Separation of populations of antibody variants by fine tuning of hydrophobic-interaction chromatography operating conditions.

The following report describes the use of hydrophobic-interaction chromatography (HIC) to separate and characterize populations of monoclonal antibodies resulting from variable N- and C-terminal processing, stressed-induced covalent modifications and conformationally altered populations present in the drug product. We investigated the use of HIC to characterize heterogeneity in the intact molecule and the Fab and Fc sub-domains resulting from papain cleavage. We found that certain classes of covalent modifications to antibodies are highly amenable to HIC separation. Specific covalent modifications occurring on antibodies could be separated into pure fractions which contained unmodified, singly modified (on 1 heavy or light chain) and doubly modified (on both heavy or light chains) molecules. This report demonstrates the utility of HIC for assessing the heterogeneity, stability and, in some cases, potency of monoclonal antibodies.

[1]  G. Hale,et al.  Non-size-based membrane chromatographic separation and analysis of monoclonal antibody aggregates. , 2006, Analytical chemistry.

[2]  S. Reed,et al.  Chemical Deglycosylation Can Induce Methylation, Succinimide Formation, and Isomerization , 2001, Journal of protein chemistry.

[3]  S. Shaltiel,et al.  Hydrophobic chromatography: use for purification of glycogen synthetase. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[4]  J. Stults,et al.  Isolation and characterization of a succinimide variant of methionyl human growth hormone. , 1991, The Journal of biological chemistry.

[5]  T. Arakawa,et al.  Preferential interactions of proteins with salts in concentrated solutions. , 1982, Biochemistry.

[6]  L. Mazzarella,et al.  Selective deamidation of ribonuclease A. Isolation and characterization of the resulting isoaspartyl and aspartyl derivatives. , 1993, The Journal of biological chemistry.

[7]  S. Cramer,et al.  Effect of pH changes on water release values in hydrophobic interaction chromatographic systems. , 2005, Journal of chromatography. A.

[8]  M. Wilchek,et al.  Stable, high capacity and non charged agarose derivatives for immobilization of biologically active compounds and for affinity chromatography , 1974, Molecular and Cellular Biochemistry.

[9]  Y. Kato.  High-performance hydrophobic interaction chromatography of proteins. , 1983, Advances in chromatography.

[10]  Alois Jungbauer,et al.  Hydrophobic interaction chromatography of proteins. I. Comparison of selectivity. , 2002, Journal of chromatography. A.

[11]  Jie Chen,et al.  Comparison of standard and new generation hydrophobic interaction chromatography resins in the monoclonal antibody purification process. , 2008, Journal of chromatography. A.

[12]  Jason C Rouse,et al.  Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain. , 2007, Analytical biochemistry.

[13]  T. Lin,et al.  Contribution of the surface free energy perturbation to protein-solvent interactions. , 1994, Biochemistry.

[14]  G. Jones.  Cell-free synthesis of amino-terminal L-pyroglutamic acid. , 1974, Biochemistry.

[15]  C. Horváth,et al.  Salt-mediated retention of proteins in hydrophobic-interaction chromatography. Application of solvophobic theory. , 1984, Journal of chromatography.

[16]  Hiroshi Nakamura,et al.  Evaluation of ammonium acetate as a volatile buffer for high-performance hydrophobic-interaction chromatography , 1990 .

[17]  R. Bischoff,et al.  Sequence-specific deamidation: isolation and biochemical characterization of succinimide intermediates of recombinant hirudin. , 1993, Biochemistry.

[18]  David R H Evans,et al.  Purification of an Fc-fusion biologic: clearance of multiple product related impurities by hydrophobic interaction chromatography. , 2008, Journal of chromatography. A.

[19]  B. Karger,et al.  Wide-pore silica-based ether-bonded phases for separation of proteins by high-performance hydrophobic-interaction and size-exclusion chromatography. , 1984, Journal of chromatography.

[20]  S. Clarke.  Propensity for spontaneous succinimide formation from aspartyl and asparaginyl residues in cellular proteins. , 2009, International journal of peptide and protein research.

[21]  J. Queiroz,et al.  Hydrophobic interaction chromatography of proteins. , 2001, Journal of biotechnology.

[22]  L. Presta,et al.  Isomerization of an aspartic acid residue in the complementarity-determining regions of a recombinant antibody to human IgE: identification and effect on binding affinity. , 1996, Biochemistry.

[23]  John F. Valliere-Douglass,et al.  Separation and characterization of an IgG2 antibody containing a cyclic imide in CDR1 of light chain by hydrophobic interaction chromatography and mass spectrometry. , 2008, Analytical chemistry.

[24]  B. Yan,et al.  Analysis of post-translational modifications in recombinant monoclonal antibody IgG1 by reversed-phase liquid chromatography/mass spectrometry. , 2007, Journal of chromatography. A.

[25]  Ronald T Borchardt,et al.  Aspartate isomerization in the complementarity-determining regions of two closely related monoclonal antibodies. , 2007, Biochemistry.

[26]  K. Inouye,et al.  Purification of monoclonal antibodies with light-chain heterogeneity produced by mouse hybridomas raised with NS-1 myelomas: application of hydrophobic interaction high-performance liquid chromatography. , 1993, Journal of biochemical and biophysical methods.

[27]  D. Aswad,et al.  Major Degradation Products of Basic Fibroblast Growth Factor: Detection of Succinimide and Iso-aspartate in Place of Aspartate15 , 1994, Pharmaceutical Research.

[28]  J. Queiroz,et al.  Comparative study on the fractionation of cellulases on some hydrophobic interaction chromatography adsorbents. , 2002, Journal of chromatography. A.

[29]  D. Chelius,et al.  Accumulation of Succinimide in a Recombinant Monoclonal Antibody in Mildly Acidic Buffers Under Elevated Temperatures , 2007, Pharmaceutical Research.

[30]  H. Ditzel,et al.  Pilot scale purification of human monoclonal IgM (COU-1) for clinical trials. , 1997, Journal of immunological methods.

[31]  A. Alpert.  High-performance hydrophobic-interaction chromatography of proteins on a series of poly(alkyl aspart-amide)-silicas , 1986 .

[32]  D. Aswad,et al.  Formation of isoaspartate at two distinct sites during in vitro aging of human growth hormone. , 1989, The Journal of biological chemistry.

[33]  Finn Hung,et al.  Purification of antibody heteropolymers using hydrophobic interaction chromatography. , 2008, Journal of chromatography. A.

[34]  F. Emmrich,et al.  Purification and analytical characterization of an anti-CD4 monoclonal antibody for human therapy. , 1994, Journal of chromatography. A.

[35]  S. Hjertén.  Some general aspects of hydrophobic interaction chromatography , 1973 .

[36]  C. Horváth,et al.  Salt effect on hydrophobic interactions in precipitation and chromatography of proteins: an interpretation of the lyotropic series. , 1977, Archives of biochemistry and biophysics.

[37]  D. Ejima,et al.  The effects of arginine on protein binding and elution in hydrophobic interaction and ion-exchange chromatography. , 2007, Protein expression and purification.

[38]  Steven M Cramer,et al.  Investigation of protein retention and selectivity in HIC systems using quantitative structure retention relationship models , 2006, Biotechnology and bioengineering.

[39]  Douglas S Rehder,et al.  Identification and characterization of deamidation sites in the conserved regions of human immunoglobulin gamma antibodies. , 2005, Analytical chemistry.

[40]  T. Arakawa,et al.  Theory of protein solubility. , 1985, Methods in enzymology.

[41]  Reed J. Harris,et al.  Identification of succinimide sites in proteins by N‐terminal sequence analysis after alkaline hydroxylamine cleavage , 1994, Protein science : a publication of the Protein Society.

[42]  B. Kabakoff,et al.  Identification of multiple sources of charge heterogeneity in a recombinant antibody. , 2001, Journal of chromatography. B, Biomedical sciences and applications.

[43]  M. Matsumura,et al.  Isomerization of a single aspartyl residue of anti-epidermal growth factor receptor immunoglobulin gamma2 antibody highlights the role avidity plays in antibody activity. , 2008, Biochemistry.

[44]  S. Clarke,et al.  Succinimide formation from aspartyl and asparaginyl peptides as a model for the spontaneous degradation of proteins. , 1989, The Journal of biological chemistry.

[45]  E. J. Fernandez,et al.  Hydrophobic interaction chromatography selectivity changes among three stable proteins: conformation does not play a major role , 2004, Biotechnology and bioengineering.

[46]  Lu Wang,et al.  Purification of humanized monoclonal antibody by hydrophobic interaction membrane chromatography. , 2006, Journal of chromatography. A.

[47]  Abraham M Lenhoff,et al.  Hydrophobic interaction chromatography of proteins. I. The effects of protein and adsorbent properties on retention and recovery. , 2007, Journal of chromatography. A.

[48]  X. Geng,et al.  Study of the retention mechanism of proteins in hydrophobic interaction chromatography , 1990 .

[49]  S C Goheen,et al.  Protein losses in ion-exchange and hydrophobic interaction high-performance liquid chromatography. , 2000, Journal of chromatography. A.

[50]  C. Horváth,et al.  Interplay of hydrophobic and electrostatic interactions in biopolymer chromatography. Effect of salts on the retention of proteins. , 1989, Journal of chromatography.

[51]  Y. Sadakane,et al.  Quantification of the isomerization of Asp residue in recombinant human alpha A-crystallin by reversed-phase HPLC. , 2003, Journal of pharmaceutical and biomedical analysis.