Skip to main content

Bioanalytical Assay for Characterization of Antibody-Drug Conjugates (ADCs)

  • Chapter
  • First Online:
Antibody-Drug Conjugates

Part of the book series: AAPS Advances in the Pharmaceutical Sciences Series ((AAPS,volume 17))

Abstract

Antibody–drug conjugates (ADCs) combine the characteristics of large-molecule biologics and small-molecule drugs. Although a number of assays have been reported to characterize the ADCs, new and comprehensive bioanalytical strategy to overcome the limitations of the current methods is required for better characterization of ADCs for clinical use. An appropriate bioanalytical assay is essential for characterization of ADCs including physiochemical and pharmacokinetic properties due to their complex and heterogeneous structures. The commonly used bioanalytical methods that are typically implemented to characterize various ADCs are summarized in this chapter. The challenges and perspectives of the assays are also discussed.

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

Access this chapter

Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  • Advani A, Coiffier B, Czuczman MS, Dreyling M, Foran J, Gine E et al (2010) Safety, pharmacokinetics, and preliminary clinical activity of inotuzumab ozogamicin, a novel immunoconjugate for the treatment of B-cell non-Hodgkin’s lymphoma: results of a phase I study. J Clin Oncol 28(12):2085–2093. doi:10.1200/JCO.2009.25.1900

    Article  CAS  PubMed  Google Scholar 

  • Alley SC, Anderson KE (2013) Analytical and bioanalytical technologies for characterizing antibody–drug conjugates. Curr Opin Chem Biol 17(3):406–411. doi:http://dx.doi.org/10.1016/j.cbpa.2013.03.022

    Article  CAS  PubMed  Google Scholar 

  • Alley SC, Benjamin DR, Jeffrey SC, Okeley NM, Meyer DL, Sanderson RJ et al (2008) Contribution of linker stability to the activities of anticancer immunoconjugates. Bioconjug Chem 19(3):759–765. doi:10.1021/bc7004329

    Article  CAS  PubMed  Google Scholar 

  • Beck A (2014) Review of antibody-drug conjugates. Methods in molecular biology series: a book edited by Laurent Ducry. mAbs 6(1):30–33

    Article  PubMed Central  PubMed  Google Scholar 

  • Boghaert ER, Khandke KM, Sridharan L, Dougher M, DiJoseph JF, Kunz A et al (2008) Determination of pharmacokinetic values of calicheamicin-antibody conjugates in mice by plasmon resonance analysis of small (5 microl) blood samples. Cancer Chemother Pharmacol 61(6):1027–1035. doi:10.1007/s00280-007-0560-2

    Article  CAS  PubMed  Google Scholar 

  • Buckwalter M, Dowell JA, Korth-Bradley J, Gorovits B, Mayer PR (2004) Pharmacokinetics of gemtuzumab ozogamicin as a single-agent treatment of pediatric patients with refractory or relapsed acute myeloid leukemia. J Clin Pharmacol 44(8):873–880. doi:10.1177/0091270004267595

    Article  CAS  PubMed  Google Scholar 

  • Carter PJ, Senter PD (2008) Antibody-drug conjugates for cancer therapy. Cancer J 14(3):154–169. doi:10.1097/PPO.0b013e318172d704

    Article  CAS  PubMed  Google Scholar 

  • Chari RV, Martell BA, Gross JL, Cook SB, Shah SA, Blattler WA et al (1992) Immunoconjugates containing novel maytansinoids: promising anticancer drugs. Cancer Res 52(1):127–131

    CAS  PubMed  Google Scholar 

  • Chen Y (2013) Drug-to-Antibody Ratio (DAR) by UV/Vis spectroscopy. In: Ducry L (ed) Antibody-drug conjugates. Humana Press, New York City, NY, USA, pp 267–273

    Google Scholar 

  • Chen Y, Clark S, Wong T, Dennis MS, Luis E, Zhong F et al (2007) Armed antibodies targeting the mucin repeats of the ovarian cancer antigen, MUC16, are highly efficacious in animal tumor models. Cancer Res 67(10):4924–4932. doi:10.1158/0008-5472.CAN-06-4512

    Article  CAS  PubMed  Google Scholar 

  • Clark T, Han X, King L, Barletta F (2013) Insights into antibody–drug conjugates: bioanalysis and biomeasures in discovery. Bioanalysis 5(9):985–987. doi:10.4155/bio.13.65

    Article  CAS  PubMed  Google Scholar 

  • Cordoba AJ, Shyong BJ, Breen D, Harris RJ (2005) Non-enzymatic hinge region fragmentation of antibodies in solution. J chromatogr B, Analyt Technol Biomed Life Sci 818(2):115–121. doi:10.1016/j.jchromb.2004.12.033

    Article  CAS  Google Scholar 

  • Dere R, Yi JH, Lei C, Saad OM, Huang C, Li Y et al (2013) PK assays for antibody-drug conjugates: case study with ado-trastuzumab emtansine. Bioanalysis 5(9):1025–1040. doi:10.4155/bio.13.72

    Article  CAS  PubMed  Google Scholar 

  • DeSilva B, Smith W, Weiner R, Kelley M, Smolec J, Lee B et al (2003) Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules. Pharm Res 20(11):1885–1900

    Article  CAS  PubMed  Google Scholar 

  • DiJoseph JF, Armellino DC, Boghaert ER, Khandke K, Dougher MM, Sridharan L et al (2004) Antibody-targeted chemotherapy with CMC-544: a CD22-targeted immunoconjugate of calicheamicin for the treatment of B-lymphoid malignancies. Blood 103(5):1807–1814. doi:10.1182/blood -2003-07-2466

    Article  CAS  PubMed  Google Scholar 

  • Dowell JA, Korth-Bradley J, Liu H, King SP, Berger MS (2001) Pharmacokinetics of gemtuzumab ozogamicin, an antibody-targeted chemotherapy agent for the treatment of patients with acute myeloid leukemia in first relapse. J Clin Pharmacol 41(11):1206–1214

    Article  CAS  PubMed  Google Scholar 

  • Francisco JA, Cerveny CG, Meyer DL, Mixan BJ, Klussman K, Chace DF et al (2003) cAC10-vcMMAE, an anti-CD30–monomethyl auristatin E conjugate with potent and selective antitumor activity. Blood 102(4):1458–1465. doi:10.1182/blood-2003-01-0039

    Article  CAS  PubMed  Google Scholar 

  • Gorovits B, Alley SC, Bilic S, Booth B, Kaur S, Oldfield P et al (2013) Bioanalysis of antibody-drug conjugates: American association of pharmaceutical scientists antibody-drug conjugate working group position paper. Bioanalysis 5(9):997–1006. doi:10.4155/bio.13.38

    Article  CAS  PubMed  Google Scholar 

  • Hamblett KJ, Senter PD, Chace DF, Sun MM, Lenox J, Cerveny CG et al (2004) Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res 10(20):7063–7070. doi:10.1158/1078-0432.CCR-04-0789

    Article  CAS  PubMed  Google Scholar 

  • Hinman LM, Hamann PR, Wallace R, Menendez AT, Durr FE, Upeslacis J (1993) Preparation and characterization of monoclonal antibody conjugates of the calicheamicins: a novel and potent family of antitumor antibiotics. Cancer Res 53(14):3336–3342

    CAS  PubMed  Google Scholar 

  • Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S et al (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nature Biotechnol 26(8):925–932. doi:10.1038/nbt.1480

    Article  CAS  Google Scholar 

  • Junutula JR, Flagella KM, Graham RA, Parsons KL, Ha E, Raab H et al (2010) Engineered thio-trastuzumab-dm1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2–positive breast cancer. Clin Cancer Res 16(19):4769–4778. doi:10.1158/1078-0432.ccr-10-0987

    Article  CAS  PubMed  Google Scholar 

  • Kaur S, Xu K, Saad OM, Dere RC, Carrasco-Triguero M (2013) Bioanalytical assay strategies for the development of antibody-drug conjugate biotherapeutics. Bioanalysis 5(2):201–226. doi:10.4155/bio.12.299

    Article  CAS  PubMed  Google Scholar 

  • King HD, Dubowchik GM, Mastalerz H, Willner D, Hofstead SJ, Firestone RA et al (2002) Monoclonal antibody conjugates of doxorubicin prepared with branched peptide linkers: inhibition of aggregation by methoxytriethyleneglycol chains. J Med Chem 45(19):4336–4343

    Article  CAS  PubMed  Google Scholar 

  • Kovtun YV, Audette CA, Ye Y, Xie H, Ruberti MF, Phinney SJ et al (2006) Antibody-drug conjugates designed to eradicate tumors with homogeneous and heterogeneous expression of the target antigen. Cancer Res 66(6):3214–3221. doi:10.1158/0008-5472.CAN-05-3973

    Article  CAS  PubMed  Google Scholar 

  • Kozak K, Raab H (2013) Assay methodologies and challenges. In: Phillips GL (ed) Antibody-drug conjugates and immunotoxins: Springer, New York, pp 41–56

    Google Scholar 

  • Lazar AC, Wang L, Blättler WA, Amphlett G, Lambert JM, Zhang W (2005) Analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometry. Rapid Commun Mass Spectrom 19(13):1806–1814. doi:10.1002/rcm.1987

    Article  CAS  PubMed  Google Scholar 

  • Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E et al (2008) Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res 68(22):9280–9290. doi:10.1158/0008-5472.CAN-08-1776

    Article  CAS  PubMed  Google Scholar 

  • Luckey JA, Drossman H, Kostichka T, Smith LM (1993) High-speed DNA sequencing by capillary gel electrophoresis. In: Ray W (ed) Methods in enzymology. Academic Press, San Diego, CA, USA, pp 154–172

    Google Scholar 

  • McDonagh CF, Turcott E, Westendorf L, Webster JB, Alley SC, Kim K et al (2006) Engineered antibody–drug conjugates with defined sites and stoichiometries of drug attachment. Protein Eng Des Sel 19(7):299–307. doi:10.1093/protein/gzl013

    Article  CAS  PubMed  Google Scholar 

  • Ouyang J (2013) Drug-to-Antibody Ratio (DAR) and drug load distribution by hydrophobic interaction chromatography and reversed phase high-performance liquid chromatography. In: Ducry L (ed) Antibody-drug conjugates. Humana Press, New York City, NY, USA, pp 275–283

    Google Scholar 

  • Polakis P (2005) Arming antibodies for cancer therapy. Curr Opin Pharmacol 5(4):382–387. doi:10.1016/j.coph.2005.04.008

    Article  CAS  PubMed  Google Scholar 

  • Pollack VA, Alvarez E, Tse KF, Torgov MY, Xie S, Shenoy SG et al (2007) Treatment parameters modulating regression of human melanoma xenografts by an antibody-drug conjugate (CR011-vcMMAE) targeting GPNMB. Cancer Chemother Pharmacol 60(3):423–435. doi:10.1007/s00280-007-0490-z

    Article  CAS  PubMed  Google Scholar 

  • Quiles S, Raisch KP, Sanford LL, Bonner JA, Safavy A (2009) Synthesis and preliminary biological evaluation of high-drug-load paclitaxel-antibody conjugates for tumor-targeted chemotherapy. J Med Chem 53(2):586–594. doi:10.1021/jm900899 g

    Article  Google Scholar 

  • Ramakrishnan NA, Drescher MJ, Sheikhali SA, Khan KM, Hatfield JS, Dickson MJ et al (2006) Molecular identification of an N-type Ca2+ channel in saccular hair cells. Neuroscience 139(4):1417–1434. doi:http://dx.doi.org/10.1016/j.neuroscience.2006.01.064

    Article  CAS  PubMed  Google Scholar 

  • Safavy A, Bonner JA, Waksal HW, Buchsbaum DJ, Gillespie GY, Khazaeli MB et al (2003) Synthesis and biological evaluation of paclitaxel-C225 conjugate as a model for targeted drug delivery. Bioconjug Chem 14(2):302–310. doi:10.1021/bc020033z

    Article  CAS  PubMed  Google Scholar 

  • Sanderson RJ, Hering MA, James SF, Sun MM, Doronina SO, Siadak AW et al (2005) In vivo drug-linker stability of an anti-CD30 dipeptide-linked auristatin immunoconjugate. Clin Cancer Res 11(2 Pt 1):843–852

    CAS  PubMed  Google Scholar 

  • Sapra P, Stein R, Pickett J, Qu Z, Govindan SV, Cardillo TM et al (2005) Anti-CD74 antibody-doxorubicin conjugate, IMMU-110, in a human multiple myeloma xenograft and in monkeys. Clin Cancer Res 11(14):5257–5264. doi:10.1158/1078-0432.CCR-05-0204

    Article  CAS  PubMed  Google Scholar 

  • Schuck P (1997) Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of interactions between biological macromolecules1. Annu Rev Biophys Biomol Struct 26(1):541–566. doi:10.1146/annurev.biophys.26.1.541

    Article  CAS  PubMed  Google Scholar 

  • Shen BQ, Xu K, Liu L, Raab H, Bhakta S, Kenrick M et al (2012) Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nature Biotechnol 30(2):184–189. doi:10.1038/nbt.2108

    Article  CAS  Google Scholar 

  • Siegel MM, Tabei K, Kunz A, Hollander IJ, Hamann RR, Bell DH et al (1997) Calicheamicin derivatives conjugated to monoclonal antibodies: determination of loading values and distributions by infrared and UV matrix-assisted laser desorption/ionization mass spectrometry and electrospray ionization mass spectrometry. Anal Chem 69(14):2716–2726

    Article  CAS  PubMed  Google Scholar 

  • Stephan JP, Chan P, Lee C, Nelson C, Elliott JM, Bechtel C et al (2008) Anti-CD22-MCC-DM1 and MC-MMAF conjugates: impact of assay format on pharmacokinetic parameters determination. Bioconjug Chem 19(8):1673–1683. doi:10.1021/bc800059t

    Article  CAS  PubMed  Google Scholar 

  • Stephan JP, Kozak KR, Wong WL (2011) Challenges in developing bioanalytical assays for characterization of antibody-drug conjugates. Bioanalysis 3(6):677–700. doi:10.4155/bio.11.30

    Article  CAS  PubMed  Google Scholar 

  • Sun MMC, Beam KS, Cerveny CG, Hamblett KJ, Blackmore RS, Torgov MY et al (2005) Reduction–alkylation strategies for the modification of specific monoclonal antibody disulfides. Bioconjug Chem 16(5):1282–1290. doi:10.1021/bc050201y

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Teicher BA, Chari RV (2011) Antibody conjugate therapeutics: challenges and potential. Clin Cancer Res 17(20):6389–6397. doi:10.1158/1078-0432.CCR-11-1417

    Article  CAS  PubMed  Google Scholar 

  • Tolcher AW, Ochoa L, Hammond LA, Patnaik A, Edwards T, Takimoto C et al (2003) Cantuzumab mertansine, a maytansinoid immunoconjugate directed to the CanAg antigen: a phase I, pharmacokinetic, and biologic correlative study. J Clin Oncol 21(2):211–222

    Article  CAS  PubMed  Google Scholar 

  • US FDA (2013) Guidance for industry: bioanalytical method validation. U.S. Food and Drug Administration, Washington, DC

    Google Scholar 

  • Vater CA, Reid K, Bartle LM, Goldmacher VS (1995) Characterization of antibody binding to cell surface antigens using a plasma membrane-bound plate assay. Anal Biochem 224(1):39–50. doi:http://dx.doi.org/10.1006/abio.1995.1006

    Article  CAS  PubMed  Google Scholar 

  • Wakankar A, Chen Y, Gokarn Y, Jacobson FS (2011) Analytical methods for physicochemical characterization of antibody drug conjugates. mAbs 3(2):161–172

    Article  PubMed Central  PubMed  Google Scholar 

  • Wang L, Amphlett G, Blättler WA, Lambert JM, Zhang W (2005) Structural characterization of the maytansinoid–monoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry. Protein Sci 14(9):2436–2446. doi:10.1110/ps.051478705

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Xie H, Audette C, Hoffee M, Lambert JM, Blattler WA (2004) Pharmacokinetics and biodistribution of the antitumor immunoconjugate, cantuzumab mertansine (huC242-DM1), and its two components in mice. J Pharmacol Exp Therap 308(3):1073–1082. doi:10.1124/jpet.103.060533

    Article  CAS  Google Scholar 

  • Xu K, Liu L, Saad OM, Baudys J, Williams L, Leipold D et al (2011) Characterization of intact antibody–drug conjugates from plasma/serum in vivo by affinity capture capillary liquid chromatography–mass spectrometry. Anal Biochem 412(1):56–66. doi:http://dx.doi.org/10.1016/j.ab.2011.01.004

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Zhijun Wang .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2015 American Association of Pharmaceutical Scientists

About this chapter

Cite this chapter

Xie, C., Wang, Z. (2015). Bioanalytical Assay for Characterization of Antibody-Drug Conjugates (ADCs). In: Wang, J., Shen, WC., Zaro, J. (eds) Antibody-Drug Conjugates. AAPS Advances in the Pharmaceutical Sciences Series, vol 17. Springer, Cham. https://doi.org/10.1007/978-3-319-13081-1_6

Download citation

Publish with us

Policies and ethics