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Site-specific glycan-conjugated NISTmAb antibody drug conjugate mimetics: synthesis, characterization, and utility

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Abstract

Antibody drug conjugates (ADCs) represent a rapidly growing modality for the treatment of numerous oncology indications. The complexity of analytical characterization method development is increased due to the potential for synthetic intermediates and process-related impurities. In addition, the cytotoxicity of such materials provides an additional challenge with regard to handling products and/or sharing materials with analytical collaborators and/or vendors for technology development. Herein, we have utilized a site-specific chemoenzymatic glycoconjugation strategy for preparing ADC mimetics composed of the NIST monoclonal antibody (NISTmAb) conjugated to non-cytotoxic payloads representing both small molecules and peptides. The materials were exhaustively characterized with high-resolution mass spectrometry-based approaches to demonstrate the utility of each analytical method for confirming the conjugation fidelity as well as deep characterization of low-abundance synthetic intermediates and impurities arising from payload raw material heterogeneity. These materials therefore represent a widely available test metric to develop novel ADC analytical methods as well as a platform to discuss best practices for extensive characterization.

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Authors and Affiliations

  1. Biosciences Division, Thermo Fisher Scientific, 29851 Willow Creek Road, Eugene, OR, 97402, USA

    Brian Agnew & Robert Aggeler

  2. Chromatography Columns and Consumables, Thermo Fisher Scientific, Sunnyvale, CA, 94085, USA

    Shanhua Lin & Terry Zhang

  3. Institute for Bioscience and Biotechnology Research, National Institute of Standards and Technology, 9600 Gudelsky Dr, Rockville, MD, 20850, USA

    Trina Mouchahoir & John Schiel

Authors
  1. Brian Agnew
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  2. Shanhua Lin
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  3. Terry Zhang
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  4. Robert Aggeler
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  5. Trina Mouchahoir
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  6. John Schiel
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Correspondence to Brian Agnew or John Schiel.

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NIST Disclaimer: Values reported herein do not supersede official NISTmAb Report of Investigation and are for informational purposes only. Users should always refer to the Report of Investigation (https://www-s.nist.gov/srmors/view_detail.cfm?srm=8671) for their specific material lot for the most up to date values and uncertainty ranges. Certain commercial equipment, instruments, or materials are identified to adequately specify the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

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Agnew, B., Lin, S., Zhang, T. et al. Site-specific glycan-conjugated NISTmAb antibody drug conjugate mimetics: synthesis, characterization, and utility. Anal Bioanal Chem 413, 4989–5001 (2021). https://doi.org/10.1007/s00216-021-03460-y

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  • DOI: https://doi.org/10.1007/s00216-021-03460-y

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