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.
Similar content being viewed by others
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
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.
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.
Guttman A. Capillary sodium dodecyl sulfate-gel electrophoresis of proteins. Electrophoresis. 1996;17:1333–41.
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.
Manabe T. Capillary electrophoresis of proteins for proteomic studies. Electrophoresis. 1999;20:3116–21.
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.
Zhu Z, Lu JJ, Liu S. Protein separation by capillary gel electrophoresis: a review. Anal Chim Acta. 2012;709:21–31.
Zhao SS, Chen DDY. Applications of capillary electrophoresis in characterizing recombinant protein therapeutics. Electrophoresis. 2014;35:96–108.
Cerutti ML, Pesce A, Bès C, Seigelchifer M. Physicochemical and biological characterization of RTXM83, a new rituximab biosimilar. BioDrugs. 2019;33:307–19.
Rustandi RR, Washabaugh MW, Wang Y. Applications of CE SDS gel in development of biopharmaceutical antibody-based products. Electrophoresis. 2008;29:3612–20.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Kahle J, Maul KJ, Wätzig H. The next generation of capillary electrophoresis instruments: performance of CE-SDS protein analysis. Electrophoresis. 2018;39:311–25.
Lee HG. High-performance sodium dodecyl sulfate-capillary gel electrophoresis of antibodies and antibody fragments. J Immunol Methods. 2000;234:71–81.
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.
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.
Mahler H-C, Friess W, Grauschopf U, Kiese S. Protein aggregation: pathways, induction factors and analysis. J Pharm Sci. 2009;98:2909–34.
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.
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.
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.
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.
Boja ES, Fales HM. Overalkylation of a protein digest with Iodoacetamide. Anal Chem. 2001;73:3576–82. https://doi.org/10.1021/ac0103423.
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.
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.
Author information
Authors and Affiliations
Corresponding author
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11095-020-02947-0