, 73:1145 | Cite as

CZE with On-line Micellar Sample Stacking for Determination of Protein Concentration of Biopharmaceuticals

  • Medhat A. Al-GhobashyEmail author
  • Martin A. K. Williams
  • Götz Laible
  • David R. K. Harding


A capillary zone electrophoresis total protein assay was developed and validated in polyethylene oxide (PEO) dynamically coated capillaries. On-line large-volume sample stacking was employed. Protein samples were denatured using SDS and then injected into PEO-filled capillaries. Such treatment enabled injection of a sample volume of ≈8% of the total capillary volume and stacking of protein-SDS molecules at the interface between the sample plug and the PEO plug. Results showed that SDS enhanced the sensitivity not only by protein denaturation but also by forming micelles, in which protein-SDS partitioned. Sensitivity of the method was further enhanced through using capillaries with (tenfold) extended detection pathlength. Such strategies resulted in a limit of detection of 0.26 μg mL−1 (3.64 nM BSA). A linear relationship between protein concentration and integrated peak area was obtained over a wide concentration range (8.49–135.87 μg mL−1R 2 = 0.995). The method is particularly useful for determination of total protein concentration in chromatography fractions. It overcomes low UV absorptivity of proteins, presence of UV absorbing additives and high salt content. Contrary to conventional methods for determination of protein concentration, this method does not involve an interaction with a dye. Thus, variations due to differences in surface properties among proteins or due to differences in posttranslational modifications of the same protein are eliminated. The protocol was successfully applied for the determination of the concentration of a biopharmaceutical protein rhMBP in chromatography fractions. This protein has been previously produced in milk of transgenic cows and several charge isoforms were detected.


Capillary zone electrophoresis Large-volume sample stacking Polyethylene oxide Biopharmaceuticals Human myelin basic protein 

Supplementary material

10337_2011_2003_MOESM1_ESM.bmp (1.5 mb)
Supplementary material 1 (BMP 1538 kb)
10337_2011_2003_MOESM2_ESM.bmp (1.9 mb)
Supplementary material 2 (BMP 1957 kb)


  1. 1.
    Zhang C, Van Cott KE (2007) In: Shukla AA, Etzel MR, Gadam S (eds) Process scale bioseparations for the biopharmaceutical industry. Taylor and Francis Group, pp 367Google Scholar
  2. 2.
    Kelner DN, Bhalgat MK (2007) In: Shukla AA, Etzel MR, Gadam S (eds) Process scale bioseparations for the biopharmaceutical industry. Taylor and Francis Group, pp 395–418Google Scholar
  3. 3.
    Al-Ghobashy MA, Williams MAK, Brophy B, Laible G, Harding DRK (2009) J Chrom B 877:1667–1677CrossRefGoogle Scholar
  4. 4.
    Al-Ghobashy MA, Williams MAK, Laible G, Harding DRK (2010) J Mol Recognit 23:84–92Google Scholar
  5. 5.
    Lutton JD, Winston R, Rodman TC (2004) Exp Biol Med 229:12–20Google Scholar
  6. 6.
    Harauz G, Ishiyama N, Hill CMD, Bates IR, Libich DS, Fares C (2004) Micron 35:503–542CrossRefGoogle Scholar
  7. 7.
    Planning S (2007) Curr Protoc Protein Sci Unit 3.4:1–29Google Scholar
  8. 8.
    Dolnik V (2008) Electrophoresis 29:143–156CrossRefGoogle Scholar
  9. 9.
    Chun M-S, Kang D, Kim Y, Chung D (2001) Microchem J 70:247–253CrossRefGoogle Scholar
  10. 10.
    Chang P-L, Chiu T-C, Chang H-T (2006) Electrophoresis 27:1922–1931CrossRefGoogle Scholar
  11. 11.
    Huang Y-F, Huang C-C, Hu C-C, Chang H-T (2006) Electrophoresis 27:3503–3522CrossRefGoogle Scholar
  12. 12.
    Horvath J, Dolnik V (2001) Electrophoresis 22:644–655CrossRefGoogle Scholar
  13. 13.
    Miksik I, Sedlakova P, Mikulikova K, Eckhardt A, Cserhati T, Horvath T (2006) Biomed Chromatogr 20:458–465CrossRefGoogle Scholar
  14. 14.
    Huang Y-F, Hsieh M-M, Tseng W-L, Chang H-T (2006) J Proteome Res 5:429–436CrossRefGoogle Scholar
  15. 15.
    Horka M, Ruzicka F, Horky J, Hola V, Slais K (2006) J Chromatogr B 841:152–159CrossRefGoogle Scholar
  16. 16.
    Kuo C-Y, Chiou S-S, Wu S-M (2006) Electrophoresis 27:2905–2909CrossRefGoogle Scholar
  17. 17.
    Lucy CA, MacDonald AM, Gulcev MD (2008) J Chromatogr A 1184:81–105Google Scholar
  18. 18.
    Hu S, Jiang J, Cook LM, Richards DP, Horlick L, Wong B, Dovichi NJ (2002) Electrophoresis 23:3136–3142CrossRefGoogle Scholar
  19. 19.
    Laemmli UK (1970) Nature 227:680–685CrossRefGoogle Scholar
  20. 20.
    Otzen DE (2002) Biophys J 83:2219–2230CrossRefGoogle Scholar
  21. 21.
    Parker W, Song PS (1992) Biophys J 61:1435–1439CrossRefGoogle Scholar
  22. 22.
    Zor T, Selinger Z (1996) Anal Biochem 236:302–308CrossRefGoogle Scholar
  23. 23.
    Al-Ghobashy MA, Williams MAK, Harding DRK (2008) Anal Lett 41:1914–1931CrossRefGoogle Scholar
  24. 24.
    Varenne A, Descroix S (2008) Anal Chem Acta 628:9–23CrossRefGoogle Scholar
  25. 25.
    Stoyanov AV, Pawliszyn J (2004) Analyst 129:979–982CrossRefGoogle Scholar
  26. 26.
    Boušková E, Presutti C, Gebauer P, Fanali S, Beckers JL, Bocek P (2004) Electrophoresis 25:355–359CrossRefGoogle Scholar
  27. 27.
    Thorsteinsson MV, Richter J, Lee AL, DePhillips P (2005) Anal Biochem 340:220–225CrossRefGoogle Scholar
  28. 28.
    Liu Y, Ling X, Fan H, Zhong Y, Li D, Wang Y (2007) J Chromatogr A 1143:284–287CrossRefGoogle Scholar
  29. 29.
    Jing P, Kaneta T, Imasaka T (2005) Anal Sci 21:37–42CrossRefGoogle Scholar
  30. 30.
    ICH (2005) Validation of analytical procedures: test and methodology. In: International conference on harmonization of technical requirements for registration of pharmaceticals for human use. Google Scholar
  31. 31.
    Goodall DM, Williams SJ, Lloyd DK (1991) Trends Anal Chem 10:272–279CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Medhat A. Al-Ghobashy
    • 1
    • 2
    Email author
  • Martin A. K. Williams
    • 3
  • Götz Laible
    • 4
  • David R. K. Harding
    • 3
  1. 1.Analytical Chemistry Department, Faculty of PharmacyCairo UniversityCairoEgypt
  2. 2.Biotechnology Centre, Faculty of PharmacyCairo UniversityCairoEgypt
  3. 3.Institute of Fundamental SciencesMassey UniversityPalmerston NorthNew Zealand
  4. 4.Agresearch, Ruakura Research CentreHamiltonNew Zealand

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