Skip to main content
SpringerLink
Log in

Physical origin of the peak tailing of monoclonal antibodies in size-exclusion chromatography using bio-compatible systems and columns

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

A Correction to this article was published on 02 February 2024

This article has been updated

Abstract

The analysis of mixtures containing monoclonal antibody (mAb) (approximately 150 kDa molecular weight) and sub-unit impurities (approximately 100 kDa) is challenging, even when adopting the latest ultra-high-pressure liquid chromatography (UHPLC) columns (4.6 mm \(\times \) 150 mm coated hardware, 1.7 \(\mu \)m 250 BEH\(^{TM}\) Surface-modified Particles) and systems (ACQUITY\(^{TM}\) UPLC\(^{TM}\) I-class Bio Plus). The main issue still encountered is a persistent tail of the mAb peak. Here, the physical origin(s) of such peak tailing in size-exclusion chromatography (SEC) are investigated from both fundamental and practical approaches. Up to five relevant physical origins are analyzed: sample heterogeneity (glycoforms), UHPLC system dispersion, strong residual binding of the mAb to the SEC particles (via hydrophobic and/or electrostatic interactions) and to the stainless steel column/system hardware, slow escape kinetics of the mAb from the SEC particles, and flow heterogeneity caused by the non-ideal slurry packing of SEC columns. Experiments (testing sample heterogeneity, system dispersion, and strong residual interactions) and calculations (predicting the transient absorption/escape kinetics in a single SEC particle and the two-dimensional peak concentration profiles) altogether unambiguously demonstrate that the observed mAb peak tailing is caused primarily by the long-range velocity biases across the SEC column combined with the slow transverse dispersion of mAbs. Therefore, improvement in the resolution between mAb and sub-unit fragment impurities can only be achieved by increasing the column length, e.g., by applying recycling chromatography at acceptable pressures.

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Change history

References

  1. Rodgers KR, Chou RC. Therapeutic monoclonal antibodies and derivatives: historical perspectives and future directions. Biotechnol Adv. 2016;34:1149–58.

    Article  CAS  PubMed  Google Scholar 

  2. Rosenberg A. Effects of protein aggregates: an immunologic perspective. AAPS J. 2006;8:E501–7.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Campieri J. Online hplc-hrms platform : the next-generation process analytical technological tool for real-time monitoring of antibody quality attributes in biopharmaceutical processes. LCGC North America. 2022;40(4):20–9.

    Article  Google Scholar 

  4. Bobály B, D’Atri V, Lauber M, Beck A, Guillarme D, Fekete S. Characterizing various monoclonal antibodies with milder reversed phase chromatography conditions. J Chromatogr B. 2018;1096:1–10.

    Article  Google Scholar 

  5. Gentiluomo L, Schneider V, Roessner D, Frieß W. Coupling multi-angle light scattering to reverse-phase ultra-high-pressure chromatography (rp-uplc-mals) for the characterization monoclonal antibodies. Sci Rep. 2019;9:1–8.

    Article  CAS  Google Scholar 

  6. Fekete S, Molnár I, Guillarme D. Separation of antibody drug conjugate species by rplc: a generic method development approach. J Pharm Biomed Anal. 2017;137:60–9.

    Article  CAS  PubMed  Google Scholar 

  7. Fekete S, Veuthey J, Beck A, Guillarme D. Hydrophobic interaction chromatography (hic) for the characterization of therapeutic monoclonal antibodies and related products. J Pharm Biomed Anal. 2016;130:3–18.

    Article  CAS  PubMed  Google Scholar 

  8. Fekete S, Murisier A, Guillarme D. Hydrophobic interaction chromatography (\(hic\)) for the characterization of therapeutic monoclonal antibodies and related products, part 1: Theoretical aspects. LCGC Europe. 2021;34:101–5.

    Google Scholar 

  9. Monograph \(<\)129\(>\). analytical procedures for recombinant therapeutic monoclonal antibodies, USP41-NF36 3.

  10. Gritti F, Guiochon G. Application of the general height equivalent to a theoretical plate equation to size exclusion chromatography. study of the mass transfer of high-molecular-mass compounds in liquid chromatography. Anal Chem. 2007;79:3188–98.

    Article  CAS  PubMed  Google Scholar 

  11. Gritti F, Guiochon G. Comparison between the loading capacities of columns packed with partially and totally porous fine particles. what is the effective surface area available for adsorption? J Chromatogr A. 2007;1176:107–22.

    Article  CAS  PubMed  Google Scholar 

  12. Gritti F, Guiochon G. Mass transfer equation for proteins in very high-pressure liquid chromatography. Anal Chem. 2009;81:2723–36.

    Article  CAS  PubMed  Google Scholar 

  13. Sepsey A, Bacskay I, Felinger A. Polydispersity in size-exclusion chromatography: A stochastic approach. J Chromatogr A. 2014;1365:156–63.

    Article  CAS  PubMed  Google Scholar 

  14. Sepsey A, Bacskay I, Felinger A. Molecular theory of size exclusion chromatography for wide pore size distributions. J Chromatogr A. 2014;1331:52–60.

    Article  CAS  PubMed  Google Scholar 

  15. Reich SJ, Svidrytski A, Höltzel A, Wang W, Kübel C, Hlushkou D, Tallarek U. Transport under confinement: Hindrance factors for diffusion in core-shell and fully porous particles with different mesopore space morphologies. Microporous Mesoporous Mater. 2019;282:188–96.

    Article  CAS  Google Scholar 

  16. Gritti F, Hochstrasser J, Svidrytsky A, Hlushkou D, Tallarek U. Morphology-transport relationships in liquid chromatography: Application to method development in size exclusion chromatography. J Chromatogr A. 2020;1620: 460991.

    Article  CAS  PubMed  Google Scholar 

  17. Dondi F, Cavazzini A, Remelli M, Felinger A, Martin M. Stochastic theory of size exclusion chromatography by the characteristic function approach. J Chromatogr A. 2002;943:185–207.

    Article  CAS  PubMed  Google Scholar 

  18. Gritti F. Kinetics of biomolecule ingress and egress from fully porous particles utilized in size exclusion chromatography. J Chromatogr A. 2023;1701: 464050.

    Article  CAS  PubMed  Google Scholar 

  19. Reising AE, Schlabach S, Baranau V, Stoeckel D, Tallarek U. Analysis of packing microstructure and wall effects in a narrow-bore ultrahigh pressure liquid chromatography column using focused ion beam scanning electron microscopy. J Chromatogr A. 2017;1513:172–82.

    Article  CAS  PubMed  Google Scholar 

  20. Gritti F, Wahab M. Understanding the science behind packing high-efficiency columns and capillaries: facts, fundamentals, challenges, and future directions. LCGC North America. 2018;36:82–98.

    CAS  Google Scholar 

  21. Gritti F. On the relationship between radial structure heterogeneities and efficiency of chromatographic columns. J Chromatogr A. 2018;1533:112–26.

    Article  CAS  PubMed  Google Scholar 

  22. Gritti F. A stochastic view on column efficiency. J Chromatogr A. 2018;1540:55–67.

    Article  CAS  PubMed  Google Scholar 

  23. Guimares G, Bartlett M. Managing nonspecific adsorption to liquid chromatography hardware: a review. Anal Chim Acta. 2023;1250:340994.

    Article  Google Scholar 

  24. Delano M, Walter T, Lauber M, Gilar M, Jung M, Nguyen J, Boissel C, Patel A, Bates-Harrison A, Wyndham K. Using hybrid organic-inorganic surface technology to mitigate analyte interactions with metal surfaces in uhplc. Anal Chem. 2021;93:5773–81.

    Article  CAS  PubMed  Google Scholar 

  25. Fekete S, Kizekai L, Sarisozen YT, Lawrence N, Shiner S, Lauber M. Investigating the secondary interactions of packing materials for size-exclusion chromatography of therapeutic proteins. J Chromatogr A. 2022;1676: 463262.

    Article  CAS  PubMed  Google Scholar 

  26. Fekete S, Delano M, Harrison A, Shiner S, Belanger J, Wyndham K, Lauber M. Size exclusion and ion exchange chromatographic hardware modified with a hydrophilic hybrid surface. Anal Chem. 2022;94:3360–7.

    Article  CAS  PubMed  Google Scholar 

  27. Reich SJ, Svidrytski A, Hlushkou D, Stoeckel D, Kübel C, Höltzel A, Tallarek U. Hindrance factor expression for diffusion in random mesoporous adsorbents obtained from pore-scale simulations in physical reconstructions. Ind Eng Chem Res. 2018;57:3031–42.

    Article  CAS  Google Scholar 

  28. Gritti F. Modeling of the transient diffusion regime in fully porous particles - application to the analysis of large biomolecules by ultra-high pressure liquid chromatography. J Chromatogr A. 2022;1679: 463362.

    Article  CAS  PubMed  Google Scholar 

  29. Gritti F. Resolution limits of size exclusion chromatography columns identified from flow reversal and overcome by recycling liquid chromatography to improve the characterization of manufactured monoclonal antibodies. J Chromatogr A. 2023;1705: 464219.

    Article  CAS  PubMed  Google Scholar 

  30. Taylor G. Dispersion of soluble matter in solvent flowing slowly through a tube. Proc R Soc Lond A. 1953;219:186–203.

    Article  ADS  CAS  Google Scholar 

  31. Taylor G. Conditions under which dispersion of a solute in a stream of solvent can be used to measure molecular diffusion. Proc R Soc Lond A. 1954;225:473–7.

    Article  ADS  CAS  Google Scholar 

  32. Crank J. The mathematics of diffusion. 2nd ed. London: Oxford University Press; 1975.

    Google Scholar 

  33. Kaczmarski K, Kotska J, Zapala W, Guiochon G. Modeling of thermal processes in high pressure liquid chromatography: I. low pressure onset of thermal heterogeneity. J Chromatogr A. 2009;1216:6560–74.

    Article  CAS  PubMed  Google Scholar 

  34. Daneyko A, Hlushkou D, Khirevich S, Tallarek U. From random sphere packings to regular pillar arrays: analysis of transverse dispersion. J Chromatogr A. 2012;1257:98–115.

    Article  CAS  PubMed  Google Scholar 

  35. Zikanov O. Essential computational fluid dynamics. 2nd ed. Hoboken, NJ, USA: John Wiley & Sons; 2010.

    Google Scholar 

  36. F. Moukalled, L. Mangani, M. Darwish, The finite volume method in computational fluid dynamics: An Advanced Introduction with OpenFOAM® and MATLAB®, Springer, London, 2016.

  37. Gritti F, Besner S, Cormier S, Gilar M. Applications of high-resolution recycling liquid chromatography : from small to large molecules. J Chromatogr A. 2017;1524:108–20.

    Article  CAS  PubMed  Google Scholar 

  38. Gritti F, Smith K. Harmonization of experimental methods used to measure the true hold-up volume of liquid chromatography columns. LCGC North America. 2023;41:28–33.

    Article  Google Scholar 

  39. mab subunit standard, Waters Care and Use Manual https://www.waters.com/webassets/cms/support/docs/720006154en.pdf (2018) 1–5.

  40. Golay M, Atwood J. Early phases of the dispersion of a sample injected in poiseuille flow. J Chromatogr. 1979;186:353–70.

    Article  CAS  Google Scholar 

  41. Golay M, Atwood J. Dispersion of peaks by short straight open tubes in liquid chromatography systems. J Chromatogr. 1981;218:97–122.

  42. Shankar A, Lenhoff AM. Dispersion and partitioning in short coated tubes. Ind Eng Chem Fundamen. 1991;30:828–35.

    Article  CAS  Google Scholar 

  43. Lau MW, Ng CO. On the early development of dispersion in flow through a tube with wall reactions. Int Schol Sci Res and Innov. 2007;9:514–9.

    Google Scholar 

  44. Gritti F, McDonald T, Gilar M. Accurate measurement of dispersion data through short and narrow tubes used in very high-pressure liquid chromatography. J Chromatogr A. 2017;1410:118–28.

    Article  Google Scholar 

  45. Fountain KJ, Neue UD, Grumbach ES, Diehl DM. Effects of extra-column band spreading, liquid chromatography system operating pressure, and column temperature on the performance of sub-2 um porous particles. J Chromatogr A. 2010;1216:5979–88.

    Article  Google Scholar 

  46. Gritti F, Guiochon G. On the minimization of the band-broadening contributions of a modern, very high pressure liquid chromatograph. J Chromatogr A. 2011;1218:4632–48.

    Article  CAS  PubMed  Google Scholar 

  47. Samuelsson J, Edström L, Forssen P, Fornstedt T. Injection profiles in liquid chromatography i. a fundamental investigation. J Chromatogr A. 2010;1217:4306–12.

    Article  CAS  PubMed  Google Scholar 

  48. Felinger A, Pasti L, Dondi F, van Hulst M, Schoenmakers PJ, Martin M. Stochastic theory of size exclusion chromatography: Peak shape analysis on single columns. Anal Chem. 2005;77:3138–48.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Waters Corporation, Instrument/Core Research/Fundamental, Milford, MA, 01757, USA

    Fabrice Gritti & Sornanathan Meyyappan

Authors
  1. Fabrice Gritti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sornanathan Meyyappan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fabrice Gritti.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

ABC Highlights: authored by Rising Stars and Top Experts.

Note: BEH, ACQUITY, UPLC, Empower, Xevo, and ACQUITY APC are trademarks of Waters Technologies Corporation. Optima is a trademark of Fisher Scientific Company L.L.C.

The original online version of this article was revised: Author name has been corrected to “Sornanathan Meyyappan”.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gritti, F., Meyyappan, S. Physical origin of the peak tailing of monoclonal antibodies in size-exclusion chromatography using bio-compatible systems and columns. Anal Bioanal Chem 416, 1281–1291 (2024). https://doi.org/10.1007/s00216-023-05119-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-023-05119-2

Keywords

Search

Navigation