Advertisement

Monitorization of α1-Acid Glycoprotein Deglycosylation Using SU-8 Microchips Electrophoresis with LIF Detection

  • María del Mar Barrios-Romero
  • Agustín G. Crevillén
  • Angel Puerta
  • Mercedes de Frutos
  • José Carlos Diez-MasaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1972)

Abstract

In the last few years, biopharmaceuticals—therapeutic drugs which are generally obtained by using molecular biology techniques—have become a major growing sector in pharmaceutical industry. A large part of these biopharmaceuticals are therapeutic glycoproteins. The production of these drugs and their purification process are implying the development of efficient analytical methods, which allow quick and reliable control of the manufacturing process and ensuring the regulatory compliance about the quality of these drugs. Capillary gel electrophoresis (CGE) in the presence of sodium dodecyl sulfate (SDS) is becoming a method of choice in the quality control of these biopharmaceuticals. On the other hand, CGE can be improved if analyses are carried out in microchip format.

This chapter reports a detailed microchips gel electrophoresis (MGE) method to separate glycosylated and deglycosylated forms of α1-acid glycoprotein (AGP) labeled with Chromeo P540, using SU-8 microchips and laser induced fluorescence detection. Due to the analogy between AGP and some therapeutic glycoproteins, we have selected AGP as a model system to illustrate the potential of MGE in the analysis of this type of biopharmaceutical compounds.

Key words

Microchip gel electrophoresis SU-8 microchips LIF detection α1-acid glycoprotein AGP Glycoprotein Deglycosylation 

Notes

Acknowledgments

The authors acknowledge the Spanish Ministries of Science and Innovation and of Economy, Industry, and Competitiveness (grants PSS-010000-2008-6 and CTQ2013-43236-R, respectively) and CSIC (joint project 2009JP003 with the Japanese Society for Promotion of Science) for financial support. M.M.B-R thanks the CSIC for a JAE-Pre contract and A.G.C. acknowledges CSIC for JAE-Doc contract. These contracts are co-financed by the European Union under the European Social Fund (ESF).

References

  1. 1.
    Walsh G (2014) Biopharmaceutical benchmarks 2014. Nat Biotech 32(10):992–1000. https://doi.org/10.1038/nbt.3040CrossRefGoogle Scholar
  2. 2.
    Malgorzata KB (2017) Progress in biopharmaceutical development. Biotechnol Appl Biochem 65:306–322. https://doi.org/10.1002/bab.1617CrossRefGoogle Scholar
  3. 3.
    Jefferis R (2017) Recombinant proteins and monoclonal antibodies. Adv Biochem Eng Biotechnol. https://doi.org/10.1007/10_2017_32Google Scholar
  4. 4.
    Little MJ, Paquette DM, Roos PK (2006) Electrophoresis of pharmaceutical proteins: status quo. Electrophoresis 27(12):2477–2485. https://doi.org/10.1002/elps.200500951CrossRefPubMedGoogle Scholar
  5. 5.
    Zhang YJ, An HJ (2017) Technologies and strategies for bioanalysis of biopharmaceuticals. Bioanalysis 9(18):1343–1347. https://doi.org/10.4155/bio-2017-4981CrossRefGoogle Scholar
  6. 6.
    O’Flaherty R, Trbojevic-Akmacic I, Greville G, Rudd PM, Lauc G (2018) The sweet spot for biologics: recent advances in characterization of biotherapeutic glycoproteins. Expert Rev Proteomics 15(1):13–29. https://doi.org/10.1080/14789450.2018.1404907CrossRefPubMedGoogle Scholar
  7. 7.
    Creamer JS, Oborny NJ, Lunte SM (2014) Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis. Anal Methods 6(15):5427–5449. https://doi.org/10.1039/c4ay00447gCrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Szekrenyes A, Roth U, Kerekgyarto M, Szekely A, Kurucz I, Kowalewski K, Guttman A (2012) High-throughput analysis of therapeutic and diagnostic monoclonal antibodies by multicapillary SDS gel electrophoresis in conjunction with covalent fluorescent labeling. Anal Bioanal Chem 404(5):1485–1494. https://doi.org/10.1007/s00216-012-6213-2CrossRefPubMedGoogle Scholar
  9. 9.
    Smith MT, Zhang S, Adams T, DiPaolo B, Dally J (2017) Establishment and validation of a microfluidic capillary gel electrophoresis platform method for purity analysis of therapeutic monoclonal antibodies. Electrophoresis 38(9–10):1353–1365. https://doi.org/10.1002/elps.201600519CrossRefPubMedGoogle Scholar
  10. 10.
    Barrios-Romero MM, Crevillén AG, Diez-Masa JC (2013) Development of an SDS-gel electrophoresis method on SU-8 microchips for protein separation with LIF detection: application to the analysis of whey proteins. J Sep Sci 36(15):2530–2537. https://doi.org/10.1002/jssc.201300275CrossRefGoogle Scholar
  11. 11.
    Ongay S, Neususs C (2010) Isoform differentiation of intact AGP from human serum by capillary electrophoresis - mass spectrometry. Anal Bioanal Chem 398(2):845–855. https://doi.org/10.1007/s00216-010-3948-5CrossRefPubMedGoogle Scholar
  12. 12.
    Fournier T, Medjoubi-N N, Porquet D (2000) Alpha-1-acid glycoprotein. Biochim Biophys Acta 1482(1–2):157–171. https://doi.org/10.1016/S0167-4838(00)00153-9CrossRefGoogle Scholar
  13. 13.
    Shakalisava Y, Poitevin M, Viovy JL, Descroix S (2009) Versatile method for electroosmotic flow measurements in microchip electrophoresis. J Chromatogr A 1216(6):1030–1033. https://doi.org/10.1016/j.chroma.2008.12.029CrossRefPubMedGoogle Scholar
  14. 14.
    Guttman A, Nolan J (1994) Comparison of the separation of proteins by sodium dodecyl-sulfate slab gel-electrophoresis and capillary sodium dodecyl-sulfate gel-electrophoresis. Anal Biochem 221(2):285–289. https://doi.org/10.1006/abio.1994.1413CrossRefPubMedGoogle Scholar
  15. 15.
    Segrest JP, Jackson RL, Andrews EP, Marchesi VT (1971) Human erythrocyte membrane glycoprotein: a re-evaluation of molecular weight as determined by SDS polyacrylamide gel electrophoresis. Biochem Biophys Res Commun 44(2):390–395. https://doi.org/10.1016/0006-291x(71)90612-7CrossRefPubMedGoogle Scholar
  16. 16.
    Engel N, Weiss VU, Wenz C, Ruefer A, Kratzmeier M, Glueck S, Marchetti-Deschmann M, Allmaier G (2015) Challenges of glycoprotein analysis by microchip capillary gel electrophoresis. Electrophoresis 36(15):1754–1758. https://doi.org/10.1002/elps.201400510CrossRefPubMedGoogle Scholar
  17. 17.
    International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use. Pharmaceutical development Q8(R2). Current step 4 version dated August 2009. https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q8_R1/Step4/Q8_R2_Guideline.pdf
  18. 18.
    Lacher NA, Garrison KE, Martin RS, Lunte SM (2001) Microchip capillary electrophoresis/electrochemistry. Electrophoresis 22(12):2526–2536. https://doi.org/10.1002/1522-2683(200107)22:12<2526::AID-ELPS2526>3.0.CO;2-KCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • María del Mar Barrios-Romero
    • 1
  • Agustín G. Crevillén
    • 1
    • 2
  • Angel Puerta
    • 1
  • Mercedes de Frutos
    • 1
  • José Carlos Diez-Masa
    • 1
    Email author
  1. 1.Institute of Organic Chemistry (IQOG-CSIC)MadridSpain
  2. 2.Science Faculty – UNEDMadridSpain

Personalised recommendations