Over the past two decades, we have seen an increase in the complexity and diversity of biotherapeutic modalities pursued by biopharmaceutical companies. These biologics are multifaceted and susceptible to post-translational modifications and in vivo biotransformation that could impose challenges for bioanalysis. It is vital to characterize the functionality, stability and biotransformation products of these molecules to enable screening, identify potential liabilities at an early stage and devise a bioanalytical strategy. This article highlights our perspective on characterization and bioanalysis of biologics using hybrid LC–MS in our global nonregulated bioanalytical laboratories. AbbVie's suite of versatile, stage-appropriate characterization assays and quantitative bioanalytical approaches are discussed, along with guidance on their utility in answering project-specific questions to aid in decision-making.

Keywords:

Papers of special note have been highlighted as: • of interest; •• of considerable interest

References

1. Zhu L, Glick J, Flarakos J. Bioanalytical challenges in support of complex modalities of antibody-based therapeutics. AAPS J. 22(6), 130 (2020). •• This review provides a very good synopsis of the landscape of complex modalities and their respective bioanalytical challenges.
Medline CASGoogle Scholar
2. Kaur S, Bateman KP, Glick J et al. IQ consortium perspective: complementary LBA and LC–MS in protein therapeutics bioanalysis and biotransformation assessment. Bioanalysis 12(4), 257–270 (2020). • This perspective article is a great reference for the two bioanalytical platform comparisons for biotherapeutic bioanalysis.
CASGoogle Scholar
3. Durbin KR, Nottoli MS, Catron ND, Richwine N, Jenkins GJ. High-throughput, multispecies, parallelized plasma stability assay for the determination and characterization of antibody–drug conjugate aggregation and drug release. ACS Omega 2(8), 4207–4215 (2017). •• This article is published by the AbbVie internal team from Drug metabolism pharmacokinetics and bioanalysis (DMPK-BA) and a routine, high-throughput, two-in-one assay platform used to assess the stability of biologics and drug conjugates.
Medline CASGoogle Scholar
4. Sydow JF, Lipsmeier F, Larraillet V et al. Structure-based prediction of asparagine and aspartate degradation sites in antibody variable regions. PLOS ONE 9(6), e100736 (2014).
MedlineGoogle Scholar
5. Jarasch A, Koll H, Regula JT, Bader M, Papadimitriou A, Kettenberger H. Developability assessment during the selection of novel therapeutic antibodies. J. Pharm. Sci. 104(6), 1885–1898 (2015).
Medline CASGoogle Scholar
6. Liu YD, Van Enk JZ, Flynn GC. Human antibody Fc deamidation in vivo. Biologicals 37(5), 313–322 (2009).
Medline CASGoogle Scholar
7. Varshavsky A. The N-end rule pathway and regulation by proteolysis. Protein Sci. 20(8), 1298–1345 (2011).
Medline CASGoogle Scholar
8. Shadid M, Bowlin S, Bolleddula J. Catabolism of antibody drug conjugates and characterization methods. Bioorg. Med. Chem. 25(12), 2933–2945 (2017).
Medline CASGoogle Scholar
9. Bessire AJ, Ballard TE, Charati M et al. Determination of antibody–drug conjugate released payload species using directed in vitro assays and mass spectrometric interrogation. Bioconjug. Chem. 27(7), 1645–1654 (2016).
Medline CASGoogle Scholar
10. Sang H, Liu J, Zhou F et al. Target-responsive subcellular catabolism analysis for early-stage antibod–drug conjugates screening and assessment. Acta Pharm. Sin. B 11(12), 4020–4031 (2021).
Medline CASGoogle Scholar
11. Tumey LN, Leverett CA, Vetelino B et al. Optimization of tubulysin antibody–drug conjugates: a case study in addressing ADC metabolism. ACS Med. Chem. Lett. 7(11), 977–982 (2016).
Medline CASGoogle Scholar
12. Cini E, Faltoni V, Petricci E et al. Antibody drug conjugates (ADCs) charged with HDAC inhibitor for targeted epigenetic modulation. Chem. Sci. 9(31), 6490–6496 (2018).
Medline CASGoogle Scholar
13. Bessire AJ, Subramanyam C. LC/MS methods for studying lysosomal ADC catabolism. Methods Mol. Biol. 2078, 341–351 (2020).
Medline CASGoogle Scholar
14. Austin CD, De Mazière AM, Pisacane PI et al. Endocytosis and sorting of ErbB2 and the site of action of cancer therapeutics trastuzumab and geldanamycin. Mol. Biol. Cell 15(12), 5268–5282 (2004).
Medline CASGoogle Scholar
15. Durbin KR, Phipps C, Liao X. Mechanistic modeling of antibody–drug conjugate internalization at the cellular level reveals inefficient processing steps. Mol. Cancer Ther. 17(6), 1341–1351 (2018). • This is an AbbVie internal publication that emphasizes the power of mechanistic modeling used with cellular disposition assay to determine antibody–drug conjugate internalization.
Medline CASGoogle Scholar
16. Durbin KR, Nottoli MS, Jenkins GJ. Effects of microtubule-inhibiting small molecule and antibody–drug conjugate treatment on differentially-sized A431 squamous carcinoma spheroids. Sci. Rep. 10(1), 907 (2020).
Medline CASGoogle Scholar
17. Bhat J, Narayan A, Venkatraman J, Chatterji M. LC–MS based assay to measure intracellular compound levels in Mycobacterium smegmatis: linking compound levels to cellular potency. J. Microbiol. Methods 94(2), 152–158 (2013).
Medline CASGoogle Scholar
18. Wu C, Wang X, Xu M, Liu Y, Di X. Intracellular accumulation as an indicator of cytotoxicity to screen hepatotoxic components of Chelidonium majus L. by LC–MS/MS. Molecules 24(13), 2410 (2019).
Medline CASGoogle Scholar
19. Singh AP, Shah DK. Measurement and mathematical characterization of cell-level pharmacokinetics of antibody–drug conjugates: a case study with trastuzumab-vc-MMAE. Drug Metab. Dispos. 45(11), 1120–1132 (2017).
Medline CASGoogle Scholar
20. Wei C, Zhang G, Clark T et al. Where did the linker-payload go? A quantitative investigation on the destination of the released linker-payload from an antibody–drug conjugate with a maleimide linker in plasma. Anal. Chem. 88(9), 4979–4986 (2016).
Medline CASGoogle Scholar
21. Shen BQ, Bumbaca D, Saad O et al. Catabolic fate and pharmacokinetic characterization of trastuzumab emtansine (T-DM1): an emphasis on preclinical and clinical catabolism. Curr. Drug Metab. 13(7), 901–910 (2012).
Medline CASGoogle Scholar
22. Kaur S, Alley SC, Szapacs M et al. 2021 white paper on recent issues in bioanalysis: mass spec of proteins, extracellular vesicles, CRISPR, chiral assays, oligos; nanomedicines bioanalysis; ICH M10 section 7.1; non-liquid & rare matrices; regulatory inputs (part 1A – recommendations on endogenous compounds, small molecules, complex methods, regulated mass spec of large molecules, small molecule, PoC & part 1B – regulatory agencies' inputs on bioanalysis, biomarkers, immunogenicity, gene & cell therapy and vaccine). Bioanalysis 14(9), 505–580 (2022).
CASGoogle Scholar
23. Van De Merbel NC. Protein quantification by LC–MS: a decade of progress through the pages of Bioanalysis. Bioanalysis 11(7), 629–644 (2019). •• This review article very well captures the progress and technology advancements in using LC–MS for protein quantification.
Google Scholar
24. Mehl JT, Landry F, Discenza L et al. Quantification of surrogate monoclonal antibodies in mouse serum using LC–MS/MS. Bioanalysis 13(3), 147–159 (2021).
CASGoogle Scholar
25. Sucharski FK, Meier S, Miess C et al. Development of an automated, interference-free, 2D-LC–MS/MS assay for quantification of a therapeutic mAb in human sera. Bioanalysis 10(13), 1023–1037 (2018).
CASGoogle Scholar
26. Jordan G, Onami I, Heinrich J, Staack RF. Evaluation of the potential use of hybrid LC–MS/MS for active drug quantification applying the ‘free analyte QC concept’. Bioanalysis 9(21), 1705–1717 (2017).
Google Scholar

Vol. 15, No. 3

Metrics

Downloaded 658 times

History

Received 11 November 2022

Accepted 8 February 2023

Published online 9 March 2023

Published in print February 2023

Information

Keywords

Author contributions

All listed authors contributed to the writing of this manuscript.

Acknowledgments

The authors acknowledge Edit Tarcsa and Yihan Li for their generous feedback and review of this article.

Financial & competing interests disclosure

All authors are employees of AbbVie and may own AbbVie stock. The design, study conduct and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review and approval of the publication. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

PDF download