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Related Concept Videos

Tandem Mass Spectrometry01:21

Tandem Mass Spectrometry

Tandem mass spectrometry is a technique that uses multiple mass analyzers in series to obtain a higher selectivity and reduce chemical noise during analyte detection. Instruments with multiple analyzers separated by an interaction cell enable secondary fragmentation and selected study of the fragment ions.Secondary fragmentations occur in the interaction cell and can be induced by various factors. Fragmentation induced by collision with inert gases, such as N2, Ar, He, etc., is called...
Peptide Identification Using Tandem Mass Spectrometry01:33

Peptide Identification Using Tandem Mass Spectrometry

Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
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Mass Spectrometry: Complex Analysis

Mass spectrometry is an important technique for the identification of pure compounds. However, it has some limitations for the analysis of complex mixtures, often due to excessive fragmentation making the spectrum too complicated to decipher. Mass spectrometry can be combined with suitable separation methods in sequence, forming hyphenated methods, which are useful in the analysis of complex mixtures.
GC–MS is a powerful hyphenated method commonly used in forensics and environmental...

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Tritosomes-Digestion for LC-MS Conjugated Payloads Quantitation: A Universal Approach for Dual-Payloads ADCs.

Francesco Molinaro1, Gabriele Sergio Colangelo1, Patrizia Cocco1

  • 1Innovative Bioanalytics, New Biological Entities, Drug Metabolism and Pharmacokinetics (NBE-DMPK), Research and Development, Merck Healthcare KGaA, RBM S.p.A.-Istituto di Ricerche Biomediche "A. Marxer", an Affiliate of Merck KGaA, Via Ribes 1, 10010 Colleretto Giacosa, TO, Italy.

International Journal of Molecular Sciences
|July 15, 2026
PubMed
Summary

A new bioanalytical method uses rat tritosomes for efficient, simultaneous quantification of dual-payload antibody-drug conjugates (ADCs). This approach simplifies analysis, saving time and resources for preclinical studies.

Keywords:
antibody-drug conjugatesbioanalysisconjugated payloaddual-payloadtritosome

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Pharmacology

Background:

  • Quantifying conjugated payloads is crucial for antibody-drug conjugate (ADC) stability and pharmacokinetics (PK).
  • Dual-payload ADCs pose analytical challenges due to diverse linker chemistries requiring complex digestion.
  • Developing individual methods for each linker is time-consuming and resource-intensive.

Purpose of the Study:

  • To develop a unified bioanalytical method for simultaneous quantitation of dual-conjugated payloads in ADCs.
  • To overcome the analytical challenges associated with dual-payload ADCs and different linker chemistries.
  • To provide an efficient platform for ADC bioanalysis supporting preclinical studies.

Main Methods:

  • Immunoaffinity purification combined with tritosome-mediated digestion.
  • Utilized rat tritosomes, a source of comprehensive enzymatic activity mimicking the lysosomal environment.
  • Applied to a model dual-payload ADC with distinct cytotoxic payloads and enzymatically cleavable linkers.

Main Results:

  • Simultaneous quantitation of both payloads with excellent accuracy (bias ± 20%) and precision (CV% ≤ 20%) across all concentration levels.
  • 100% of quality control samples met acceptance criteria for hybrid LC-MS/MS quantitation.
  • The method demonstrated robustness across 4 different model ADCs and in vivo samples.

Conclusions:

  • The tritosome-based bioanalytical method offers a unified and efficient platform for multi-payload ADC analysis.
  • This approach eliminates the need for linker-specific method optimization, saving time and resources.
  • The validated method provides reliable data for preclinical PK studies of ADCs.