Improved antibody aggregate removal by hydroxyapatite chromatography in the presence of polyethylene glycol.

This paper reports a method for improving the effectiveness of aggregate removal from monoclonal antibodies by the use of hydroxyapatite (HA) in the presence of polyethylene glycol (PEG). PEG preferentially enhances aggregate retention, thereby increasing the degree of separation between aggregated and non-aggregated antibodies. PEG has a similar effect on ion exchangers but only half the enhancement observed with HA. The HA method is suitable for preparation of aggregate-free IgG and IgM monoclonal antibodies for research, diagnostic, or therapeutic applications.

[1]  S. Hjertén,et al.  Calcium phosphate chromatography of normal human serum and of electrophoretically isolated serum proteins. , 1959, Biochimica et biophysica acta.

[2]  I. Marison,et al.  Ceramic Hydroxyapatite: A New Tool for Separation and Analysis of IGA Monoclonal Antibodies , 1997 .

[3]  S. N. Timasheff,et al.  The interaction of proteins with hydroxyapatite. III. Mechanism. , 1984, Analytical biochemistry.

[4]  M. Hearn,et al.  High-performance liquid chromatography of amino acids, peptides and proteins , 1981 .

[5]  F. Regnier,et al.  High-performance liquid chromatography: effective protein purification by various chromatographic modes. , 1990, Methods in enzymology.

[6]  J. Chiba,et al.  Separation of different molecular forms of mouse IgA and IgM monoclonal antibodies by high-performance liquid chromatography on spherical hydroxyapatite beads. , 1993, Journal of immunological methods.

[7]  B. Skoog.  Determination of Polyethylene Glycols 4000 and 6000 in Plasma Protein Preparations , 1979, Vox sanguinis.

[8]  R. Freitag,et al.  Development of a downstream process for the isolation and separation of monoclonal immunoglobulin A monomers, dimers and polymers from cell culture supernatant. , 1998, Journal of chromatography. A.

[9]  M. Carrondo,et al.  Animal Cell Technology , 1997, Springer Netherlands.

[10]  R. Freitag,et al.  Isolation of a recombinant antibody from cell culture supernatant: continuous annular versus batch and expanded-bed chromatography. , 2001, Biotechnology and bioengineering.

[11]  S. Ho,et al.  Ion-exchange chromatography of proteins : The effect of neutral polymers in the mobile phase , 1989 .

[12]  T. Arakawa.  The mechanism of increased elution volume of proteins by polyethylene glycol. , 1985, Analytical biochemistry.

[13]  M. Hearn,et al.  High-performance liquid chromatography of amino acids, peptides and proteins. XCIII. Comparison of methods for the purification of mouse monoclonal immunoglobulin M autoantibodies. , 1989, Journal of chromatography.

[14]  T. Kawasaki.  Hydroxyapatite as a liquid chromatographic packing , 1991 .

[15]  T. Arakawa,et al.  Mechanism of poly(ethylene glycol) interaction with proteins. , 1985, Biochemistry.

[16]  F. Regnier,et al.  Flow-through particles for the high-performance liquid chromatographic separation of biomolecules: perfusion chromatography. , 1990, Journal of chromatography.

[17]  M. J. Gorbunoff,et al.  The interaction of proteins with hydroxyapatite. II. Role of acidic and basic groups. , 1984, Analytical biochemistry.

[18]  P. Gagnon,et al.  Method for obtaining unique selectivities in ion-exchange chromatography by addition of organic polymers to the mobile phase , 1996 .

[19]  J. Chiba,et al.  High Performance Liquid Chromatography of Mouse Monoclonal Antibodies on Spherical Hydroxyapatite Beads , 1988 .

[20]  M. J. Gorbunoff.  The interaction of proteins with hydroxyapatite. I. Role of protein charge and structure. , 1984, Analytical biochemistry.

[21]  R. Freitag,et al.  Comparison of ceramic hydroxy- and fluoroapatite versus Protein A/G-based resins in the isolation of a recombinant human antibody from cell culture supernatant. , 2007, Journal of chromatography. A.

[22]  D. Josić,et al.  Purification of monoclonal antibodies by hydroxylapatite HPLC and size exclusion HPLC. , 1991, Biological chemistry Hoppe-Seyler.