Retention Mechanism of Proteins in Hydroxyapatite Chromatography – Multimodal Interaction Based Protein Separations: A Model Study

Author(s): Daisuke Itoh, Noriko Yoshimoto, Shuichi Yamamoto*

Journal Name: Current Protein & Peptide Science

Volume 20 , Issue 1 , 2019

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Graphical Abstract:


Background: Retention mechanism of proteins in hydroxyapatite chromatography (HAC) was investigated by linear gradient elution experiments (LGE).

Materials and Methods: Several mobile phase (buffer) solution strategies and solutes were evaluated in order to probe the relative contributions of two adsorption sites of hydroxyapatite (HA) particles, C-site due to Ca (metal affinity) and P-site due to PO4 (cation-exchange). When P-site was blocked, two basic proteins, lysozyme (Lys) and ribonuclease A(RNase), were not retained whereas cytochrome C(Cyt C) and lactoferrin (LF) were retained and also retention of acidic proteins became stronger as the repulsion due to P-site was eliminated. The number of the binding site B values determined from LGE also increased, which also showed reduction of repulsion forces.

Conclusion: The selectivity (retention) of four basic proteins (RNase, Lys, Cyt C, LF) in HAC was different from that in ion-exchange chromatography. Moreover, it was possible to tune the selectivity by using NaCl gradient.

Keywords: Hydroxyapatite chromatography, linear gradient elution, protein retention, binding site, LGE, LF.

Karlson, E.; Hirsch, I. Ion Exchange Chromatography, In Janson, J.-C. (Ed.). Protein Purification: Principles, High Resolution Methods, and Applications 3rd Ed.; , 2011, 4, pp. 93-133. John Wiley & Sons, Hoboken, USA
Carta, G.; Jungbauer, A. Protein Chromatography: Process Development and Scale-Up; Wiley-VCH: Weinheim, 2010.
Ladisch, M.R. Bioseparations Engineering: Principles Practice, and Economics, 452-513, 540-555; Wiley: New York, 2001.
Harison, R.G.; Todd, P.; Rudge, S.R.; Petrides, D.P. Bioseparation Science and Engineering; Oxford University Press: New York, 2003.
Yamamoto, S.; Nakanishi, K.; Matsuno, R. Ion-Exchange Chromatography of Proteins; Marcel Dekker: New York, 1988.
Tiselius, A.; Hjerten, S.; Levin, O. Protein chromatography on calcium phosphate columns. Arch. Biochem. Biophys., 1956, 65, 132-155.
Bernardi, G. Chromatography of proteins on hydroxyapatite. Methods Enzymol., 1971, 22, 325-342.
Gorbunoff, M.J. Protein chromatography on hydroxyapatite columns. Methods Enzymol., 1990, 182, 329-339.
Kawasaki, T. Hydroxyapatite as a liquid chromatographic packing. J. Chromatogr. A, 1991, 544, 147-184.
John, M.; Schmidt, J. High-resolution hydroxyapatite chromatography of proteins. Anal. Biochem., 1984, 141, 466-471.
Gagnon, P. Hydroxyapatite chromatography. In: Purification tools for monoclonal antibodies; Validated Biosystems, 1996; pp. 87-102.
Cummings, L.J.; Snyder, M.A.; Brisack, K. Protein chromatography on hydroxyapatite columns. Methods Enzymol., 2009, 463, 387-404.
Gagnon, P. Monoclonal antibody purification with hydroxyapatite. New. Biotechnol., 2009, 25, 287-293.
Gagnon, P.; Beam, K. Antibody aggregate removal by hydroxyapatite chromatography. Curr. Pharm. Biotechnol., 2009, 10, 440-446.
Morrison, C.J.; Gagnon, P.; Cramer, S.M. Purification of monomeric mAb from associated aggregates using selective desorption chromatography in hydroxyapatite systems. Biotech. Bioeng., 2011, 108, 813-821.
Gagnon, P.; Cheung, C-W.; Yazaki, P.J. Cooperative multimodal retention of IgG, fragments, and aggregates on hydroxyapatite. J. Sep. Sci., 2009, 32, 3857-3865.
Wensel, D.L.; Kelley, B.D.; Coffman, J.L. High-throughput screening of chromatographic separations: III. monoclonal antibodies on ceramic hydroxyapatite. Biotech. Bioeng., 2008, 100, 839-854.
Morrison, C.J.; Gagnon, P.; Cramer, S.M. Unique selectivity windows using selective displacers/eluents and mobile phase modifiers on hydroxyapatite. J. Chromatogr. A, 2010, 1217, 6484-6495.
Hou, Y.; Morrison, C.J.; Cramer, S.M. Classification of protein binding in hydroxyapatite chromatography: Synergistic interactions on the molecular scale. Anal. Chem., 2011, 83, 3709-3716.
Hilbrig, F.; Freitag, R. Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatograph. Biotech. J., 2012, 7, 90-102.
Nakagawa, T.; Ishihara, T.; Yoshida, H.; Yoneya, T.; Wakamatsu, K.; Kadoya, T. Relationship between human IgG structure and retention time in hydroxyapatite chromatography with sodium-phosphate gradient elution. J. Sep. Sci., 2010, 33, 37-45.
Nakagawa, T.; Ishihara, T.; Yoshida, H.; Yoneya, T.; Wakamatsu, K.; Kadoya, T. Relationship between human IgG structure and retention time in hydroxyapatite chromatography with sodium chloride gradient elution. J. Sep. Sci., 2010, 33, 2045-2051.
Bankston, T.E.; Dattolo, L.; Carta, G. pH Transients in hydroxyapatite chromatography columns-Experimental evidence and phenomenological modeling. J. Chromatogr. A, 2010, 1217, 2123-2131.
Yamasaki, Y.; Yokoyama, A.; Ohnaka, A.; Kato, Y.; Murotsu, T.; Matsubara, K-I. High-performance hydroxyapatite chromatography of nucleic acids. J. Chromatogr. A, 1989, 467, 299-303.
Yamamoto, S. Electrostatic interaction chromatography process for protein separations: The impact of the engineering analysis of biorecognition mechanism on the process optimization. Chem. Eng. Technol., 2005, 28, 1387-1393.
Gagnon, P. Purification of Monoclonal Antibodies by Mixed-Mode Chromatography, In Gottschalk, U. (Ed.), Process Scale Purification of Antibodies; , 2008, 6, pp. 125-143. John Wiley & Sons, Hoboken, USA.
Chen, J.; Tetrault, J.; Zhang, Y.; Wasserman, A.; Conley, G.; DiLeo, M.; Haimesa, E.; Nixon, A.E.; Ley, A. The distinctive separation attributes of mixed-mode resins and their application in monoclonal antibody downstream purification process. J. Chromatogr. A, 2010, 1217, 216-224.
Karkov, H.S.; Sejergaard, L.; Cramer, S.M. Methods development in multimodal chromatography with mobile phase modifiers using the steric mass action model. J. Chromatogr. A, 2013, 1318, 149-155.
Righetti, P.G.; Caravaggio, T. Isoelectric points and molecular weights of proteins. A table. J. Chromatogr. A, 1976, 127, 1-28.
Righetti, P.G.; Tudor, G.; Ek, K. Isoelectric points and molecular weights of proteins. A new table. J. Chromatogr. A, 1981, 220, 115-194.
Yamamoto, S.; Ishihara, T. Ion-exchange chromatography of proteins near the isoelectric points. J. Chromatogr. A, 1999, 852, 31-36.
Yamamoto, S.; Ishihara, T. Resolution and retention of proteins near isoelectric points in ion-exchange chromatography. Molecular recognition in electrostatic interaction chromatography. Separ. Sci. Technol., 2000, 35, 1707-1717.
Barut, M.; Podgornik, A.; Merhar, M.; Strancar, A. In: ; Svec, F.; Tennikova, T.B.; Deyl, Z., Eds.; Monolithic Materials: Preparation, Properties and Applications, vol. 3, Elsevier, Amsterdam,, 2003; p. 51.
Yoshimoto, N.; Isakari, Y.; Itoh, D.; Yamamoto, S.; Yoshimoto, N. PEG chain length impacts yield of solid-phase protein PEGylation and efficiency of PEGylated protein separation by ion-exchange chromatography: Insights of mechanistic models. Biotech. J., 2013, 31, 1-7.
Ng, P.K.; Yoshitake, T. Purification of lactoferrin using hydroxyapatite. J. Chromatogr.B , 2010, 878, 976-980.
Gagnon, P. Personal communication.
Arakawa, T.; Timasheff, S.N. Abnormal solubility behavior of beta-lactoglobulin: Salting-in by glycine and sodium chloride. Biochemistry, 1987, 26, 5147-5153.

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Article Details

Year: 2019
Published on: 09 November, 2018
Page: [75 - 81]
Pages: 7
DOI: 10.2174/1389203718666171024122106

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