Abstract
UV/Vis spectroscopy is a simple and convenient method to determine protein concentrations as well as the average number of drugs that are conjugated to the antibody in an antibody–drug conjugate (ADC). Using the measured absorbances of the ADC and the extinction coefficients of the antibody and the drug, the average drug-to-antibody ratio (DAR) can be determined.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wakankar A, Chen Y, Gokarn Y (2011) Analytical methods for physicochemical characterization of antibody drug conjugates. MAbs 3:161–172
Hamblett KJ, Senter PD, Chace DF et al (2004) Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res 10:7063–7070
Hinman LM, Hamann PR, Wallace R et al (1993) Preparation and characterization of monoclonal antibody conjugates of the calicheamicins: a novel and potent family of antitumor antibiotics. Cancer Res 53:3336–3342
Siegel MM, Hollander IJ, Hamann PR et al (1991) Matrix-assisted UV-laser desorption/ionization mass spectrometric analysis of monoclonal antibodies for the determination of carbohydrate, conjugated chelator and conjugated drug content. Anal Chem 63:2470–2481
Lazar AC, Wang L, Blattler WA et al (2005) Analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometry. Rapid Commun Mass Spectrom 19:1806–1814
Schoonheydt RA (2010) UV-VIS-NIR spectroscopy and microscopy of heterogeneous catalysts. Chem Soc Rev 39:5051–5066
Pretsch E, Bühlmann P, Badertscher M (2009) Structure determination of organic compounds, tables of spectral data, 4th edn. Springer, New York, pp 401–420
Schmid F (2001) Biological macromolecules: UV-visible spectrophotometry. Encyclopedia Life sci. doi:10.1038/npg.els.0003142
Wang L, Amphlett G, Blattler WA et al (2005) Structural characterization of the maytansinoid-monoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry. Protein Sci 14:2436–2446
Hudecz F, Garnett MC, Khan T et al (1992) The influence of synthetic conditions on the stability of methotrexate-monoclonal antibody conjugates determined by reversed phase high performance liquid chromatography. Biomed Chromatogr 6:128–132
Chari RV, Jackel KA, Bourret LA et al (1995) Enhancement of the selectivity and antitumor efficacy of a CC-1065 analogue through immunoconjugate formation. Cancer Res 55:4079–4084
Greenfield RS, Kaneko T, Daues A et al (1990) Evaluation in vitro of adriamycin immunoconjugates synthesized using an acid-sensitive hydrazone linker. Cancer Res 50:6600–6607
Willner D, Trail PA, Hofstead SJ et al (1993) (6-Maleimidocaproyl)hydrazone of doxorubicin—a new derivative for the preparation of immunoconjugates of doxorubicin. Bioconjug Chem 4:521–527
Moran J (2002) Presentation at 224th American Chemical Society National Meeting, 18–22 Aug, Boston, MA
Laguzza BC, Nichols CL, Briggs SL et al (1989) New antitumor monoclonal antibody-vinca conjugates LY203725 and related compounds: design, preparation and representative in vivo activity. J Med Chem 32:548–555
Benson AM, Suruda AJ, Talalay P (1975) Concentration-dependent association of A-3-ketosteroid isomerase of Pseudomonas testosteroni. J Biol Chem 250:276–280
Jaenicke L (1974) A rapid micromethod for the determination of nitrogen and phosphate in biological material. Anal Biochem 61:623–627
Pace CN, Vajdos F, Fee L et al (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci 4:2411–2423
Acknowledgements
We thank Pat Rancatore, Richard Seipert, Boyan Zhang, and Andrea Ji for comments and help in preparing this review.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Chen, Y. (2013). Drug-to-Antibody Ratio (DAR) by UV/Vis Spectroscopy. In: Ducry, L. (eds) Antibody-Drug Conjugates. Methods in Molecular Biology, vol 1045. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-541-5_16
Download citation
DOI: https://doi.org/10.1007/978-1-62703-541-5_16
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-540-8
Online ISBN: 978-1-62703-541-5
eBook Packages: Springer Protocols