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Chromatographia

, Volume 82, Issue 4, pp 767–776 | Cite as

Stability-Indicating Size Exclusion Chromatography Method for the Analysis of IgG mAb-Cetuximab

  • Afsaneh Farjami
  • Parvin Akbarzadehlaleh
  • Ommoleila Molavi
  • Mohammadreza Siahi-ShadbadEmail author
Original
  • 38 Downloads

Abstract

A simple and sensitive stability-indicating size exclusion chromatography method was developed and validated for the quantitative analysis of cetuximab. The effect of variety of parameters including mobile phase composition, pH, flow rate and injection volume was investigated to achieve acceptable peak resolution and the optimum condition was selected. The proposed method was validated in accordance with the International Conference on Harmonization guidelines. Method validation showed good linearity over the concentration range of 1.56–250 µg mL−1 (r2 = 0.9997), acceptable precision (relative standard deviations < 2.8%) and accuracy (recovery of 97.6–99.5%). The limits of detection and quantitation were 0.34 µg mL−1 and 1.03 µg mL−1, respectively. The robustness of the method was evaluated by small variation in buffer composition, buffer pH and flow rate and was determined to be acceptable. Assessment of the specificity and stability-indicating capability of the method using thermally stressed, photo degraded, acidic and oxidative stressed samples revealed no interference between cetuximab and excipients or force degradation products. Furthermore, evaluation of bioactivity of stressed samples showed significant differences (p < 0.05). The proposed method could be utilized as a precise and robust stability-indicating method which can be reproduced in any labs for high-throughput quantitative analysis, stability monitoring and quality control of cetuximab in pharmaceutical formulation.

Keywords

Size exclusion chromatography Stability-indicating method Method validation Monoclonal antibody Cetuximab 

Notes

Acknowledgements

This work is a part of A. Farjami’s thesis, submitted for PhD degree (no. 117) and supported by Research Council, Tabriz University of Medical Sciences and we would like to thank the CinnaGen Medical Biotechnology Center for kindly providing all of the cetuximab medicinal samples.

Compliance with Ethical Standards

Conflict of Interest

The authors state no conflict of interest.

Research Involving Human Participants and/or Animals

This article does not contain any studies with human participants or animals performed by any of the authors.

References

  1. 1.
    Nicolaides NC, Sass PM, Grasso L (2006) Monoclonal antibodies: a morphing landscape for therapeutics. Drug Dev Res 67(10):781–789.  https://doi.org/10.1002/ddr.20149 CrossRefGoogle Scholar
  2. 2.
    Oliva A, Llabres M, Farina JB (2015) Fitting bevacizumab aggregation kinetic data with the Finke–Watzky two-step model: effect of thermal and mechanical stress. Eur J Pharm Sci 77:170–179.  https://doi.org/10.1016/j.ejps.2015.06.011 CrossRefGoogle Scholar
  3. 3.
    Mitragotri S, Burke PA, Langer R (2014) Overcoming the challenges in administering biopharmaceuticals: formulation and delivery strategies. Nat Rev Drug Discov 13(9):655–672.  https://doi.org/10.1038/nrd4363 CrossRefGoogle Scholar
  4. 4.
    Martinelli E, De Palma R, Orditura M, De Vita F, Ciardiello F (2009) Anti-epidermal growth factor receptor monoclonal antibodies in cancer therapy. Clin Exp Immunol 158(1):1–9.  https://doi.org/10.1111/j.1365-2249.2009.03992.x CrossRefGoogle Scholar
  5. 5.
    Seshacharyulu P, Ponnusamy MP, Haridas D, Jain M, Ganti AK, Batra SK (2012) Targeting the EGFR signaling pathway in cancer therapy. Expert Opin Ther Targets 16(1):15–31.  https://doi.org/10.1517/14728222.2011.648617 CrossRefGoogle Scholar
  6. 6.
    Dai J, Zhang Y (2018) A middle-up approach with online capillary isoelectric focusing/mass spectrometry for in-depth characterization of cetuximab charge heterogeneity. Anal Chem 90(24):14527–14534.  https://doi.org/10.1021/acs.analchem.8b04396 CrossRefGoogle Scholar
  7. 7.
    Sundaram S, Matathia A, Qian J, Zhang J, Hsieh M-C, Liu T, Crowley R, Parekh B, Zhou Q (2011) An innovative approach for the characterization of the isoforms of a monoclonal antibody product. In: MAbs, vol 6. Taylor & Francis, pp 505–512Google Scholar
  8. 8.
    Ayoub D, Jabs W, Resemann A, Evers W, Evans C, Main L, Baessmann C, Wagner-Rousset E, Suckau D, Beck A (2013) Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques. In: MAbs, vol 5. Taylor & Francis, pp 699–710Google Scholar
  9. 9.
    Liu S, Gao W, Wang Y, He Z, Feng X, Liu BF, Liu X (2017) Comprehensive N-glycan profiling of cetuximab biosimilar candidate by NP-HPLC and MALDI-MS. PLoS One 12(1):e0170013.  https://doi.org/10.1371/journal.pone.0170013 CrossRefGoogle Scholar
  10. 10.
    Biacchi M, Said N, Beck A, Leize-Wagner E, Francois YN (2017) Top-down and middle-down approach by fraction collection enrichment using offline capillary electrophoresis–mass spectrometry coupling: application to monoclonal antibody Fc/2 charge variants. J Chromatogr A 1498:120–127.  https://doi.org/10.1016/j.chroma.2017.02.064 CrossRefGoogle Scholar
  11. 11.
    Daugherty AL, Mrsny RJ (2006) Formulation and delivery issues for monoclonal antibody therapeutics. Adv Drug Deliv Rev 58(5–6):686–706.  https://doi.org/10.1016/j.addr.2006.03.011 CrossRefGoogle Scholar
  12. 12.
    Tokhadze N, Chennell P, Le Basle Y, Sautou V (2018) Stability of infliximab solutions in different temperature and dilution conditions. J Pharm Biomed Anal 150:386–395.  https://doi.org/10.1016/j.jpba.2017.12.012 CrossRefGoogle Scholar
  13. 13.
    Farjami A, Siahi-Shadbad M, Akbarzadehlaleh P, Molavi O (2018) Development and validation of salt gradient CEX chromatography method for charge variants separation and quantitative analysis of the IgG mAb-cetuximab. Chromatographia.  https://doi.org/10.1007/s10337-018-3627-9 Google Scholar
  14. 14.
    Vergote V, Burvenich C, Van de Wiele C, De Spiegeleer B (2009) Quality specifications for peptide drugs: a regulatory-pharmaceutical approach. J Pept Sci 15(11):697–710.  https://doi.org/10.1002/psc.1167 CrossRefGoogle Scholar
  15. 15.
    Staub A, Guillarme D, Schappler J, Veuthey JL, Rudaz S (2011) Intact protein analysis in the biopharmaceutical field. J Pharm Biomed Anal 55(4):810–822.  https://doi.org/10.1016/j.jpba.2011.01.031 CrossRefGoogle Scholar
  16. 16.
    Frokjaer S, Otzen DE (2005) Protein drug stability: a formulation challenge. Nat Rev Drug Discov 4(4):298–306.  https://doi.org/10.1038/nrd1695 CrossRefGoogle Scholar
  17. 17.
    Shah DD, Zhang J, Hsieh MC, Sundaram S, Maity H, Mallela KMG (2018) Effect of peroxide- versus alkoxyl-induced chemical oxidation on the structure, stability, aggregation, and function of a therapeutic monoclonal antibody. J Pharm Sci 107(11):2789–2803.  https://doi.org/10.1016/j.xphs.2018.07.024 CrossRefGoogle Scholar
  18. 18.
    Lu X, Nobrega RP, Lynaugh H, Jain T, Barlow K, Boland T, Sivasubramanian A, Vasquez M, Xu Y (2019) Deamidation and isomerization liability analysis of 131 clinical-stage antibodies. MAbs 11(1):45–57.  https://doi.org/10.1080/19420862.2018.1548233 CrossRefGoogle Scholar
  19. 19.
    Nowak C, Katiyar JKC,SMD, Bhat A, Sun R, Ponniah J, Neill G, Mason A, Beck B, Liu A H (2017) Forced degradation of recombinant monoclonal antibodies: a practical guide. MAbs 9(8):1217–1230.  https://doi.org/10.1080/19420862.2017.1368602 CrossRefGoogle Scholar
  20. 20.
    Xu Y, Wang D, Mason B, Rossomando T, Li N, Liu D, Cheung JK, Xu W, Raghava S, Katiyar A, Nowak C, Xiang T, Dong DD, Sun J, Beck A, Liu H (2018) Structure, heterogeneity and developability assessment of therapeutic antibodies. MAbs.  https://doi.org/10.1080/19420862.2018.1553476 Google Scholar
  21. 21.
    Rathore AS, Winkle H (2009) Quality by design for biopharmaceuticals. Nat Biotechnol 27(1):26–34.  https://doi.org/10.1038/nbt0109-26 CrossRefGoogle Scholar
  22. 22.
    Mahler HC, Friess W, Grauschopf U, Kiese S (2009) Protein aggregation: pathways, induction factors and analysis. J Pharm Sci 98(9):2909–2934.  https://doi.org/10.1002/jps.21566 CrossRefGoogle Scholar
  23. 23.
    Ehkirch A, Goyon A, Hernandez-Alba O, Rouviere F, D’Atri V, Dreyfus C, Haeuw JF, Diemer H, Beck A, Heinisch S, Guillarme D, Cianferani S (2018) A novel online four-dimensional SECxSEC-IMxMS methodology for characterization of monoclonal antibody size variants. Anal Chem.  https://doi.org/10.1021/acs.analchem.8b03333 Google Scholar
  24. 24.
    ICH: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (2005) Topic Q2 (R1): validation of analytical methods—text and methodology. http://www.ich.org/fleadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1_Guideline.pdf. Accessed 30 Oct 2018
  25. 25.
    Rambla-Alegre M, Esteve-Romero J, Carda-Broch S (2012) Is it really necessary to validate an analytical method or not? That is the question. J Chromatogr A 1232:101–109.  https://doi.org/10.1016/j.chroma.2011.10.050 CrossRefGoogle Scholar
  26. 26.
    Shah VP, Midha KK, Findlay JW, Hill HM, Hulse JD, McGilveray IJ, McKay G, Miller KJ, Patnaik RN, Powell ML, Tonelli A, Viswanathan CT, Yacobi A (2000) Bioanalytical method validation—a revisit with a decade of progress. Pharm Res 17(12):1551–1557CrossRefGoogle Scholar
  27. 27.
    Ozkan SA (2018) Analytical method validation: the importance for pharmaceutical analysis. Pharm Sci 24:1–2CrossRefGoogle Scholar
  28. 28.
    Riley CM, Rosanske TW (1996) Development and validation of analytical methods, vol 3. Elsevier, OxfordGoogle Scholar
  29. 29.
    Hawe A, Wiggenhorn M, van de Weert M, Garbe JH, Mahler HC, Jiskoot W (2012) Forced degradation of therapeutic proteins. J Pharm Sci 101(3):895–913.  https://doi.org/10.1002/jps.22812 CrossRefGoogle Scholar
  30. 30.
    Maggio RM, Vignaduzzo SE, Kaufman TS (2013) Practical and regulatory considerations for stability-indicating methods for the assay of bulk drugs and drug formulations. Trends Anal Chem 49:57–70.  https://doi.org/10.1016/j.trac.2013.05.008 CrossRefGoogle Scholar
  31. 31.
    Lahlou A, Blanchet B, Carvalho M, Paul M, Astier A (2009) Mechanically-induced aggregation of the monoclonal antibody cetuximab. Ann Pharm Fr 67(5):340–352.  https://doi.org/10.1016/j.pharma.2009.05.008 CrossRefGoogle Scholar
  32. 32.
    Hernandez-Jimenez J, Salmeron-Garcia A, Cabeza J, Velez C, Capitan-Vallvey LF, Navas N (2016) The effects of light-accelerated degradation on the aggregation of marketed therapeutic monoclonal antibodies evaluated by size-exclusion chromatography with diode array detection. J Pharm Sci 105(4):1405–1418.  https://doi.org/10.1016/j.xphs.2016.01.012 CrossRefGoogle Scholar
  33. 33.
    Farrell A, Bones J, Cook K (2017) Optimizing protein aggregate analysis by SEC. Biopharm Int 30(10):46–46+Google Scholar
  34. 34.
    Martínez-Ortega A, Herrera A, Salmerón-García A, Cabeza J, Cuadros-Rodríguez L, Navas N (2016) Study and ICH validation of a reverse-phase liquid chromatographic method for the quantification of the intact monoclonal antibody cetuximab. J Pharm Anal 6(2):117–124CrossRefGoogle Scholar
  35. 35.
    Stahl M (2003) Peak purity analysis in HPLC and CE using diode-array technology. Agilent Technologies, WaldbronnGoogle Scholar
  36. 36.
    ICH: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (1995) Topic Q5C: stability testing of biotechnological/biological products. http://www.ich.org/fleadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q5C/Step4/Q5C_Guideline.pdf. Accessed 30 Oct 2018
  37. 37.
    Chi EY, Krishnan S, Randolph TW, Carpenter JF (2003) Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation. Pharm Res 20(9):1325–1336CrossRefGoogle Scholar
  38. 38.
    Philo JS (2006) Is any measurement method optimal for all aggregate sizes and types? Aaps j 8(3):E564–E571.  https://doi.org/10.1208/aapsj080365 CrossRefGoogle Scholar
  39. 39.
    Vermeer AW, Norde W, van Amerongen A (2000) The unfolding/denaturation of immunogammaglobulin of isotype 2b and its F(ab) and F(c) fragments. Biophys J 79(4):2150–2154.  https://doi.org/10.1016/s0006-3495(00)76462-9 CrossRefGoogle Scholar
  40. 40.
    Paul M, Vieillard V, Jaccoulet E, Astier A (2012) Long-term stability of diluted solutions of the monoclonal antibody rituximab. Int J Pharm 436(1–2):282–290.  https://doi.org/10.1016/j.ijpharm.2012.06.063 CrossRefGoogle Scholar
  41. 41.
    ICH: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (1996) Topic Q1B: stability testing: photostability testing of new drug substances and products. https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q1B/Step4/Q1B_Guideline.pdf. Accessed 30 Oct 2018
  42. 42.
    Luo Q, Joubert MK, Stevenson R, Ketchem RR, Narhi LO, Wypych J (2011) Chemical modifications in therapeutic protein aggregates generated under different stress conditions. J Biol Chem 286(28):25134–25144.  https://doi.org/10.1074/jbc.M110.160440 CrossRefGoogle Scholar
  43. 43.
    Yan B, Yates Z, Balland A, Kleemann GR (2009) Human IgG1 hinge fragmentation as the result of H2O2-mediated radical cleavage. J Biol Chem 284(51):35390–35402.  https://doi.org/10.1074/jbc.M109.064147 CrossRefGoogle Scholar
  44. 44.
    De Groot AS, Scott DW (2007) Immunogenicity of protein therapeutics. Trends Immunol 28(11):482–490.  https://doi.org/10.1016/j.it.2007.07.011 CrossRefGoogle Scholar
  45. 45.
    Wang W, Nema S, Teagarden D (2010) Protein aggregation—pathways and influencing factors. Int J Pharm 390(2):89–99.  https://doi.org/10.1016/j.ijpharm.2010.02.025 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Afsaneh Farjami
    • 1
    • 2
    • 3
  • Parvin Akbarzadehlaleh
    • 1
  • Ommoleila Molavi
    • 1
    • 4
  • Mohammadreza Siahi-Shadbad
    • 1
    • 2
    Email author
  1. 1.Faculty of PharmacyTabriz University of Medical SciencesTabrizIran
  2. 2.Food and Drug Safety Research CenterTabriz University of Medical SciencesTabrizIran
  3. 3.Student Research CommitteeTabriz University of Medical SciencesTabrizIran
  4. 4.Biotechnology Research CentreTabriz University of Medical SciencesTabrizIran

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