Pharmaceutical Research

, Volume 29, Issue 1, pp 209–224 | Cite as

Elucidation of Degradants in Acidic Peak of Cation Exchange Chromatography in an IgG1 Monoclonal Antibody Formed on Long-Term Storage in a Liquid Formulation

  • Sejal Gandhi
  • Da Ren
  • Gang Xiao
  • Pavel Bondarenko
  • Christopher Sloey
  • Margaret Speed Ricci
  • Sampathkumar Krishnan
Research Paper



An IgG1 therapeutic monoclonal antibody showed an increase in acidic or pre-peak by cation exchange chromatography (CEX) at elevated temperatures, though stable at 2–8°C long-term storage in a liquid formulation. Characterization effort was undertaken to elucidate the degradants in CEX pre-peak and effect on biological activity.


Purified CEX fractions were collected and analyzed by peptide mapping, size exclusion, intact and reduced-alkylated reversed phase techniques. Biophysical characterization, isoelectric focusing and Isoquant analysis were also performed to determine nature of degradants. Bioassay and surface plasmon resonance experiments were performed to determine the impact on biological activity of the degradants.


No major degradation due to oxidation, clipping or aggregation was detected; conformational differences between purified fractions observed were not significant. Sialic acid, N-terminal glutamine cyclization and glycation differences contributed to the CEX pre-peak in the mAb control sample; increase in CEX pre-peak at 25°C and higher was caused by additive degradation pathways of deamidation, related isomerization and clipping.


The observed CEX pre-peak increase was caused by multiple degradations, especially deamidation and clipping. This elucidation of degradants in CEX peaks may apply to other therapeutic IgG1 monoclonal antibodies.


acidic-peak cation exchange chromatography deamidation IgG1monoclonal antibody protein formulation 





aspartic acid


4,4′-dianilino-1,1′-binapthyl-5,5′-disulfonic acid dipotassium salt


circular dichroism


complementarity-determining region


cation exchange chromatography


capillary isoelectric focusing


neonatal Fc receptor


Fourier transform infrared spectroscopy


hydrophobic interaction chromatography


high performance liquid chromatography


immunoglobulin gamma 1


monoclonal antibody


protein L-isoaspartyl methyltransferase




S-adenosyl homocysteine


size exclusion chromatography


surface plasmon resonance


tangential flow filtration



Our sincere thanks to Scott Smallwood, Jeffrey Reichert, Himanshu Gadgil, Gary Pipes, Ramil Latypov, David Hambly, Jaymi Lee, Lynn Peabody, Renuka Thirumangalathu, Thomas Dillon and process team members all within Amgen for their technical support and discussion.

Supplementary material

11095_2011_536_MOESM1_ESM.doc (110 kb)
ESM 1 (DOC 110 kb)


  1. 1.
    Liu H, Gaza-Bulseco G, Faldu D, Chumsae C, Sun J. Heterogeneity of monoclonal antibodies. J Pharm Sci. 2007;97:2426–47.CrossRefGoogle Scholar
  2. 2.
    Wang W. Instability, stabilization, and formulation of liquid protein pharmaceuticals. Int J Pharm. 1999;185:129–88.PubMedCrossRefGoogle Scholar
  3. 3.
    Manning MC, Patel K, Borchardt R. Stability of protein pharmaceuticals. Pharm Res. 1989;6:903–18.PubMedCrossRefGoogle Scholar
  4. 4.
    Liu D. Deamidation: a source of microheterogeneity in pharmaceutical proteins. Trends Biotechnol. 1992;10:364–9.PubMedCrossRefGoogle Scholar
  5. 5.
    Aswad D, Paranandi M, Schuter B. Isoaspartate in peptides and proteins: formation, significance, and analysis. J Pharm Biomed Anal. 2000;21:1129–36.PubMedCrossRefGoogle Scholar
  6. 6.
    Johnson K, Paisley-Flango K, Tangarone B, Porter T, Rouse J. Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain. Anal Biochem. 2006;360:75–83.PubMedCrossRefGoogle Scholar
  7. 7.
    Perkins M, Theiler R, Lunte S, Jeschke M. Determination of the origin of charge heterogeneity in a murine monoclonal antibody. Pharm Res. 2000;17:1110–7.PubMedCrossRefGoogle Scholar
  8. 8.
    Weitzhandler M, Farnan D, Rohrer J, Avdalovic N. Protein variant separations using cation exchange chromatography on grafted, polymeric stationary phases. Proteomics. 2001;1:179–85.PubMedCrossRefGoogle Scholar
  9. 9.
    Weitzhandler M, Farnan D, Horwath J, Rohrer J, Slingsby R, Avdalovic N, et al. Protein variant separations by cation-exchange chromatography on tentacle-type polymeric stationary phases. J Chromatogr A. 1998;828:365–72.PubMedCrossRefGoogle Scholar
  10. 10.
    Harris RJ, Kabakoff B, Macchi FD, Shen FJ, Kwong M, Andya JD, et al. Identification of multiple sources of charge heterogeneity in a recombinant antibody. J Chromatogr B. 2001;752:233–45.CrossRefGoogle Scholar
  11. 11.
    Vlasak J, Ionescu R. Heterogeneity of monoclonal antibodies revealed by charge-sensitive methods. Curr Pharm Biotechnol. 2008;9:468–81.PubMedCrossRefGoogle Scholar
  12. 12.
    Wakankar A, Borchardt R. Formulation considerations for proteins susceptible to asparagine deamidation and aspartate isomerization. J Pharm Sci. 2006;95:2321–36.PubMedCrossRefGoogle Scholar
  13. 13.
    Capasso S, Mazzarella L, Sica F, Zagari A, Salvadori S. Kinetics and mechanism of succinimide ring formation in the deamidation process of asparagine residues. J Chem Soc, Perkin Trans. 1993;2:679–82.Google Scholar
  14. 14.
    Patel K, Borchardt R. Chemical pathways of peptide degradation. III. Effect of primary sequence on the pathways of deamidation of asparaginyl residues in hexapeptides. Pharm Res. 1990;7:787–93.PubMedCrossRefGoogle Scholar
  15. 15.
    Oliyai C, Borchardt R. Chemical pathways of peptide degradation. IV. Pathways, kinetics, and mechanism of degradation of an aspartyl residue in a model hexapeptide. Pharm Res. 1993;10:95–102.PubMedCrossRefGoogle Scholar
  16. 16.
    Song Y, Schowen R, Borchardt R, Topp E. Effect of ‘pH’ on the rate of asparagine deamidation in polymeric formulations: ‘pH’-rate profile. J Pharm Sci. 2001;90:141–56.PubMedCrossRefGoogle Scholar
  17. 17.
    Geiger T, Clarke S. Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation. J Biol Chem. 1987;262:785–94.PubMedGoogle Scholar
  18. 18.
    Patel K, Borchardt RT. Chemical pathways of peptide degradation: II. Kinetics of deamidation of an asparaginyl residue in a model hexapeptide. Pharm Res. 1990;7:703–11.PubMedCrossRefGoogle Scholar
  19. 19.
    Kosky AA, Razzaq UO, Treuheit MJ, Brems DN. The effects of α-helix on the stability of Asn residues: deamidation rates in peptides of varying helicity. Protein Sci. 1999;8:2519–23.PubMedCrossRefGoogle Scholar
  20. 20.
    Robinson NE, Robinson AB. Prediction of protein deamidation rates from primary and three-dimensional structure. Proc Natl Acad Sci USA. 2001;98:4367–72.PubMedCrossRefGoogle Scholar
  21. 21.
    Capasso S. Estimation of the deamidation rate of asparagine side chains. J Pept Res. 2000;55:224–9.PubMedCrossRefGoogle Scholar
  22. 22.
    Oliyai C, Borchardt R. Chemical pathways of peptide degradation. VI. Effect of the primary sequence on the pathways of degradation of aspartyl residues in model hexapeptides. Pharm Res. 1994;11:751–8.PubMedCrossRefGoogle Scholar
  23. 23.
    Li B, Gorman E, Moore K, Williams T, Schowen R, Topp E, et al. Effects of acidic N + 1 residues on asparagine deamidation rates in solution and in the solid state. J Pharm Sci. 2005;94:666–75.PubMedCrossRefGoogle Scholar
  24. 24.
    Yan B, Steen S, Hambly D, Valliere-Douglass J, Bos T, Smallwood S, et al. Succinimide formation at Asn 55 in the complementarity determining region of a recombinant monoclonal antibody IgG1 heavy chain. J Pharm Sci. 2009;98:3509–21.PubMedCrossRefGoogle Scholar
  25. 25.
    Zhang Y, Martinez T, Woodruff B, Goetze A, Bailey R, Pettit D, et al. Hydrophobic interaction chromatography of soluble interleukin I receptor type II to reveal chemical degradations resulting in loss of potency. Anal Chem. 2008;80:7022–8.PubMedCrossRefGoogle Scholar
  26. 26.
    Paranandi M, Guzzetta A, Hancock W, Aswad D. Deamidation and isoaspartate formations during in vitro aging of recombinant tissue plasminogen activator. J Biol Chem. 1994;269:243–53.PubMedGoogle Scholar
  27. 27.
    Stevenson C, Anderegg R, Borchardt R. Comparison of separation and detection techniques for human growth hormone releasing factor (hGRF) and the products from deamidation. J Pharm Biomed Anal. 1993;11:367–73.PubMedCrossRefGoogle Scholar
  28. 28.
    Bongers J, Cummings J, Ebert M, Federici M, Gledhill L, Gulati D, et al. Validation of a peptide mapping method for a therapeutic monoclonal antibody: what could we possibly learn about a method we have run 100 times? J Pharm Biomed Anal. 2000;21:1099–128.PubMedCrossRefGoogle Scholar
  29. 29.
    Chelius D, Rehder D, Bondarenko P. Identification and characterization of deamidation sites in the conserved regions of human immunoglobulin gamma antibodies. Anal Chem. 2005;77:6004–11.PubMedCrossRefGoogle Scholar
  30. 30.
    Terashima I, Koga A, Nagai H. Identification of deamidation and isomerization sites on pharmaceutical recombinant antibody using H218O. Anal Biochem. 2007;368:49–60.PubMedCrossRefGoogle Scholar
  31. 31.
    Ren D, Pipes GD, Liu D, Shih L, Nichols AC, Treuheit MJ, et al. An improved trypsin digestion method minimizes digestion-induced modifications on proteins. Anal Biochem. 2009;393:12–21.CrossRefGoogle Scholar
  32. 32.
    Dong A, Caughey WS. Infrared methods for study of hemoglobin reactions and structures. Meth Enzymol. 1994;232:139–75.PubMedCrossRefGoogle Scholar
  33. 33.
    Kendrick BS, Dong A, Allison SD, Manning MC, Carpenter JF. Quantitation of the area of overlap between second-derivative amide I infrared spectra to determine the structural similarity of a protein in different states. J Pharm Sci. 1996;85:155–8.PubMedCrossRefGoogle Scholar
  34. 34.
    Zhang Z. Prediction of low-energy collision-induced dissociation spectra of peptides. Anal Chem. 2004;76:3908–22.PubMedCrossRefGoogle Scholar
  35. 35.
    Zhang Z. De novo peptide sequencing based on a divide-and-conquer algorithm and peptide tandem spectrum simulation. Anal Chem. 2004;76:6374–83.PubMedCrossRefGoogle Scholar
  36. 36.
    Zhang Z. Large-scale identification and modification of covalent modifications on therapeutic proteins. Anal Chem. 2009;81:8354–64.PubMedCrossRefGoogle Scholar
  37. 37.
    Mario N, Baudin B, Aussel C, Giboudeau J. Capillary isoelectric focusing and high-performance cation-exchange chromatography compared for qualitative and quantitative analysis of hemoglobin variants. Clin Chem. 1997;43:2137–42.PubMedGoogle Scholar
  38. 38.
    Rehder DS, Chelius D, McAuley A, Dillon TM, Xiao G, Crousse-Zeineddini J, et al. Isomerization of a single aspartyl residue of anti-epidermal growth factor receptor immunoglobulin gamma 2 antibody highlights the role avidity plays in antibody activity. Biochemistry. 2008;47:2518–30.PubMedCrossRefGoogle Scholar
  39. 39.
    Martin W, Bjorkman P. Characterization of the 2:1 complex between the class I MHC-related Fc receptor and its Fc ligand in solution. Biochemistry. 1999;38:12639–47.PubMedCrossRefGoogle Scholar
  40. 40.
    Hambly DM, Banks DD, Scavezze JL, Siska CC, Gadgil HS. Detection and quantitation of IgG 1 hinge aspartate isomerization: the fastest degradation in stressed stability studies. Anal Chem. 2009;81:7454–9.PubMedCrossRefGoogle Scholar
  41. 41.
    Dick Jr LW, Qiu D, Cheng K. Identification and measurement of isoaspartic acid formation in the complementarity determining region of a fully human monoclonal antibody. J Chromatogr B. 2009;877:3841–9.CrossRefGoogle Scholar
  42. 42.
    Lau H, Pace D, Yan B, McGrath T, Smallwood S, Patel K, et al. Investigation of degradation processes in IgG1 monoclonal antibodies by limited proteolysis coupled with weak cation exchange HPLC. J Chromatogr B. 2010;878:868–76.CrossRefGoogle Scholar
  43. 43.
    Quan C, Alcala E, Petkovska I, Matthews D, Canova-Davis E, Taticek R, et al. A study in glycation of a therapeutic recombinant humanized monoclonal antibody: where it is, how it got there, and how it affects charge-based behavior. Anal Biochem. 2008;373:179–91.PubMedCrossRefGoogle Scholar
  44. 44.
    Robinson NE, Robinson AB. Molecular clocks. Proc Natl Acad Sci USA. 2001;98:944–9.PubMedCrossRefGoogle Scholar
  45. 45.
    Reissner KJ, Aswad DW. Deamidation and isoaspartate formation in proteins: unwanted alterations or surreptitious signals? Cell Mol Life Sci. 2003;60:1281–95.PubMedCrossRefGoogle Scholar
  46. 46.
    Weintraub SJ, Manson SR. Asparagine deamidation: a regulatory hourglass. Mech Ageing Dev. 2004;125:255–7.PubMedCrossRefGoogle Scholar
  47. 47.
    Vlasak J, Bussat M, Wang S, Wagner-Rousset E, Schaefer M, Klinguer-Hamour C, et al. Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody. Anal Biochem. 2009;392:145–54.PubMedCrossRefGoogle Scholar
  48. 48.
    Yeung YA, Leabman MK, Marvin JS, Qiu J, Adams CW, Lien S, et al. Engineering human IgG1 affinity to human neonatal Fc receptor: impact of affinity improvement on pharmacokinetics in primates. J Immunol. 2009;182:7663–71.PubMedCrossRefGoogle Scholar
  49. 49.
    Israel EJ, Wilsker DF, Hayes KC, Schoenfeld D, Simister NE. Increased clearance pf IgG in mice that lack β2-microglobulin: possible protective role of FcRn. Immunology. 1996;89:573–8.PubMedCrossRefGoogle Scholar
  50. 50.
    Capasso S, Di Cerbo P. Kinetic and thermodynamic control of the relative yield of the deamidation of asparagine and isomerization of aspartic acid residues. J Pept Res. 2000;56:382–7.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Sejal Gandhi
    • 1
  • Da Ren
    • 1
  • Gang Xiao
    • 1
  • Pavel Bondarenko
    • 1
  • Christopher Sloey
    • 1
  • Margaret Speed Ricci
    • 1
  • Sampathkumar Krishnan
    • 1
    • 2
  1. 1.Formulation and Analytical Resources Process and Product DevelopmentAmgen Inc.Thousand OaksUSA
  2. 2.Amgen Inc.Thousand OaksUSA

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