaceuticals using two-dimensional liquid chromatography hyphenated to mass spectrometry.J. Chromatogr. A2021163646178610.1016/j.chroma.2020.46178633326927
     [Google Scholar]
  14. LeblancY. RamonC. BihoreauN. ChevreuxG. Charge variants characterization of a monoclonal antibody by ion exchange chromatography coupled on-line to native mass spectrometry: Case study after a long-term storage at +5 °C.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.2017104813013910.1016/j.jchromb.2017.02.01728242492
     [Google Scholar]
  15. VlasakJ. IonescuR. Heterogeneity of monoclonal antibodies revealed by charge-sensitive methods.Curr. Pharm. Biotechnol.20089646848110.2174/13892010878678640219075686
     [Google Scholar]
  16. BeckA. BussatM.C. ZornN. RobillardV. Klinguer-HamourC. ChenuS. GoetschL. CorvaïaN. Van DorsselaerA. HaeuwJ.F. Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.2005819220321810.1016/j.jchromb.2004.06.05215833284
     [Google Scholar]
  17. De LucaC. LievoreG. BozzaD. BurattiA. CavazziniA. RicciA. MacisM. CabriW. FellettiS. CataniM. Downstream processing of therapeutic peptides by means of preparative liquid chromatography.Molecules20212615468810.3390/molecules2615468834361839
     [Google Scholar]
  18. MantC.T. HodgesR.S. Separation of peptides by strong cation-exchange high-performance liquid chromatography.J. Chromatogr. A198532714715510.1016/S0021‑9673(01)81643‑54030954
     [Google Scholar]
  19. EdelmannM.J. Strong cation exchange chromatography in analysis of posttranslational modifications: Innovations and perspectives.BioMed Res. Int.20112011193650810.1155/2011/93650822174558
     [Google Scholar]
  20. Cation exchange chromatography.Available from: https://www.bio-rad.com/en-us/applications-technologies/cation-exchange-chromatography?ID=MWHB018UU
  21. D’AddioS.M. BotheJ.R. NeriC. WalshP.L. ZhangJ. PiersonE. MaoY. GindyM. LeoneA. TempletonA.C. New and evolving techniques for the characterization of peptide therapeutics.J. Pharm. Sci.2016105102989300610.1016/j.xphs.2016.06.01127499338
     [Google Scholar]
  22. StoyanovA.V. RohlfingC.L. ConnollyS. RobertsM.L. NauserC.L. LittleR.R. Use of cation exchange chromatography for human C-peptide isotope dilution – Mass spectrometric assay.J. Chromatogr. A20111218519244924910.1016/j.chroma.2011.10.08022098929
     [Google Scholar]
  23. Exploring the principle of ion exchange chromatography and its applications.Available from: https://www.technologynetworks.com/analysis/articles/exploring-the-principle-of-ion-exchange-chromatography-and-its-applications-357491
  24. Ion exchange chromatography principles and methods.Available from: https://research.fredhutch.org/content/dam/stripe/hahn/methods/biochem/Ion_Exchange_Chromatography_Handbook.pdf
  25. 3.4.3. Ion exchange chromatography.Available from: https://chem.libretexts.org/Courses/University_of_Arkansas_Little_Rock/CHEM_4320_5320:_Biochemistry_1/03:_Methods_of_Protein_Purification_and_Characterization/3.4:_Chromatography/3.4.3._Ion_Exchange_Chromatography
  26. Technical note on gradient elution in ion exchange chromatography from thermo scientific.Available from: https://assets.thermofisher.com/TFS-Assets/CMD/Technical-Notes/tn-19-ic-gradient-elution-lpn032834-en.pdf
  27. KurilA.K. SaravananK. High-throughput method for peptide mapping and amino acid sequencing for calcitonin salmon in calcitonin salmon injection using ultra high performance liquid chromatography – High resolution mass spectrometry (UHPLC-HRMS) with the application of bioinformatic tools.J. Pharm. Biomed. Anal.202424311609410.1016/j.jpba.2024.11609438479303
     [Google Scholar]
  28. KurilA.K. SaravananK. Method development and validation for the determination of higher molecular weight species of calcitonin salmon in calcitonin salmon injection by high performance size exclusion chromatography.Int. J. Pharm. Sci. Drug Res.202426026610.25004/IJPSDR.2024.160216
     [Google Scholar]
  29. KurilA.K. Navigating mass spectrometry: A comprehensive guide to basic concepts and techniques.SSRN Electronic J.202496354710.2139/ssrn.4879107
     [Google Scholar]
  30. RathoreA.S. HebbiV. Ion exchange chromatographic methods for purification of therapeutic antibodies.Therapeutic Antibodies. Methods in Molecular Biology. HouenG. New York, NYHumana2022231310.1007/978‑1‑0716‑1450‑1_10
     [Google Scholar]
  31. Ankur VashiA.K.K. Identifying trending issues in assay of peptide therapeutics during stability study.Am. J. Biomed. Sci. Res.20242204501504
     [Google Scholar]
  32. Agilent Technologies.Available from: https://www.agilent.com/cs/library/primers/public/59913775EN_BioIEX_HowTo_LR.pdf
  33. Combining ion exchange and reverse phase chromatography for highly efficient and cost effective peptide purification.2024Available from: https://downstreamcolumn.com/combining-ion-exchange-and-reverse-phase-chromatography-for-highly-efficient-and-cost-effective-peptide-purification/
  34. Available from: https://www.bioworks.com/hubfs/Documents/AN40200010BA_Peptide_purification_by_CIEX.pdf?hsLang=en
  35. HardinA.M. HarinarayanC. MalmquistG. AxénA. van ReisR. Ion exchange chromatography of monoclonal antibodies: Effect of resin ligand density on dynamic binding capacity.J. Chromatogr. A20091216204366437110.1016/j.chroma.2008.08.04719344903
     [Google Scholar]
  36. FarmerieL. RustandiR.R. LoughneyJ.W. DawodM. Recent advances in isoelectric focusing of proteins and peptides.J. Chromatogr. A2021165146227410.1016/j.chroma.2021.46227434090060
     [Google Scholar]
  37. KhawliL.A. GoswamiS. HutchinsonR. KwongZ.W. YangJ. WangX. YaoZ. SreedharaA. CanoT. TesarD.B. NijemI. AllisonD.E. WongP.Y. KaoY.H. QuanC. JoshiA. HarrisR.J. MotchnikP. Charge variants in IgG1.MAbs20102661362410.4161/mabs.2.6.1333320818176
     [Google Scholar]
  38. KašičkaVáclav Recent developments in capillary and microchip electroseparations of peptides (2021-mid-2023).Electrophoresis2024451-216519810.1002/elps.202300152
     [Google Scholar]
  39. ŠtěpánováS. KašičkaV. Applications of capillary electromigration methods for separation and analysis of proteins (2017–mid 2021) – A review.Analytica Chimica Acta2022120933944710.1016/j.aca.2022.339447
     [Google Scholar]
  40. WuJ. McElroyW. PawliszynJ. HegerC.D. Imaged capillary isoelectric focusing: Applications in the pharmaceutical industry and recent innovations of the technology.Trends Analyt. Chem.202215011656710.1016/j.trac.2022.116567
     [Google Scholar]
  41. BahadirO. Ion-exchange chromatography and its applications.Column ChromatographyInTech2013
     [Google Scholar]
  42. KovácsP. SchäferT. HádaV. HevérH. KlingelhöferS. NebelM. StadieT. KissR. UrbányiZ. Comparative physicochemical and biological characterisation of the similar biological medicinal product teriparatide and its reference medicinal product.BioDrugs2020341657510.1007/s40259‑019‑00386‑x31595483
     [Google Scholar]
  43. FüsslF. TrappeA. CookK. SchefflerK. FitzgeraldO. BonesJ. Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry.MAbs201911111612810.1080/19420862.2018.153166430296204
     [Google Scholar]
  44. CaoM. De MelN. ShannonA. ProphetM. WangC. XuW. NiuB. KimJ. AlbarghouthiM. LiuD. MeinkeE. LinS. WangX. WangJ. Charge variants characterization and release assay development for co-formulated antibodies as a combination therapy.MAbs201911348949910.1080/19420862.2019.157813730786796
     [Google Scholar]
  45. BaranK. ZimochP. StańczakA. PiątkowskiW. AntosD. Separation of charge variants of a monoclonal antibody by overloaded ion exchange chromatography.J. Chromatogr. A2021165846260710.1016/j.chroma.2021.46260734656842
     [Google Scholar]
  46. KhanHidayat Ullah The role of ion exchange chromatography in purification and characterization of molecules.Ion Exchange Technologies.InTech201210.5772/52537
     [Google Scholar]
  47. AudainE. RamosY. HermjakobH. FlowerD.R. Perez-RiverolY. Accurate estimation of isoelectric point of protein and peptide based on amino acid sequences.Bioinformatics201632682182710.1093/bioinformatics/btv67426568629
     [Google Scholar]
  48. Separation of proteins with cation exchange chromatography on Sepapure SP and CM.Available from: https://www.knauer.net/Application/application_notes/VBS0072_Separation%20of%20proteins%20with%20cation%20exchange%20chromatography%20on%20sepapure%20SM%20and%20CM_Final%20-web-.pdf
  49. MantC.T. HodgesR.S. Mixed‐mode hydrophilic interaction/cation‐exchange chromatography (HILIC/CEX) of peptides and proteins.J. Sep. Sci.200831152754277310.1002/jssc.20080024318668504
     [Google Scholar]
  50. KurilA.K. Differential scanning calorimetry: A powerful and versatile tool for analyzing proteins and peptides.J. Pharm. Res. Int.202436717918710.9734/jpri/2024/v36i77549
     [Google Scholar]
  51. FornstedtT. EnmarkM. Separation of therapeutic oligonucleotides using ion-pair reversed-phase chromatography based on fundamental separation science.J. Chromatogr. Open.2023310007910.1016/j.jcoa.2023.100079
     [Google Scholar]
  52. SharmaN. KukrejaD. GiriT. KumarS. ShahR.P. Synthetic pharmaceutical peptides characterization by chromatography principles and method development.J. Sep. Sci.202245132200221610.1002/jssc.20210103435460196
     [Google Scholar]
  53. Di GrandiD. DayehD.M. KaurK. ChenY. HendersonS. MoonY. BhowmickA. IhnatP.M. FuY. MuthusamyK. PalackalN. PylesE.A. A single-nucleotide resolution capillary gel electrophoresis workflow for poly(A) tail characterization in the development of mRNA therapeutics and vaccines.J. Pharm. Biomed. Anal.202323611569210.1016/j.jpba.2023.11569237696189
     [Google Scholar]
  54. LeA. T. H. KrylovaS. M. KrylovS. N. Kinetic capillary electrophoresis in screening oligonucleotide libraries for protein binders.Trends Anal. Chem.202316211706110.1016/j.trac.2023.117061
     [Google Scholar]
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Ion Exchange Chromatography: A Versatile Technique for Comprehensive Characterization of Therapeutic Proteins and Peptides
Current Analytical Chemistry 21, 795 (2025); https://doi.org/10.2174/0115734110337889240920090945
/content/journals/cac/10.2174/0115734110337889240920090945
/content/journals/cac/10.2174/0115734110337889240920090945
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  • Article Type:     Review Article
Keyword(s): anion exchange chromatography; cation exchange chromatography; charged base variants; Ion exchange chromatography; isoelectric point; purification

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