Abstract
Bispecific antibodies and fusion proteins are part of the next generation of biotherapeutics that contain more than one functional domain. These biotherapeutic modalities are structurally complex and require unique approaches to bioanalysis. For their pharmacokinetic analysis, multiple assays may be required to characterize the disposition of the whole drug molecule as well as the individual functional domains. The immunogenicity analysis of these biotherapeutics also requires additional tiers of domain-specific characterization. The bioanalytical approaches for these modalities thus require additional considerations due to the complexity of their structure, additional domain-specific critical reagent generation, and increased assay complexities due to interferences observed by individual functional domains.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Broders O, Wessels U, Zadak M, Beckmann R, Stubenrach K. Novel bioanalytical method for the characterization of the immune response directed against a bispecific F(ab) fragment. Bioanalysis. 2020;12(8):509–17. https://doi.org/10.4155/bio-2020-0064.
Caliceti P. Pharmacokinetic and biodistribution properties of poly(ethylene glycol)–protein conjugates. Adv Drug Deliv Rev. 2003;55(10):1261–1277. https://doi.org/10.1016/S0169-409X(03)00108-X.
Cao M, Wang C, Chung WK, Motabar D, Wang J, Christian E, Lin S, Hunter A, Wang X, Liu D. Characterization and analysis of scFv-IgG bispecific antibody size variants. MAbs. 2018;10(8):1236–47. https://doi.org/10.1080/19420862.2018.1505398.
Chen X, Lee HF, Zaro JL, Shen WC. Effects of receptor binding on plasma half-life of bifunctional transferrin fusion proteins. Molecular Pharmaceutics 2011;8(2):457–465. https://doi.org/10.1021/mp1003064.
Conrad U, Plagmann I, Malchow S, Sack M, Floss DM, Kruglov AA, Nedospasov SA, Rose-John S, Jürgen, J. ELPylated anti-human TNF therapeutic single-domain antibodies for prevention of lethal septic shock. Plant Biotechnol J. 2011;9(1):22–31. https://doi.org/10.1111/j.1467-7652.2010.00523.x.
Datta-Mannan A, Croy JE, Schirtzinger L, Torgerson S, Breyer M, Wroblewski VJ. Aberrant bispecific antibody pharmacokinetics linked to liver sinusoidal endothelium clearance mechanism in cynomolgus monkeys. MAbs. 2016;8(5):969–82. https://doi.org/10.1080/19420862.2016.1178435.
Fares FA, Suganuma N, Nishimori K, LaPolt PS, Hsueh AJ, Boime I. Design of a long-acting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin beta subunit to the follitropin beta subunit. Proc Natl Acad Sci. 1992;89(10):4304–4308. https://doi.org/10.1073/pnas.89.10.4304.
FDA Guidance for industry: Bispecific Antibody Development Programs, DHHS FDA Docket No. FDA-2019-D-0621; 2019.
Fischer SK, Yang J, Anand B, Cowan K, Hendricks R, Li J, Nakamura G, Song A. (2014) The assay design used for measurement of therapeutic antibody concentrations can affect pharmacokinetic parameters. mAbs. 2012;4(5):623–631. https://doi.org/10.4161/mabs.20814.
Giorgetti J, Beck A, Leize-Wagner E, François YN. Combination of intact, middle-up and bottom-up levels to characterize 7 therapeutic monoclonal antibodies by capillary electrophoresis—mass spectrometry. J Pharm Biomed Anal. 2020;182. https://doi.org/10.1016/j.jpba.2020.113107.
Haraya K, Tachibana T, Igawa T. Improvement of pharmacokinetic properties of therapeutic antibodies by antibody engineering. Drug Metab Pharmacokinet. 2019;34(1):25–41. https://doi.org/10.1016/j.dmpk.2018.10.003.
Husain B, Ellerman D. Expanding the boundaries of biotherapeutics with bispecific antibodies. BioDrugs. 2018;32(5):441–64. https://doi.org/10.1007/s40259-018-0299-9.
International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. ICH harmonized tripartite guideline on preclinical safety evaluation of biotechnology-derived pharmaceuticals S6(R1), Step 4 Version; 2011.
Iwamoto N, Shimada T. Regulated LC-MS/MS bioanalysis technology for therapeutic antibodies and Fc-fusion proteins using structure-indicated approach. Drug Metab Pharmacokinet. 2019;34(1):19–24. https://doi.org/10.1016/j.dmpk.2018.10.002.
Iwamoto N, Yokoyama K, Takanashi M, Yonezawa A, Matsubara K, Shimada T. Application of nSMOL coupled with LC-MS bioanalysis for monitoring the fc-fusion biopharmaceuticals etanercept and abatacept in human serum. Pharmacol Res Perspect. 2018;6(4):1–10. https://doi.org/10.1002/prp2.422.
Jeremy Woods R, **e MH, Kreudenstein TSV, Ng GY, Dixit SB. LC-MS characterization and purity assessment of a prototype bispecific antibody. MAbs. 2013;5(5):711–22. https://doi.org/10.4161/mabs.25488.
Kaur S, Liu L, Cortes DF, Shao J, Jenkins R, Mylott WR, Xu K. Validation of a biotherapeutic immunoaffinity-LC-MS/MS assay in monkey serum: “plug-and-play” across seven molecules. Bioanalysis. 2016;8(15):1565–77. https://doi.org/10.4155/bio-2016-0117.
Kaur S, Xu K, Saad OM, Dere RC, Carrasco-Triguero M. Bioanalytical assay strategies for the development of antibody–drug conjugate biotherapeutics. Bioanalysis. 2013;5(2):201–226. https://doi.org/10.4155/bio.12.299.
Kellie JF, Pannullo KE, Li Y, Fraley K, Mayer A, Sychterz CJ, Szapacs ME, Karlinsey MZ. Antibody subunit LC-MS analysis for pharmacokinetic and biotransformation determination from in-life studies for complex biotherapeutics. Anal Chem. 2020;92(12):8268–77. https://doi.org/10.1021/acs.analchem.0c00520.
Kernstock R, Sperinde G, Finco D, Davis R, Montgomery D. Clinical immunogenicity risk assessment strategy for a low risk monoclonal antibody. AAPS J. 2020;22(3):60. Published 2020 Mar 17. https://doi.org/10.1208/s12248-020-00440-5.
Knight T, Callaghan MU. The role of emicizumab, a bispecific factor IXa- and factor X-directed antibody, for the prevention of bleeding episodes in patients with hemophilia A. Ther Adv Hematol. 2018;9(10):319–34. https://doi.org/10.1177/2040620718799997. Published 2018 Oct 10
Kontermann R, Brinkmann U. Bispecific antibodies. Drug Discov Today. 2015;20(7):838–47. https://doi.org/10.1016/j.drudis.2015.02.008.
Labrijn AF, Janmaat ML, Reichert JM, Parren PWHI. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov. 2019;18(8):585–608. https://doi.org/10.1038/s41573-019-0028-1.
Li H, Ortiz R, Tran L, Hall M, Spahr C, Walker K, Laudemann J, Miller S, Salimi-Moosavi H, Lee JW. General LC-MS/MS method approach to quantify therapeutic monoclonal antibodies using a common whole antibody internal standard with application to preclinical studies. Anal Chem. 2012;84(3):1267–73. https://doi.org/10.1021/ac202792n.
Ma M, Colletti K, Yang TY, Leung S, Pederson S, Hottenstein CS, Xu X, Warrino D, Wakshull E. Bioanalytical challenges and unique considerations to support pharmacokinetic characterization of bispecific biotherapeutics. Bioanalysis. 2019;11(5):427–35. https://doi.org/10.4155/bio-2018-0146.
Manikwar P, Mulagapati SHR, Kasturirangan S, Moez K, Rainey GJ, Lobo B. Characterization of a novel bispecific antibody with improved conformational and chemical stability. J Pharm Sci. 2020;109(1):220–32. https://doi.org/10.1016/j.xphs.2019.06.025.
Mullard A. FDA approves first bispecific. Nat Rev Drug Discov. 2015;14:7. https://doi.org/10.1038/nrd4531.
Neubert H, Olah T, Lee A, Fraser S, Dodge R, Laterza O, Szapacs M, Alley SC, Saad OM, Amur S, Chen L, Cherry E, Cho SJ, Cludts I, Donato LD, Edmison A, Ferrari L, Garofolo F, Haidar S, Hopper S, Hottenstein S, Ishii-Watabe A, Kassim S, Kurki P, Lima Santos GM, Miscoria G, Palandra J, Pedras-Vasconcelos J, Piccoli S, Rogstad S, Saito Y, Savoie N, Sikorski T, Spitz S, Staelens L, Verthelyi D, Vinter S, Wadhwa M, Wang YM, Welink J, Weng N, Whale E, Woolf E, Wu J, Yan H, Yu H, Zhou S. 2018 White Paper on Recent Issues in Bioanalysis: focus on immunogenicity assays by hybrid LBA/LCMS and regulatory feedback (part 2—PK, PD & ADA assays by hybrid LBA/LCMS & regulatory agencies' inputs on bioanalysis, biomarkers and immunogenicity). Bioanalysis. 2018;10(23):1897–917. https://doi.org/10.4155/bio-2018-0285. Epub 2018 Nov 29
Roopenian DC, Shreeram, S. FcRn: the neonatal Fc receptor comes of age. Nat Rev Immunol. 2007;7(9):715–725. https://doi.org/10.1038/nri2155.
Runcie K, Budman DR, John V, et al. Bi-specific and tri-specific antibodies-the next big thing in solid tumor therapeutics. Mol Med. 2018;24:50. https://doi.org/10.1186/s10020-018-0051-4.
Sawyer WS, Srikumar N, Carver J, Chu PY, Shen A, Xu A, Williams AJ, Spiess C, Wu C, Liu Y, Tran JC. High-throughput antibody screening from complex matrices using intact protein electrospray mass spectrometry. Proc Natl Acad Sci U S A. 2020;117(18):9851–6. https://doi.org/10.1073/pnas.1917383117.
Schellenberger V, Wang C, Geething NC, Spink BJ, Campbell A, To W, Scholle MD, Yin Y, Yao Y, Bogin O, Cleland JL, Silverman J, Stemmer WPC. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nature Biotechnology 2009;27(12):1186–1190. https://doi.org/10.1038/nbt.1588.
Stevenson L, Kelley M, Gorovits B, Kingsley C, Myler H, Österlund K, Muruganandam A, Minamide Y, Dominguez M. Large molecule specific assay operation: recommendation for best practices and harmonization from the global bioanalysis consortium harmonization team. AAPS J. 2014;16(1):83–88. https://doi.org/10.1208/s12248-013-9542-y.
Shi J, Chen X, Diao J, et al. Bioanalysis in the age of new drug modalities. AAPS J. 2021;23:64. https://doi.org/10.1208/s12248-021-00594-w.
Subklewe M. BiTEs better than CAR T cells. Blood Adv. 2021;5(2):607–12. https://doi.org/10.1182/bloodadvances.2020001792.
Swann PG, Shapiro MA (2011) Regulatory considerations for development of bioanalytical assays for biotechnology products. Bioanalysis. 2011;3(6):597–603. https://doi.org/10.4155/bio.11.27.
Tibbitts J, Canter D, Graff R, Smith A, Khawli LA. Key factors influencing ADME properties of therapeutic proteins: a need for ADME characterization in drug discovery and development. MAbs. 2016;8(2):229–45. https://doi.org/10.1080/19420862.2015.1115937.
Trivedi A, Stienen S, Zhu M, Li H, Yuraszeck T, Gibbs J, Heath T, Loberg R, Kasichayanula S. Clinical pharmacology and translational aspects of bispecific antibodies. Clin Transl Sci. 2017;10:147–62. https://doi.org/10.1111/cts.12459.
Voronin K, Allentoff AJ, Bonacorsi SJ, Mapelli C, Gong SX, Lee V, Riexinger D, Sanghvi N, Jiang H, Zeng J. Synthesis of a stable isotopically labeled universal surrogate peptide for use as an internal standard in LC-MS/MS bioanalysis of human IgG and Fc-fusion protein drug candidates. J Label Compd Radiopharm. 2014;57(9):579–83. https://doi.org/10.1002/jlcr.3218.
Wang C, Vemulapalli B, Cao M, Gadre D, Wang J, Hunter A, Wang X, Liu D. A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture. MAbs. 2018;10(8):1226–35. https://doi.org/10.1080/19420862.2018.1511198.
Wang, YMC, Jawa V, Ma M. Immunogenicity and PK/PD evaluation in biotherapeutic drug development: scientific considerations for bioanalytical methods and data analysis. Bioanalysis 2014;6(1):79–87.
Wang Y, Li X, Liu YH, Richardson D, Li H, Shameem M, Yang X. Simultaneous monitoring of oxidation, deamidation, isomerization, and glycosylation of monoclonal antibodies by liquid chromatography-mass spectrometry method with ultrafast tryptic digestion. MAbs. 2016;8(8):1477–86. https://doi.org/10.1080/19420862.2016.1226715.
Yang W, Luong M, Guadiz C, Zhang M, Gorovits B. Addressing soluble target interference in the development of a functional assay for the detection of neutralizing antibodies against a BCMA-CD3 bispecific antibody. J Immunol Methods. 2019;474 https://doi.org/10.1016/j.jim.2019.112642.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Ethics declarations
Any opinions or forward-looking statements expressed are those of the authors and may not reflect views held by their employers (Janssen Biotherapeutics for Sanjeev Bhardwaj and EMD Serono Research & Development Institute, Inc./Merck KGaA, Darmstadt, Germany, for Kelly Covert and Hongmei Niu).
Rights and permissions
Copyright information
© 2022 American Association of Pharmaceutical Scientists
About this chapter
Cite this chapter
Covert, K., Niu, H., Bhardwaj, S. (2022). An Introduction to Bioanalysis of Bispecific and Fusion Proteins. In: Kumar, S. (eds) An Introduction to Bioanalysis of Biopharmaceuticals. AAPS Advances in the Pharmaceutical Sciences Series, vol 57. Springer, Cham. https://doi.org/10.1007/978-3-030-97193-9_4
Download citation
DOI: https://doi.org/10.1007/978-3-030-97193-9_4
Published:
Publisher Name: Springer, Cham
Print ISBN: 978-3-030-97192-2
Online ISBN: 978-3-030-97193-9
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)