Glythera Limited is a biotechnology company specializing in the development of next generation biotherapeutics, through the application of their advanced proprietary linker and stable glycan technologies: PermaLink™ and PermaCarb™.
Glythera requested an assessment of DARs for an antibody-drug-conjugate (ADC) product in plasma samples derived from mouse Pharmacokinetic (PK) studies. The purpose of the project was to use a customized method to provide quantitative data for each PK time point in order to calculate the corresponding DARs.
- Q-TOF LC-MS instrumentation was used for the initial structural characterization of the ADC to determine the DAR.
- Standards were made by spiking mouse plasma with reference/client ADC material
- Sample purification and separation of ADC light and heavy chains
- Proteolytic digestion of sample components
- Selection of marker peptides and drug-conjugated peptides from light and heavy chains
- MS/MS detection with Multiple-Reaction-Monitoring transitions (MRM) for all peptides
- Analysis conducted using UPLC and Triple Quad Mass Spectrometry
Technical Considerations – Candidate Peptide Selection
A method development phase using high-resolution Q-TOF instrumentation was used to first select candidate marker and drug-conjugated peptides that were structurally stable and could be consistently followed in the PK assessment. Cold isotope-labelling is the method of choice for validating the PK data sets derived from MS studies.
“The specific expertise surrounding quantitative mass spectrometry at BioPharmaSpec is excellent and Glythera benefitted greatly from the technical approaches used during method development and optimization” – Senior Development Scientist, Glythera