Formation of an Unprecedented Impurity during CE-SDS Analysis of a Recombinant Protein

Abstract

Purposes

The main purposes of this article are to describe an unprecedented phenomenon in which significant amount of a shoulder peak impurity was observed during normal non-reducing capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis of a recombinant fusion protein X, and to evaluate the root cause for this phenomenon.

Methods

A series of experiments were conducted to study the nature of this degradation. Effects of iodoacetamide (IAM), heating temperature, duration, and SDS on the formation of this specific impurity were evaluated using a variety of characterization techniques.

Results

The formation of the impurity as observed in CE-SDS was actually due to alkylation of lysine and serine residues with IAM, as confirmed by peptide mapping and LC-MS/MS, which increased the molecular weight and therefore decreased the electrophoretic mobility. The amount of impurity was also strongly dependent on sample preparation conditions including the presence or absence of SDS.

Conclusions

Our study clearly suggested that even though IAM has been used extensively as an alkylation reagent in the traditional non-reducing CE-SDS analysis of monoclonal antibodies and other proteins, alkylation with IAM could potentially lead to additional impurity peak, and therefore complicating analysis. Therefore, before performing CE-SDS and other analyses, the effects of sample preparation procedures on analytical results must be evaluated. For protein X, IAM should be excluded for CE-SDS analysis.

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Abbreviations

CD:

Circular dichroism

CE-SDS:

Capillary electrophoresis-sodium dodecyl sulfate

IAM:

Iodoacetamide

icIEF:

Imaged capillary isoelectric focusing

LC-MS:

Liquid chromatography coupled with mass spectrometry

mAb:

Monoclonal antibody

MALDI-TOF-MS:

Matrix-assisted laser desorption/ionization time of flight mass spectrometry

RP-HPLC:

Reversed phase-high performance liquid chromatography

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SE-HPLC:

Size exclusion-high performance liquid chromatography

References

  1. 1.

    Guttman A, Nolan J. Comparison of the separation of proteins by sodium dodecyl sulfate-slab gel electrophoresis and capillary sodium dodecyl sulfate-gel electrophoresis. Anal Biochem. 1994;221:285–9.

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Shieh PCH, Hoang D, Guttman A, Cooke N. Capillary sodium dodecyl sulfate gel electrophoresis of proteins I. Reproducibility and stability. J Chromatogr A. 1994;676:219–26.

  3. 3.

    Guttman A. Capillary sodium dodecyl sulfate-gel electrophoresis of proteins. Electrophoresis. 1996;17:1333–41.

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Jo Schmerr M, Jenny A, Cutlip RC. Use of capillary sodium dodecyl sulfate gel electrophoresis to detect the prion protein extracted from scrapie-infected sheep. J Chromatogr B Analyt Technol Biomed Life Sci. 1997;697:223–9.

    Article  Google Scholar 

  5. 5.

    Manabe T. Capillary electrophoresis of proteins for proteomic studies. Electrophoresis. 1999;20:3116–21.

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Hu S, Jiang J, Cook LM, Richards DP, Horlick L, Wong B, et al. Capillary sodium dodecyl sulfate-DALT electrophoresis with laser-induced fluorescence detection for size-based analysis of proteins in human colon cancer cells. Electrophoresis. 2002;23:3136–42.

  7. 7.

    Zhu Z, Lu JJ, Liu S. Protein separation by capillary gel electrophoresis: a review. Anal Chim Acta. 2012;709:21–31.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Zhao SS, Chen DDY. Applications of capillary electrophoresis in characterizing recombinant protein therapeutics. Electrophoresis. 2014;35:96–108.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Cerutti ML, Pesce A, Bès C, Seigelchifer M. Physicochemical and biological characterization of RTXM83, a new rituximab biosimilar. BioDrugs. 2019;33:307–19.

    CAS  Article  Google Scholar 

  10. 10.

    Rustandi RR, Washabaugh MW, Wang Y. Applications of CE SDS gel in development of biopharmaceutical antibody-based products. Electrophoresis. 2008;29:3612–20.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Chen Y, Kim MT, Zheng L, Deperalta G, Jacobson F. Structural characterization of cross-linked species in Trastuzumab Emtansine (Kadcyla). Bioconjug Chem. 2016;27:2037–47.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Chen T, Chen Y, Stella C, Medley CD, Gruenhagen JA, Zhang K. Antibody-drug conjugate characterization by chromatographic and electrophoretic techniques. J Chromatogr B Analyt Technol Biomed Life Sci. 2016;1032:39–50.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Dada OO, Rao R, Jones N, Jaya N, Salas-Solano O. Comparison of SEC and CE-SDS methods for monitoring hinge fragmentation in IgG1 monoclonal antibodies. J Pharm Biomed Anal. 2017;145:91–7.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Esterman AL, Katiyar A, Krishnamurthy G. Implementation of USP antibody standard for system suitability in capillary electrophoresis sodium dodecyl sulfate (CE-SDS) for release and stability methods. J Pharm Biomed Anal. 2016;128:447–54.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Li W, Yang B, Zhou D, Xu J, Li W, Suen W-C. Identification and characterization of monoclonal antibody fragments cleaved at the complementarity determining region using orthogonal analytical methods. J Chromatogr B Analyt Technol Biomed Life Sci. 2017;1048:121–9.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Rustandi RR, Wang Y. Use of CE-SDS gel for characterization of monoclonal antibody hinge region clipping due to copper and high pH stress. Electrophoresis. 2011;32:3078–84.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Strand J, Huang C-T, Xu J. Characterization of Fc-fusion protein aggregates derived from extracellular domain disulfide bond rearrangements. J Pharm Sci. 2013;102:441–53.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Wang T, Fodor S, Hapuarachchi S, Jiang XG, Chen K, Apostol I, et al. Analysis and characterization of aggregation of a therapeutic fc-fusion protein. J Pharm Biomed Anal. 2013;72:59–64.

  19. 19.

    Gahoual R, Beck A, Leize-Wagner E, François Y-N. Cutting-edge capillary electrophoresis characterization of monoclonal antibodies and related products. J Chromatogr B Analyt Technol Biomed Life Sci. 2016;1032:61–78.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Hapuarachchi S, Fodor S, Apostol I, Huang G. Use of capillary electrophoresis-sodium dodecyl sulfate to monitor disulfide scrambled forms of an fc fusion protein during purification process. Anal Biochem. 2011;414:187–95.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Hunt G, Nashabeh W. Capillary electrophoresis sodium dodecyl sulfate nongel sieving analysis of a therapeutic recombinant monoclonal antibody: a biotechnology perspective. Anal Chem. 1999;71:2390–7.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Krull IS, Liu X, Dai J, Gendreau C, Li G. HPCE methods for the identification and quantitation of antibodies, their conjugates and complexes. J Pharm Biomed Anal. 1997;16:377–93.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Lee HG, Chang S, Fritsche E. Rational approach to quantitative sodium dodecyl sulfate capillary gel electrophoresis of monoclonal antibodies. J Chromatogr A. 2002;947:143–9.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Tous GI, Wei Z, Feng J, Bilbulian S, Bowen S, Smith J, et al. Characterization of a novel modification to monoclonal antibodies: thioether cross-link of heavy and light chains. Anal Chem. 2005;77:2675–82.

  25. 25.

    Kubota K, Kobayashi N, Yabuta M, Ohara M, Naito T, Kubo T, et al. Identification and characterization of a thermally cleaved fragment of monoclonal antibody-a detected by sodium dodecyl sulfate-capillary gel electrophoresis. J Pharm Biomed Anal. 2017;140:98–104.

  26. 26.

    Arrell MS, Kálmán F. Estimation of protein concentration at high sensitivity using SDS-capillary gel electrophoresis-laser induced fluorescence detection with 3-(2-furoyl)quinoline-2-carboxaldehyde protein labeling. Electrophoresis. 2016;37:2913–21.

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Kahle J, Maul KJ, Wätzig H. The next generation of capillary electrophoresis instruments: performance of CE-SDS protein analysis. Electrophoresis. 2018;39:311–25.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Lee HG. High-performance sodium dodecyl sulfate-capillary gel electrophoresis of antibodies and antibody fragments. J Immunol Methods. 2000;234:71–81.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Salas-Solano O, Tomlinson B, Du S, Parker M, Strahan A, Ma S. Optimization and validation of a quantitative capillary electrophoresis sodium dodecyl sulfate method for quality control and stability monitoring of monoclonal antibodies. Anal Chem. 2006;78:6583–94.

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Zhang J, Burman S, Gunturi S, Foley JP. Method development and validation of capillary sodium dodecyl sulfate gel electrophoresis for the characterization of a monoclonal antibody. J Pharm Biomed Anal. 2010;53:1236–43.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Mahler H-C, Friess W, Grauschopf U, Kiese S. Protein aggregation: pathways, induction factors and analysis. J Pharm Sci. 2009;98:2909–34.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Kaschak T, Boyd D, Yan B. Characterization of glycation in an IgG1 by capillary electrophoresis sodium dodecyl sulfate and mass spectrometry. Anal Biochem. 2011;417:256–63.

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Lacher NA, Wang Q, Roberts RK, Holovics HJ, Aykent S, Schlittler MR, et al. Development of a capillary gel electrophoresis method for monitoring disulfide isomer heterogeneity in IgG2 antibodies. Electrophoresis. 2010;31:448–58.

  34. 34.

    Martinez T, Guo A, Allen MJ, Han M, Pace D, Jones J, et al. Disulfide connectivity of human immunoglobulin G2 structural isoforms. Biochemistry. 2008;47:7496–508.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Wypych J, Li M, Guo A, Zhang Z, Martinez T, Allen MJ, et al. Human IgG2 antibodies display disulfide-mediated structural isoforms. J Biol Chem. 2008;283:16194–205.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Boja ES, Fales HM. Overalkylation of a protein digest with Iodoacetamide. Anal Chem. 2001;73:3576–82. https://doi.org/10.1021/ac0103423.

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Sreejit G, Ahmed A, Parveen N, Jha V, Valluri VL, Ghosh S, et al. The ESAT-6 protein of mycobacterium tuberculosis interacts with beta-2-microglobulin (β2M) affecting antigen presentation function of macrophage. PLoS Pathog. 2014;10:e1004446.

    Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Zheng H-J, Shen B-B, Wang J, Wang H, Huo G-L, Huang L-R, et al. Uncommon peptide bond cleavage of glucagon from a specific vendor under near neutral to basic conditions. Pharm Res. 2019;36:118.

    CAS  Article  PubMed  Google Scholar 

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Correspondence to Wei-Jie Fang.

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Shen, BB., Zhang, Z., Yuan, JJ. et al. Formation of an Unprecedented Impurity during CE-SDS Analysis of a Recombinant Protein. Pharm Res 37, 228 (2020). https://doi.org/10.1007/s11095-020-02947-0

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Key words

  • alkylation
  • CE-SDS
  • impurity
  • LC-MS
  • peptide mapping
  • sample preparation