Skip to main content
SpringerLink
Log in

Assessment for quantification of biopharmaceutical protein using a microvolume spectrometer on microfluidic slides

  • Original Article
  • Published:
BioChip Journal Aims and scope Submit manuscript

Abstract

Spectrophotometric procedure is a traditional method for quantifying protein concentration in solution. The benefit of this method is simple and direct, not requiring standard materials, sample incubation, and exogenous chromophore addition. There are also some drawbacks, low detection limits and high amounts of protein needed. The recent advances in fiber optics and nano-technology enable to overcome those drawbacks by using microvolume sampling and extremely short pathlengths. In this study, the utility of the microvolume system with microfluidic carriers was assessed for concentration measurement of biopharmaceutical protein using absorbance at 280 nm and also compared to a traditional instrument. The results show a high correlation of estimation by Cohen’s d effect size and Pearson correlation coefficient between two different spectrometers. For UV spectral analysis, there are no statistically significant difference between those spectra from a microvolume spectrometer and a traditional one. For the extinction coefficient determination, two different spectrometers also give quite comparable and similar values. Thus a microvolume spectrometer can be used as an alternative over a traditional one to determine the concentration of biopharmaceutical protein. Moreover, the dynamic range for protein quantification by a microvolume spectrometer is over 10-fold higher than that of a traditional one.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Song, K.E. et al. Structural identification of modified amino acids on the interface between EPO and its receptor from EPO BRP, human recombinant erythropoietin by LC/MS analysis. Mol. Cells. 37, 819–826 (2014).

    Article  Google Scholar 

  2. Byeon, J., Choi Y.J. & Suh J.K. Structural Characterization of Modification on the Interface between a Ligand and its Receptor for Biopharmaceuticals. Receptors Clin. Investig. 2, 1–5 (2014).

    Google Scholar 

  3. Greer, F., Reason, A. & Rogers, M. Post-translational modifications of biopharmaceuticals-a challenge for analytical characterisation. European BioPharm. Rev. 106-111 (2002).

  4. Walsh, G. Post-translational modifications of protein biopharmaceuticals. Drug Discov. Today 15, 773–780 (2010).

    Article  CAS  Google Scholar 

  5. Diepold, K. et al. Simultaneous assessment of Asp isomerization and Asn deamidation in recombinant antibodies by LC-MS following incubation at elevated temperatures. PLoS One 7, e30295 (2012).

    Article  CAS  Google Scholar 

  6. Warburg, O. & Christian, W. Insulation and crystalisation of the Fermenting process of enolase. Biochem. Z. 310, 384–421 (1942).

    CAS  Google Scholar 

  7. Layne, E. Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymol. 3, 447–454 (1957).

    Article  Google Scholar 

  8. Grimsley, G.R. & Pace C.N. Spectrophotometric determination of protein concentration. Curr. Protoc. Protein Sci. 33, 3.1.1–3.1.9 (2004).

    Google Scholar 

  9. Pace, C.N., Vajdos, F., Fee, L., Grimsley, G. & Gray, T. How to measure and predict the molar absorption coefficient of a protein. Prot. Sci. 4, 2411–2423 (1995).

    Article  CAS  Google Scholar 

  10. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254 (1976).

    Article  CAS  Google Scholar 

  11. Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275 (1951).

    CAS  Google Scholar 

  12. Desjardins, P., Hansen, J.B. & Allen, M. Microvolume spectrophotometric and fluorometric determination of protein concentration. Curr. Protoc. Protein Sci. 55, 3.10.1–3.10.16 (2009).

    Article  Google Scholar 

  13. Pace, C.N. & Schmid, F.X. How to determine the molar absorbance coefficient of a protein. In Protein Structure: A Practical Approach, T.E. Creighton, eds. (Oxford, UK: IRL Press), 253–259 (1997).

    Google Scholar 

  14. Sittampalam, G.S., Ellis, R.M., Miner, D.J., Rickard, E.C. & Clodfelter, D.K. Evaluation of Amino Acid Analysis as Reference Method to Quantitate Highly Purified Proteins. J. Assoc. Offic. Anal. Chem. 71, 833–838 (1988).

    CAS  Google Scholar 

  15. Juris, O. Amino acid analysis. Methods Enzymol. 182, 587–601 (1990).

    Article  Google Scholar 

  16. Dunn, B. Quantitative amino acid analysis. Curr. Protoc. Mol. Biol. 76, 10.1B (2006).

    Google Scholar 

  17. Anders, J.C., Parten, B.F., Petrie, G.E., Marlowe, R.L. & McEntire, J.E. Using amino acid analysis to determine absorptivity constants: A validation case study using bovine serum albumin. BioPharm Int. 2, 30–37 (2003).

    Google Scholar 

  18. Gill, S.C. & Hippel, P.H. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319–326 (1989).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Bioanalysis, BIOnSYSTEMS, Inc., Seoul, Korea

    Jaehee Byeon & Kui Hyun Kang

  2. Newmedia Business, Seoul Media Institute of Technology, Seoul, Korea

    Hoe-Kyung Jung

  3. Newmedia Content, Seoul Media Institute of Technology, Seoul, Korea

    Jung-Keun Suh

Authors
  1. Jaehee Byeon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kui Hyun Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hoe-Kyung Jung
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jung-Keun Suh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jung-Keun Suh.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Byeon, J., Kang, K.H., Jung, HK. et al. Assessment for quantification of biopharmaceutical protein using a microvolume spectrometer on microfluidic slides. BioChip J 11, 21–29 (2017). https://doi.org/10.1007/s13206-016-1104-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13206-016-1104-9

Keywords

Search

Navigation