Chemical Papers

, Volume 64, Issue 4, pp 461–468 | Cite as

Impact of ionic strength on adsorption capacity of chromatographic particles employed in separation of monoclonal antibodies

  • Katarzyna Wrzosek
  • Michal Gramblička
  • Darina Tóthová
  • Monika Antošová
  • Milan PolakovičEmail author
Original Paper


The influence of ionic strength on the adsorption capacity of seven commercial adsorbents used in downstream processing of monoclonal antibodies was examined. Affinity (MabSelect, Poros 50A High Capacity, ProSep-vA High Capacity), hydrophobic charge-induction (MEP HyperCel), and cation exchange adsorbents (FractoGel EMD SE Hicap (M), SP Sepharose Fast Flow, Ceramic HyperD F) were used to study the adsorption of polyclonal human immunoglobulin G at optimal pH values. The ionic strength, adjusted by sodium chloride concentrations in the range of 0–225 mM, strongly decreased the adsorption capacity of the cation exchangers. Equilibrium data were described in the form of the dependence of the ratio of protein concentrations in the solid and liquid phases on the total concentration of cation counter ions. They were successfully fitted and interpreted through a stoichiometric ion-exchange model.


monoclonal antibody adsorption capacity protein A chromatography cation-exchange chromatography hydrophobic charge-induction chromatography ionic strength pH effect stoichiometric model 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Barrande, M., Beurroies, I., Denoyel, R., Tatárová, I., Gramblička, M., Polakovič, M., Joehnck, M., & Schulte, M. (2009). Characterisation of porous materials for bioseparation. Journal of Chromatography A, 1216, 6906–6916. DOI: 10.1016/j.chroma.2009.07.075.CrossRefGoogle Scholar
  2. Boschetti, E. (2002). Antibody separation by hydrophobic charge induction chromatography. Trends in Biotechnology, 20, 333–337. DOI: 10.1016/S0167-7799(02)01980-7.CrossRefGoogle Scholar
  3. Burton, S. C., & Harding, D. R. K. (1998). Hydrophobic charge induction chromatography: salt independent protein adsorption and facile elution with aqueous buffers. Journal of Chromatography A, 814, 71–81. DOI: 10.1016/S0021-9673(98)00436-1.CrossRefGoogle Scholar
  4. Carter-Franklin, N., Victa, C., McDonald, P., & Fahrner, R. (2007). Fragments of protein A eluted during protein A affinity chromatography. Journal of Chromatography A, 1163, 105–111. DOI: 10.1016/j.chroma.2007.06.012.CrossRefGoogle Scholar
  5. Chen, J., Tetrault, J., & Ley, A. (2008). Comparison of standard and new generation hydrophobic interaction chromatography resins in the monoclonal antibody purification process. Journal of Chromatography A, 1177, 272–281. DOI: 10.1016/j.chroma.2007.07.083.CrossRefGoogle Scholar
  6. Denton, G., Murray, A., Price, M. R., & Levison, P. R. (2001). Direct isolation of monoclonal antibodies from tissue culture supernatant using the cation-exchange cellulose Express-Ion S. Journal of Chromatography A, 908, 223–234. DOI: 10.1016/S0021-9673(00)00834-7.CrossRefGoogle Scholar
  7. Faude, A., Zacher, D., Müller, E., & Böttinger, H. (2007). Fast determination of conditions for maximum dynamic capacity in cation-exchange chromatography of human monoclonal antibodies. Journal of Chromatography A, 1161, 29–35 DOI: 10.1016/j.chroma.2007.03.114.CrossRefGoogle Scholar
  8. Follman, D. K., & Fahrner, R. L. (2004). Factorial screening of antibody purification processes using three chromatography steps without protein A. Journal of Chromatography A, 1024, 79–85. DOI: 10.1016/j.chroma.2003.10.060.CrossRefGoogle Scholar
  9. Forrer, N., Butté, A., & Morbidelli, M. (2008). Chromatographic behavior of a polyclonal antibody mixture on a strong cation exchanger column. Part I: Adsorption characterization Journal of Chromatography A, 1214, 59–70. DOI: 10.1016/j.chroma.2008.10.048.CrossRefGoogle Scholar
  10. Ghose, S., Hubbard, B., & Cramer, S. M. (2006). Evaluation and comparison of alternatives to Protein A chromatography: Mimetic and hydrophobic charge induction chromatographic stationary phases. Journal of Chromatography A, 1122, 144–152. DOI: 10.1016/j.chroma.2006.04.083.CrossRefGoogle Scholar
  11. Gramblička, M., Tóthová, D., Antošová, M., & Polakovič, M. (2008). Influence of pH on adsorption of human immunoglobulin gamma, human serum albumin and horse myoglobin by commercial chromatographic materials designed for downstream processing of monoclonal antibodies. Acta Chimica Slovaca, 1(1) 85–94.Google Scholar
  12. Guerrier, L., Flayeux, I., & Boschetti, E. (2001). A dual-mode approach to the selective separation of antibodies and their fragments. Journal of Chromatography B, 755, 37–46. DOI: 10.1016/S0378-4347(00)00598-3.CrossRefGoogle Scholar
  13. Hahn, R., Schlegel, R., & Jungbauer, A. (2003). Comparison of protein A affinity sorbents. Journal of Chromatography B, 790, 35–51. DOI: 10.1016/S1570-0232(03)00092-8.CrossRefGoogle Scholar
  14. Hahn, R., Shimahara, K., Steindl, F., & Jungbauer, A. (2006). Comparison of protein A affinity sorbents III. Life time study. Journal of Chromatography A, 1102, 224–231. DOI: 10.1016/j.chroma.2005.10.083.Google Scholar
  15. Hober, S., Nord, K., & Linhult, M. (2007). Protein A chromatography for antibody purification. Journal of Chromatography B, 848, 40–47. DOI: 10.1016/j.jchromb.2006.09. 030.CrossRefGoogle Scholar
  16. Huse, K., Böhme, H.-J., & Scholz, G. H. (2002). Purification of antibodies by affinity chromatography. Journal of Biochemical and Biophysical Methods, 51, 217–231. DOI: 10.1016/S0165-022X(02)00017-9.CrossRefGoogle Scholar
  17. Jungbauer, A. (2005). Chromatographic media for bioseparation. Journal of Chromatography A, 1065, 3–12. DOI: 10.1016/j.chroma.2004.08.162.CrossRefGoogle Scholar
  18. Low, D., O’Leary, R., & Pujar, N. S. (2007). Future of antibody purification. Journal of Chromatography B, 848, 48–63. DOI: 10.1016/j.jchromb.2006.10.033.CrossRefGoogle Scholar
  19. Mollerup, J. M. (2006). Applied thermodynamics: A new frontier for biotechnology. Fluid Phase Equilibria, 241, 205–215. DOI: 10.1016/j.fluid.2005.12.037.CrossRefGoogle Scholar
  20. Necina, R., Amatschek, K., & Jungbauer, A. (1998). Capture of human monoclonal antibodies from cell culture supernatant by ion exchange media exhibiting high charge density. Biotechnology and Bioengineering, 60, 689–698. DOI: 10.1002/(SICI)1097-0290(19981220)60:6〈689::AID-BIT6〉3.0.CO;2-M.CrossRefGoogle Scholar
  21. Okay, O. (2000). Macroporous copolymer networks. Progress in Polymer Science, 25, 711–779. DOI: 10.1016/S0079-6700(00)00015-0.CrossRefGoogle Scholar
  22. Roque, A. C. A., Silva, C. S. O., & Taipa, M. Á. (2007). Affinitybased methodologies and ligands for antibody purification: Advances and perspectives. Journal of Chromatography A, 1160, 44–55. DOI: 10.1016/j.chroma.2007.05.109.CrossRefGoogle Scholar
  23. Schwartz, W., Judd, D., Wysocki, M., Guerrier, L., Birck-Wilson, E., & Boschetti, E. (2001). Comparison of hydrophobic charge induction chromatography with affinity chromatography on protein A for harvest and purification of antibodies. Journal of Chromatography A, 908, 251–263. DOI: 10.1016/S0021-9673(00)01013-X.CrossRefGoogle Scholar
  24. Shukla, A. A., Hubbard, B., Tressel, T., Guhan, S., & Low, D. (2007). Downstream processing of monoclonal antibodies—Application of platform approaches. Journal of Chromatography B, 848, 28–39. DOI: 10.1016/j.jchromb.2006.09.026.CrossRefGoogle Scholar
  25. Smith, A. W. (1948). Elements of physics (5th ed.). New York, NY, USA: McGraw-Hill.Google Scholar
  26. Staby, A., Jacobsen, J. H., Hansen, R. G., Bruus, U. K., & Holm Jensen, I. (2006). Comparison of chromatographic ion-exchange resins: V. Strong and weak cation-exchange resins. Journal of Chromatography A, 1118, 168–179. DOI: 10.1016/j.chroma.2006.03.116.CrossRefGoogle Scholar
  27. Staby, A., Sand, M.-B., Hansen, R. G., Jacobsen, J. H., Andersen, L. A., Gerstenberg, M., Bruus, U. K., & Holm Jensen, I. (2005). Comparison of chromatographic ion-exchange resins: IV. Strong and weak cation-exchange resins and heparin resins. Journal of Chromatography A, 1069, 65–77. DOI: 10.1016/j.chroma.2004.11.094.CrossRefGoogle Scholar
  28. Stone, M. C., Tao, Y., & Carta, G. (2009). Protein adsorption and transport in agarose and dextran-grafted agarose media for ion exchange chromatography: Effect of ionic strength and protein characteristics. Journal of Chromatography A, 1216, 4465–4474. DOI: 10.1016/j.chroma.2009.03.044.CrossRefGoogle Scholar
  29. Tatárová, I., Gramblička, M., Antošová, M., & Polakovič, M. (2008). Characterization of pore structure of chromatographic adsorbents employed in separation of monoclonal antibodies using size-exclusion techniques. Journal of Chromatography A, 1193, 129–135. DOI: 10.1016/j.chroma.2008.04.023.CrossRefGoogle Scholar
  30. Tugcu, N., Bae, S. S., Moore, J. A., & Cramer, S. M. (2002). Stationary phase effects on the dynamic affinity of lowmolecular-mass displacers. Journal of Chromatography A, 954, 127–135. DOI: 10.1016/S0021-9673(02)00164-4.CrossRefGoogle Scholar
  31. Zhu-Shimoni, J., Gunawan, F., Thomas, A., Vanderlaan, M., & Stults, J. (2009). Trace level analysis of leached Protein A in bioprocess samples without interference from the large excess of rhMAb IgG. Journal of Immunological Methods, 341, 59–67. DOI: 10.1016/j.jim.2008.10.015.CrossRefGoogle Scholar

Copyright information

© Institute of Chemistry, Slovak Academy of Sciences 2010

Authors and Affiliations

  • Katarzyna Wrzosek
    • 1
  • Michal Gramblička
    • 1
  • Darina Tóthová
    • 1
  • Monika Antošová
    • 1
  • Milan Polakovič
    • 1
    Email author
  1. 1.Department of Chemical and Biochemical Engineering, Faculty of Chemical and Food TechnologySlovak University of TechnologyBratislavaSlovak Republic

Personalised recommendations