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2000
Volume 20, Issue 8
  • ISSN: 1573-4129
  • E-ISSN: 1875-676X

Abstract

Background

Atypical peaks were observed in capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) during the development of therapeutic monoclonal IgG4 antibodies (mAb-X). Based on the previous literature reports, the atypical peak may be caused by various factors such as post-translational modifications (PTMs), method-induced artifacts, sample degradation and sequence variants. Due to the high complexity structure of mAbs and the limitations of CE-SDS, acquiring comprehensive profiling of atypical peaks has historically been challenging.

Objective

Here we developed a strategy utilizing complementary analytical methods to identify the cause of atypical peak.

Methods

This strategy includes optimizing reduced CE-SDS method to evaluate artifacts induced by the analytical method, excluding potential glycation modifications, and utilizing Liquid Chromatograph Mass Spectrometer (LC-MS) to characterize mAb-X.

Results

Our study demonstrates that the atypical peaks of mAb-X are a mixture of method-induced artifacts and variants in the C-terminal extension sequence of the light chain.

Conclusion

Strategy for complementary analytical methods tools helps to identify the cause of unknown species and plays a key role in product and process characterization.

© 2024 Bentham Science Publishers
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2024-09-04
2025-08-27

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References

  1. Millán-MartínS. JakesC. CarilloS. RogersR. RenD. BonesJ. Comprehensive multi-attribute method workflow for biotherapeutic characterization and current good manufacturing practices testing.Nat. Protoc.20231841056108910.1038/s41596‑022‑00785‑536526726
     [Google Scholar]
  2. CrescioliS. KaplonH. ChenowethA. WangL. VisweswaraiahJ. ReichertJ.M. Antibodies to watch in 2024.MAbs2024161229745010.1080/19420862.2023.229745038178784
     [Google Scholar]
  3. BeckA. Wagner-RoussetE. AyoubD. Van DorsselaerA. Sanglier-CianféraniS. Characterization of therapeutic antibodies and related products.Anal. Chem.201385271573610.1021/ac303235523134362
     [Google Scholar]
  4. AlhazmiH.A. AlbrattyM. Analytical Techniques for the Characterization and Quantification of Monoclonal Antibodies.Pharmaceuticals (Basel)202316229110.3390/ph1602029137259434
     [Google Scholar]
  5. GiorgettiJ. BeckA. Leize-WagnerE. FrançoisY.N. Combination of intact, middle-up and bottom-up levels to characterize 7 therapeutic monoclonal antibodies by capillary electrophoresis – Mass spectrometry.J. Pharm. Biomed. Anal.202018211310710.1016/j.jpba.2020.11310732004767
     [Google Scholar]
  6. FeketeS. GuillarmeD. SandraP. SandraK. Chromatographic, Electrophoretic, and Mass Spectrometric Methods for the Analytical Characterization of Protein Biopharmaceuticals.Anal. Chem.201688148050710.1021/acs.analchem.5b0456126629607
     [Google Scholar]
  7. ChakrabartiA. Separation of Monoclonal Antibodies by Analytical Size Exclusion Chromatography.Antibody EngineeringInTech BöldickeT. 201810.5772/intechopen.73321
     [Google Scholar]
  8. YangR. TangY. ZhangB. LuX. LiuA. ZhangY.T. High resolution separation of recombinant monoclonal antibodies by size-exclusion ultra-high performance liquid chromatography (SE-UHPLC).J. Pharm. Biomed. Anal.2015109526110.1016/j.jpba.2015.02.03225766848
     [Google Scholar]
  9. Eon-DuvalA. BrolyH. GleixnerR. Quality attributes of recombinant therapeutic proteins: An assessment of impact on safety and efficacy as part of a quality by design development approach.Biotechnol. Prog.201228360862210.1002/btpr.154822473974
     [Google Scholar]
  10. ChirinoA.J. Mire-SluisA. Characterizing biological products and assessing comparability following manufacturing changes.Nat. Biotechnol.200422111383139110.1038/nbt103015529163
     [Google Scholar]
  11. FeketeS. BeckA. VeutheyJ.L. GuillarmeD. Theory and practice of size exclusion chromatography for the analysis of protein aggregates.J. Pharm. Biomed. Anal.201410116117310.1016/j.jpba.2014.04.01124816223
     [Google Scholar]
  12. GilardoniE. RegazzoniL. Liquid phase separation techniques for the characterization of monoclonal antibodies and bioconjugates.Journal of Chromatography Open2022210003410.1016/j.jcoa.2022.100034
     [Google Scholar]
  13. LechnerA. GiorgettiJ. GahoualR. BeckA. Leize-WagnerE. FrançoisY.N. Insights from capillary electrophoresis approaches for characterization of monoclonal antibodies and antibody drug conjugates in the period 2016–2018.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.20191122-112311710.1016/j.jchromb.2019.05.01431128359
     [Google Scholar]
  14. KumarR. GuttmanA. RathoreA.S. Applications of capillary electrophoresis for biopharmaceutical product characterization.Electrophoresis.2022431-214316610.1002/elps.20210018234591322
     [Google Scholar]
  15. DadouchM. LadnerY. PerrinC. Analysis of monoclonal antibodies by capillary electrophoresis: Sample preparation, separation, and detection.Separations202181410.3390/separations8010004
     [Google Scholar]
  16. ShenB.B. YuanJ.J. QianC. GaoH. FangW.J. Investigation of an artifact during non-reduced capillary electrophoresis with sodium dodecyl sulfate analysis utilizing N-ethylmaleimide as an alkylation reagent.Anal. Biochem.202265511483310.1016/j.ab.2022.11483335961398
     [Google Scholar]
  17. HuttererK.M. HongR.W. LullJ. ZhaoX. WangT. PeiR. LeM.E. BorisovO. PiperR. LiuY.D. PettyK. ApostolI. FlynnG.C. Monoclonal antibody disulfide reduction during manufacturing.MAbs20135460861310.4161/mabs.2472523751615
     [Google Scholar]
  18. WagnerE. ColasO. ChenuS. GoyonA. MurisierA. CianferaniS. FrançoisY. FeketeS. GuillarmeD. D’AtriV. BeckA. Determination of size variants by CE-SDS for approved therapeutic antibodies: Key implications of subclasses and light chain specificities.J. Pharm. Biomed. Anal.202018411316610.1016/j.jpba.2020.11316632113118
     [Google Scholar]
  19. GahoualR. BeckA. Leize-WagnerE. FrançoisY.N. Cutting-edge capillary electrophoresis characterization of monoclonal antibodies and related products.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.20161032617810.1016/j.jchromb.2016.05.02827265157
     [Google Scholar]
  20. Sánchez-HernándezL. MontealegreC. KiessigS. MoritzB. NeusüßC. In-capillary approach to eliminate SDS interferences in antibody analysis by capillary electrophoresis coupled to mass spectrometry.Electrophoresis20173871044105210.1002/elps.20160046428008632
     [Google Scholar]
  21. CamperiJ. GoyonA. GuillarmeD. ZhangK. StellaC. Multi-dimensionalL.C-M.S. Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches.Analyst (Lond.)2021146374776910.1039/D0AN01963A33410843
     [Google Scholar]
  22. YangB. LiW. ZhaoH. WangA. LeiY. XieQ. XiongS. Discovery and characterization of CHO host cell protease-induced fragmentation of a recombinant monoclonal antibody during production process development.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.2019111211010.1016/j.jchromb.2019.02.02030836312
     [Google Scholar]
  23. DadaO.O. RaoR. JonesN. JayaN. Salas-SolanoO. Comparison of SEC and CE-SDS methods for monitoring hinge fragmentation in IgG1 monoclonal antibodies.J. Pharm. Biomed. Anal.2017145919710.1016/j.jpba.2017.06.00628654781
     [Google Scholar]
  24. WangD. NowakC. MasonB. KatiyarA. LiuH. Analytical artifacts in characterization of recombinant monoclonal antibody therapeutics.J. Pharm. Biomed. Anal.202018311313110.1016/j.jpba.2020.11313132058288
     [Google Scholar]
  25. ZhuZ.C. ChenY. AckermanM.S. WangB. WuW. LiB. Obenauer-KutnerL. ZhaoR. TaoL. IhnatP.M. LiuJ. GandhiR.B. QiuB. Investigation of monoclonal antibody fragmentation artifacts in non-reducing SDS-PAGE.J. Pharm. Biomed. Anal.201383899510.1016/j.jpba.2013.04.03023708435
     [Google Scholar]
  26. DuhamelL. GuY. BarnettG. TaoY. VoronovS. DingJ. MussaN. LiZ.J. Therapeutic protein purity and fragmented species characterization by capillary electrophoresis sodium dodecyl sulfate using systematic hybrid cleavage and forced degradation.Anal. Bioanal. Chem.2019411215617562910.1007/s00216‑019‑01942‑831214752
     [Google Scholar]
  27. KaschakT. BoydD. YanB. Characterization of glycation in an IgG1 by capillary electrophoresis sodium dodecyl sulfate and mass spectrometry.Anal. Biochem.2011417225626310.1016/j.ab.2011.06.02421756870
     [Google Scholar]
  28. RustandiR.R. WashabaughM.W. WangY. Applications of CE SDS gel in development of biopharmaceutical antibody‐based products.Electrophoresis200829173612362010.1002/elps.20070095818803223
     [Google Scholar]
  29. TousG.I. WeiZ. FengJ. BilbulianS. BowenS. SmithJ. StrouseR. McGeehanP. Casas-FinetJ. SchenermanM.A. Characterization of a novel modification to monoclonal antibodies: thioether cross-link of heavy and light chains.Anal. Chem.20057792675268210.1021/ac050058215859580
     [Google Scholar]
  30. WangW.H. Cheung-LauJ. ChenY. LewisM. TangQ.M. Specific and high-resolution identification of monoclonal antibody fragments detected by capillary electrophoresis–sodium dodecyl sulfate using reversed-phase HPLC with top-down mass spectrometry analysis.MAbs20191171233124410.1080/19420862.2019.164655431348730
     [Google Scholar]
  31. Gaza-BulsecoG. LiuH. Fragmentation of a recombinant monoclonal antibody at various pH.Pharm. Res.20082581881189010.1007/s11095‑008‑9606‑318473123
     [Google Scholar]
  32. GuanQ. KnihtilaR. AtsmaJ. TulsanR. SinghS. KarS. BeckmanJ. DingJ. LiZ.J. Enhancement of covalent aggregate quantification of protein therapeutics by non-reducing capillary gel electrophoresis using sodium hexadecyl sulfate (CE-SHS).J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.2020115212223010.1016/j.jchromb.2020.12223032585496
     [Google Scholar]
  33. BeckmanJ. SongY. GuY. VoronovS. ChennamsettyN. KrystekS. MussaN. LiZ.J. Purity Determination by Capillary Electrophoresis Sodium Hexadecyl Sulfate (CE-SHS): A Novel Application For Therapeutic Protein Characterization.Anal. Chem.20189042542254710.1021/acs.analchem.7b0383129357216
     [Google Scholar]
  34. GuanQ. AtsmaJ. TulsanR. VoronovS. DingJ. BeckmanJ. LiZ.J. Minimization of artifact protein aggregation using tetradecyl sulfate and hexadecyl sulfate in capillary gel electrophoresis under reducing conditions.Electrophoresis20204113-141245125210.1002/elps.20190043532297333
     [Google Scholar]
  35. ZhangL. FeiM. TianY. LiS. ZhuX. WangL. XuY. XieM.H. Characterization and elimination of artificial non-covalent light Chain dimers in reduced CE-SDS analysis of pertuzumab.J. Pharm. Biomed. Anal.202019011352710.1016/j.jpba.2020.11352732911382
     [Google Scholar]
  36. ShenB.B. ZhangZ. YuanJ.J. ZhengA. ZengS. GaoJ.Q. BaoW. BarnardJ. WangH. FangW.J. Formation of an Unprecedented Impurity during CE-SDS Analysis of a Recombinant Protein.Pharm. Res.2020371122810.1007/s11095‑020‑02947‑033098017
     [Google Scholar]
  37. LiW. YangB. ZhouD. XuJ. LiW. SuenW.C. Identification and characterization of monoclonal antibody fragments cleaved at the complementarity determining region using orthogonal analytical methods.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.2017104812112910.1016/j.jchromb.2017.02.01928242491
     [Google Scholar]
  38. LinJ. XieM. LiuD. GaoZ. ZhaoX. MaH. DingS. LiS. LiS. LiuY. ZhouF. HuH. ChenT. ChenH. XieM. YangB. ChengJ. MaM. NanY. JuD. Characterization of light chain c-terminal extension sequence variant in one bispecific antibody.Front Chem.20221099447210.3389/fchem.2022.99447236204149
     [Google Scholar]
  39. DelmarJ.A. HarrisC. GrassiL. MacapagalN. WangJ. HattonD. XuW. Monoclonal Antibody Sequence Variants Disguised as Fragments: Identification, Characterization, and Their Removal by Purification Process Optimization.J. Pharm. Sci.2022111113009301610.1016/j.xphs.2022.08.00235940243
     [Google Scholar]
  40. LiM. YuC. WangW. WuG. WangL. Interlaboratory method validation of capillary electrophoresis sodium dodecyl sulfate (CE-SDS) methodology for analysis of mAbs.Electrophoresis202142191900191310.1002/elps.20200039634240427
     [Google Scholar]
  41. KrebsF. ZagstH. SteinM. RatihR. MinknerR. OlabiM. HartungS. SchellerC. Lapizco-EncinasB.H. Sänger-van de GriendC. GarcíaC.D. WätzigH. Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications-Updated and completely revised edition.Electrophoresis.20234417-181279134110.1002/elps.202300158
     [Google Scholar]
  42. WangS.T. GaoH. ShenB.B. WangH. FangW.J. Elimination of light chain tailing in reducing capillary electrophoresis with sodium dodecyl sulfate analysis of a monoclonal antibody.Electrophoresis20224318-191850185810.1002/elps.20220013435776503
     [Google Scholar]
  43. WeiB. BerningK. QuanC. ZhangY.T. Glycation of antibodies: Modification, methods and potential effects on biological functions.MAbs20179458659410.1080/19420862.2017.130021428272973
     [Google Scholar]
  44. MoJ. JinR. YanQ. SokolowskaI. LewisM.J. HuP. Quantitative analysis of glycation and its impact on antigen binding.MAbs201810340641510.1080/19420862.2018.143879629436927
     [Google Scholar]
  45. LhotaG. SissolakB. StriednerG. SommereggerW. Vorauer-UhlK. Quantification of glycated IgG in CHO supernatants: A practical approach.Biotechnol. Prog.2021373e312410.1002/btpr.312433428326
     [Google Scholar]
  46. ZhangB. YangY. YukI. Unveiling a glycation hot spot in a recombinant humanized monoclonal antibody.Anal Chem. 2008 Apr 1;80(7):2379-90200880723792390
     [Google Scholar]
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Identification of Method-Induced Artifacts and Light Chain C-terminal Extension Sequence Variants in Therapeutic Monoclonal Antibodies by Complementary Analytical Methods
Current Pharmaceutical Analysis 20, 779 (2024); https://doi.org/10.2174/0115734129317679240829055442
/content/journals/cpa/10.2174/0115734129317679240829055442
/content/journals/cpa/10.2174/0115734129317679240829055442
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  • Article Type:     Research Article
Keyword(s): CE-SDS; LC-MS; Method-induced artifacts, light chain C-terminal extension sequence variants, mAbs, complementary analytical methods

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