Bioanalytical Challenges in Support of Complex Modalities of Antibody-Based Therapeutics


Antibody-based therapeutic classes are evolving from monoclonal antibodies to antibody derivatives with complex structures to achieve advanced therapeutic effect. These antibody derivatives may contain multiple functional domains and are often vulnerable to in vivo biotransformation. Understanding the pharmacokinetics of these antibody derivatives requires a sophisticated bioanalytical approach to carefully characterize the whole drug and each functional domain with respect to quantity, functionality enabled by biotransformation, and corresponding immune responses. Ligand binding assays and liquid chromatography-mass spectrometry assays are predominantly used in bioanalytical support of monoclonal antibodies and are continuously used for antibody derivatives such as antibody drug conjugate and bispecific antibodies. However, they become increasingly cumbersome in coping with increased complexity of drug modality and associated biotransformation. In this mini-review, we examined the current pharmacokinetic assays in the literature for antibody drug conjugate and bispecific antibodies, and presented our view of promising bioanalytical technologies to address the distinct bioanalytical needs of complex modalities.

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  1. 1.

    Lu RM, Hwang YC, Liu IJ, Lee CC, Tsai HZ, Li HJ, et al. Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci. 2020;27(1).

  2. 2.

    Goulet DR, Atkins WM. Considerations for the design of antibody-based therapeutics. J Pharm Sci-US. 2020;109(1):74–103.

    CAS  Article  Google Scholar 

  3. 3.

    Mokhtari RB, Homayouni TS, Baluch N, Morgatskaya E, Kumar S, Das B, et al. Combination therapy in combating cancer. Oncotarget. 2017;8(23):38022–43.

    Article  PubMed Central  Google Scholar 

  4. 4.

    Lee JW, Kelley M, King LE, Yang JH, Salimi-Moosavi H, Tang MT, et al. Bioanalytical approaches to quantify “Total” and “Free” therapeutic antibodies and their targets: technical challenges and PK/PD applications over the course of drug development. Aaps Journal. 2011;13(1):99–110.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Wadhwa M, Knezevic I, Kang HN, Thorpe R. Immunogenicity assessment of biotherapeutic products: an overview of assays and their utility. Biologicals. 2015;43(5):298–306.

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Schadt S, Hauri S, Lopes F, Edelmann MR, Staack RF, Villasenor R, et al. Are biotransformation studies of therapeutic proteins needed? Scientific considerations and technical challenges. Drug Metab Dispos. 2019;47(12):1443–56.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Cong YT, Zhang Z, Zhang S, Hu LH, Gu JK. Quantitative MS analysis of therapeutic mAbs and their glycosylation for pharmacokinetics study. Proteom Clin Appl. 2016;10(4):303–14.

    CAS  Article  Google Scholar 

  8. 8.

    Jiang H, Myler H, Zeng JN, Mora J, Kolaitis G, Pillutla R. Perspectives on exploring hybrid LBA/LC-MS approach for clinical immunogenicity testing. Bioanalysis. 2019;11(17):1605–17.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Fandozzi C, Evans C, Wilson A, Su D, Anderson M, Clausen V, et al. 2019 white paper on recent issues in bioanalysis: chromatographic assays (part 1-innovation in small molecules and oligonucleotides & mass spectrometric method development strategies for large molecule bioanalysis). Bioanalysis. 2019;11(22):2029–48.

    Article  PubMed  Google Scholar 

  10. 10.

    Purushothama S, Dysinger M, Chen Y, Osterlund K, Mora J, Chunyk AG, et al. Emerging technologies for biotherapeutic bioanalysis from a high-throughput and multiplexing perspective: insights from an AAPS emerging technology action program committee. Bioanalysis. 2018;10(3):181–94.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Todoroki K, Mizuno H, Sugiyama E, Toyo'oka T. Bioanalytical methods for therapeutic monoclonal antibodies and antibody-drug conjugates: a review of recent advances and future perspectives. J Pharm Biomed Anal. 2020;179:112991.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Dere R, Yi JH, Lei C, Saad OM, Huang C, Li YH, et al. PK assays for antibody-drug conjugates: case study with ado-trastuzumab emtansine. Bioanalysis. 2013;5(9):1025–40.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Faria M, Peay M, Lam B, Ma E, Yuan M, Waldron M, et al. Multiplex LC-MS/MS assays for clinical bioanalysis of MEDI4276, an antibody-drug conjugate of tubulysin analogue attached via cleavable linker to a biparatopic humanized antibody against HER-2. Antibodies (Basel). 2019;8(1).

  14. 14.

    Myler H, Rangan VS, Wang J, Kozhich A, Cummings JA, Neely R, et al. An integrated multiplatform bioanalytical strategy for antibody-drug conjugates: a novel case study. Bioanalysis. 2015;7(13):1569–82.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    He JT, Su D, Ng C, Liu LN, Yu SF, Pillow TH, et al. High-resolution accurate-mass mass spectrometry enabling in-depth characterization of in vivo biotransformations for intact antibody-drug conjugates. Anal Chem. 2017;89(10):5476–83.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Kontermann RE, Brinkmann U. Bispecific antibodies. Drug Discov Today. 2015;20(7):838–47.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    FDA. Bispecific Antibody Development Programs Guidance 2019.

  18. 18.

    Silacci M, Lembke W, Woods R, Attinger-Toller I, Baenziger-Tobler N, Batey S, et al. Discovery and characterization of COVA322, a clinical-stage bispecific TNF/IL-17A inhibitor for the treatment of inflammatory diseases. Mabs. 2016;8(1):141–9.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Ma M, Colletti K, Yang TY, Leung S, Pederson S, Hottenstein CS, et al. Bioanalytical challenges and unique considerations to support pharmacokinetic characterization of bispecific biotherapeutics. Bioanalysis. 2019;11(5):427–35.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Trivedi A, Stienen S, Zhu M, Li H, Yuraszeck T, Gibbs J, et al. Clinical pharmacology and translational aspects of Bispecific antibodies. Cts-Clin Transl Sci. 2017;10(3):147–62.

    CAS  Article  Google Scholar 

  21. 21.

    Murphy RE, Kinhikar AG, Shields MJ, Del Rosario J, Preston R, Levin N, et al. Combined use of immunoassay and two-dimensional liquid chromatography mass spectrometry for the detection and identification of metabolites from biotherapeutic pharmacokinetic samples. J Pharmaceut Biomed. 2010;53(3):221–7.

    CAS  Article  Google Scholar 

  22. 22.

    Schaller TH, Foster MW, Thompson JW, Spasojevic I, Normantaite D, Moseley MA, et al. Pharmacokinetic analysis of a novel human EGFRvIII:CD3 bispecific antibody in plasma and whole blood using a high-resolution targeted mass spectrometry approach. J Proteome Res. 2019;18(8):3032–41.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Woodbury N, Bald E, Geist B, Yang TY. Application of multiplexed pharmacokinetic immunoassay to quantify in vivo drug forms and coadministered biologics. Bioanalysis. 2019;11(24):2251–68.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Xu KY, Liu LN, Maia M, Li J, Lowe J, Song A, et al. A multiplexed hybrid LC-MS/MS pharmacokinetic assay to measure two co-administered monoclonal antibodies in a clinical study. Bioanalysis. 2014;6(13):1781–94.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Kang L, Weng N, Jian W. LC-MS bioanalysis of intact proteins and peptides. Biomed Chromatogr. 2020;34(1):e4633.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Jian WY, Kang LJ, Burton L, Weng ND. A workflow for absolute quantitation of large therapeutic proteins in biological samples at intact level using LC-HRMS. Bioanalysis. 2016;8(16):1679–91.

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Qiu X, Kang LJ, Case M, Weng ND, Jian WY. Quantitation of intact monoclonal antibody in biological samples: comparison of different data processing strategies. Bioanalysis. 2018;10(13):1055–67.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Kang LJ, Camacho RC, Li WY, D'Aquino K, You S, Chuo V, et al. Simultaneous catabolite identification and quantitation of large therapeutic protein at the intact level by Immunoaffinity capture liquid chromatography-high-resolution mass spectrometry. Anal Chem. 2017;89(11):6066–76.

    CAS  Article  Google Scholar 

  29. 29.

    Lanshoeft C, Cianferani S, Heudi O. Generic hybrid ligand binding assay liquid chromatography high resolution mass spectrometry-based workflow for multiplexed human immunoglobulin G1 quantification at the intact protein level: application to preclinical pharmacokinetic studies. Anal Chem. 2017;89(4):2628–35.

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Jin W, Burton L, Moore I. LC-HRMS quantitation of intact antibody drug conjugate trastuzumab emtansine from rat plasma. Bioanalysis. 2018;10(11):851–62.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Zhang LY, Vasicek LA, Hsieh S, Zhang SL, Bateman KP, Henion J. Top-down LC-MS quantitation of intact denatured and native monoclonal antibodies in biological samples. Bioanalysis. 2018;10(13):1039–54.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Chen JQ, Wakefield LM, Goldstein DJ. Capillary nano-immunoassays: advancing quantitative proteomics analysis, biomarker assessment, and molecular diagnostics. J Transl Med. 2015;13:182.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Misiewicz-Krzeminska I, Corchete LA, Rojas EA, Martinez-Lopez J, Garcia-Sanz R, Oriol A, et al. A novel nano-immunoassay method for quantification of proteins from CD138-purified myeloma cells: biological and clinical utility. Haematologica. 2018;103(5):880–9.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Beekman C, Janson AA, Baghat A, van Deutekom JC, Datson NA. Use of capillary Western immunoassay (Wes) for quantification of dystrophin levels in skeletal muscle of healthy controls and individuals with Becker and Duchenne muscular dystrophy. Plos One. 2018;13(4).

  35. 35.

    Nariai Y, Kamino H, Obayashi E, Kato H, Sakashita G, Sugiura T, et al. Generation and characterization of antagonistic anti-human interleukin (IL)-18 monoclonal antibodies with high affinity: two types of monoclonal antibodies against full-length IL-18 and the neoepitope of inflammatory caspase-cleaved active IL-18. Arch Biochem Biophys. 2019;663:71–82.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Wirths O, Walter S, Kraus I, Klafki HW, Stazi M, Oberstein TJ, et al. N-truncated Abeta4-x peptides in sporadic Alzheimer’s disease cases and transgenic Alzheimer mouse models. Alzheimers Res Ther. 2017;9(1):80.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Wu J, Haitjema CH, Heger CD, Boge A. A proof-of-concept analysis of carbohydrate-deficient transferrin by imaged capillary isoelectric focusing and in-capillary immunodetection. Biotechniques. 2020;68(2):85–90.

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Li Y, Duah E, Long N, Persaud A, VanGundy Z, Magliery T, et al. An efficient and quantitative assay for epitope-tagged therapeutic protein development with a capillary western system. Bioanalysis. 2019;11(6):471–84.

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Kodani M, Martin M, de Castro VL, Drobeniuc J, Kamili S. An automated immunoblot method for detection of IgG antibodies to hepatitis C virus: a potential supplemental antibody confirmatory assay. J Clin Microbiol. 2019;57(3).

  40. 40.

    Sokolowska I, Mo J, Rahimi Pirkolachahi F, McVean C, Meijer LAT, Switzar L, et al. Implementation of a high-resolution liquid chromatography-mass spectrometry method in quality control laboratories for release and stability testing of a commercial antibody product. Anal Chem. 2020;92(3):2369–73.

    CAS  Article  PubMed  Google Scholar 

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Correspondence to Liang Zhu.

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Zhu, L., Glick, J. & Flarakos, J. Bioanalytical Challenges in Support of Complex Modalities of Antibody-Based Therapeutics. AAPS J 22, 130 (2020).

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  • antibody drug conjugate
  • bioanalytical
  • bispecific antibody
  • ligand binding assay
  • liquid chromatograph-mass spectrometry