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    <publishedYear>2024</publishedYear>
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    <pageLast>22</pageLast>
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    <title language="eng">Challenges and Insights in Absolute Quantification of Recombinant Therapeutic Antibodies by Mass Spectrometry: An Introductory Review</title>
    <abstract language="eng">This review describes mass spectrometry (MS)-based approaches for the absolute quantification of therapeutic monoclonal antibodies (mAbs), focusing on technical challenges in sample treatment and calibration. Therapeutic mAbs are crucial for treating cancer, inflammatory, infectious, and autoimmune diseases. We trace their development from hybridoma technology and the first murine mAbs in 1975 to today’s chimeric and fully human mAbs. With increasing commercial relevance, absolute quantification of mAbs, traceable to SI units, has attracted attention from science, industry, and national metrology institutes (NMIs). Quantification of proteotypic peptides after enzymatic digestion using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as the most viable strategy, though methods targeting intact mAbs are still being explored. We review peptide-based quantification, focusing on critical experimental steps like denaturation, reduction, alkylation, choice of digestion enzyme, and selection of signature peptides. Challenges in amino acid analysis (AAA) for quantifying pure mAbs and peptide calibrators, along with software tools for targeted MS data analysis, are also discussed. Short explanations within each chapter provide newcomers an overview of the field’s challenges. Finally, we discuss prospects and limitations of developing standardized protocols and certified reference materials (CRMs) and suggest future applications of newer technologies for the absolute quantification of therapeutic antibodies.</abstract>
    <parentTitle language="eng">Prerpints.org</parentTitle>
    <identifier type="doi">10.20944/preprints202412.1081.v1</identifier>
    <identifier type="urn">urn:nbn:de:kobv:b43-621841</identifier>
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    <licence>Creative Commons - CC BY - Namensnennung 4.0 International</licence>
    <author>Sarah Döring</author>
    <author>Michael G. Weller</author>
    <author>Yvonne Reinders</author>
    <author>Zoltán Konthur</author>
    <author>Carsten Jaeger</author>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Antibody</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Mass spectrometry</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Absolute quantification</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Metrology</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Reference products</value>
    </subject>
    <collection role="institutes" number="">1 Analytische Chemie; Referenzmaterialien</collection>
    <collection role="institutes" number="">1.5 Proteinanalytik</collection>
    <collection role="institutes" number="">1.8 Umweltanalytik</collection>
    <collection role="themenfelder" number="">Chemie und Prozesstechnik</collection>
    <collection role="themenfelder" number="">Chemische Charakterisierung und Spurenanalytik</collection>
    <collection role="fulltextaccess" number="">Datei für die Öffentlichkeit verfügbar ("Open Access")</collection>
    <collection role="literaturgattung" number="">Graue Literatur</collection>
    <collection role="unnumberedseries" number="">Preprints der BAM</collection>
    <thesisPublisher>Bundesanstalt für Materialforschung und -prüfung (BAM)</thesisPublisher>
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