Abstract
Antibody-drug conjugates (ADC) are made up of three components: (1) a mAb specific to cells of choice, (2) a small molecule with desired end goal, and (3) a linker to covalently link drug molecule to the antibody. Bringing together the mAb, drug molecule, and the linker results in the formation of an immunoconjugate designed to selectively deliver the drug molecule to a cell of interest. Synergic effects of the mAb and drug molecule lead to destroying the target tumor cells while leaving the normal cells unharmed. However, the development of ADCs is associated with challenges due to the heterogeneity of the ADC molecules created from the conjugation process. Addition of the linker and drug moieties during processing as well as the hydrophobicity of the drug itself can lead to structural changes that may affect the stability and functional profile of the conjugated molecule. Furthermore, linkers site of attachment plays a major role in determining the conformational and colloidal properties of the ADCs. In this chapter, several characterization methods are introduced to determine the biophysical characteristics of the ADC. Protocols, data analysis as well as notes for circular dichroism, intrinsic fluorescence, ANS fluorescence, differential scanning calorimetry, and dynamic scanning fluorimetry are outlined in detail.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Chari RVJ (2008) Targeted Cancer therapy: conferring specificity to cytotoxic drugs. Acc Chem Res 41:98–107
Wu AM, Senter PD (2005) Arming antibodies: prospects and challenges for immunoconjugates. Nat Biotechnol 23:1137
Sun MMC, Beam KS, Cerveny CG et al (2005) Reduction−alkylation strategies for the modification of specific monoclonal antibody disulfides. Bioconjug Chem 16:1282–1290
Baldwin AD, Kiick KL (2011) Tunable degradation of Maleimide–Thiol adducts in reducing environments. Bioconjug Chem 22:1946–1953
Chih H-W, Gikanga B, Yang Y et al (2011) Identification of amino acid residues responsible for the release of free drug from an antibody–drug conjugate utilizing lysine–Succinimidyl Ester chemistry. J Pharm Sci 100:2518–2525
Johnson WC Jr (1988) Secondary structure of proteins through circular dichroism spectroscopy. Annu Rev Biophys Biophys Chem 17:145–166
Venyaminov SY, Vassilenko KS (1994) Determination of protein tertiary structure class from circular dichroism spectra. Anal Biochem 222:176–184
Johnson WC (1999) Analyzing protein circular dichroism spectra for accurate secondary structures. Proteins Struct Funct Genet 35:307–312
Levitt M, Chothia C (1976) Structural patterns in globular proteins. Nature 261:552–558
Rodger A, Marrington R, Roper D et al (2005) Circular Dichroism spectroscopy for the study of protein-ligand interactions. In: Ulrich Nienhaus G (ed) Protein-ligand interactions: methods and applications. Humana Press, Totowa, NJ, pp 343–363
Sreerama N, Venyaminov S, Woody RW (2001) Analysis of protein circular Dichroism spectra based on the tertiary structure classification. Anal Biochem 299:271–274
Garidel P, Hegyi M, Bassarab S et al (2008) A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy. Biotechnol J 3:1201–1211
Guo J, Kumar S, Chipley M et al (2016) Characterization and higher-order structure assessment of an Interchain cysteine-based ADC: impact of drug loading and distribution on the mechanism of aggregation. Bioconjug Chem 27:604–615
Guo J, Kumar S, Prashad A et al (2014) Assessment of physical stability of an antibody drug conjugate by higher order structure analysis: impact of Thiol- Maleimide chemistry. Pharm Res 31(7):1710–1723
Kubista M, Sjöback R, Eriksson S et al (1994) Experimental correction for the inner-filter effect in fluorescence spectra. Analyst 119:417–419
Anonymous (2006) Protein Fluorescence. In: Lakowicz JR (ed) Principles of fluorescence spectroscopy. Springer US, Boston, MA, pp 529–575
Wakankar AA, Feeney MB, Rivera J et al (2010) Physicochemical stability of the antibody−drug conjugate Trastuzumab-DM1: changes due to modification and conjugation processes. Bioconjug Chem 21:1588–1595
Wen J, Arthur K, Chemmalil L et al (2012) Applications of differential scanning calorimetry for thermal stability analysis of proteins: qualification of DSC. J Pharm Sci 101:955–964
Freire E (1995) Differential scanning Calorimetry. In: Shirley BA (ed) Protein stability and folding: theory and practice. Humana Press, Totowa, NJ, pp 191–218
Bastiansen O (1977) J. J. Christensen, L. D. Hansen, and R. M. Izatt: Handbook of proton ionization heats and related thermodynamic quantities. J. Wiley and Sons, New York 1976. 269 Seiten, Preis: $28.50. Berichte der Bunsengesellschaft für physikalische Chemie 81:540–540
He F, Hogan S, Latypov RF et al (2010) High throughput thermostability screening of monoclonal antibody formulations. J Pharm Sci 99:1707–1720
Samra HS, He F (2012) Advancements in high throughput biophysical technologies: applications for characterization and screening during early formulation development of monoclonal antibodies. Mol Pharm 9:696–707
Kung CE, Reed JK (1989) Fluorescent molecular rotors: a new class of probes for tubulin structure and assembly. Biochemistry 28:6678–6686
Lindgren M, Sorgjerd K, Hammarstrom P (2005) Detection and characterization of aggregates, prefibrillar amyloidogenic oligomers, and protofibrils using fluorescence spectroscopy. Biophys J 88:4200–4212
Nelson R, Sawaya MR, Balbirnie M et al (2005) Structure of the cross-beta spine of amyloid-like fibrils. Nature 435:773–778
Sawaya MR, Sambashivan S, Nelson R et al (2007) Atomic structures of amyloid cross-beta spines reveal varied steric zippers. Nature 447:453–457
Kayser V, Chennamsetty N, Voynov V et al (2011) Conformational stability and aggregation of therapeutic monoclonal antibodies studied with ANS and Thioflavin T binding. MAbs 3:408–411
Brummitt RK, Nesta DP, Chang L et al (2011) Nonnative aggregation of an IgG1 antibody in acidic conditions, part 2: nucleation and growth kinetics with competing growth mechanisms. J Pharm Sci 100:2104–2119
Ablinger E, Leitgeb S, Zimmer A (2012) Differential scanning fluorescence approach using a fluorescent molecular rotor to detect thermostability of proteins in surfactant-containing formulations. Int J Pharm 441(1-2):255–260
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Mudhivarthi, V.K., Guo, J. (2020). Biophysical Methods for Characterization of Antibody-Drug Conjugates. In: Tumey, L. (eds) Antibody-Drug Conjugates. Methods in Molecular Biology, vol 2078. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9929-3_22
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9929-3_22
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9928-6
Online ISBN: 978-1-4939-9929-3
eBook Packages: Springer Protocols