Purpose: With more monoclonal antibody (mAb) drug conjugates (ADC’s) under preclinical development and in clinical studies, comprehensive and reliable pharmacokinetic evaluation of ADC moieties is increasingly demanded. Liquid chromatography coupled to mass spectrometry (LC-MS) methods have served as sensitive, selective, and versatile tools for ADC PK profiling. Presented here are ADC LC-MS methodology approaches used at Frontage.
Methods: 1) “Top-down” Intact mAb
• Intact “top-down” approach for mAb quantitation and ADC characterization using high resolution mass spectrometry (HRMS).
• The biological samples were pre-treated by immunoprecipitation (Dynabeads from Thermo Fisher Scientific)
• LC-HRMS analysis (Sciex TripleTOF® 6600).
• A specific charge state or the deconvoluted peak was selected for mAb quantitation or calculation of drug-to-antibody (DAR) ratio.
2) “Bottom-up” mAb surrogate peptide
• Surrogate peptide “bottom-up” approach utilized either immunoprecipitation or pellet digestion for sample pretreatment.
• After immunoprecipitation, the captured antibody or ADC was subject to protease digestion to generate the surrogate peptide.
• For the pellet digestion, the sample was precipitated by using an organic solvent and then reconstituted in a neutral buffer for digestion.
• LC-MS/MS analysis (Sciex)
3) “Bottom-up” small molecule drug
• In addition to monitoring the surrogate peptide for total mAb quantitation, another step was added to the sample pretreatment to release the small molecule warhead from the captured ADC.
• In the final solution for LC-MS/MS analysis, both surrogate peptide and the warhead were present and respectively used for total mAb and conjugated warhead quantitation. The stable isotope labeled surrogate peptide and warhead were used as internal standards.
Method Development Challenges:
1. Sensitivity improvement,
2. Selection of proper surrogate peptides,
3. Sample pretreatment optimization to combine digestion & chemical reactions that release small molecule warhead from captured ADC (for multiplexing approach).
Results: Sensitivity for the quantitation methods are generally below:
• 1 µg/mL for the intact “top-down” intact mAb approach,
• 10-100 ng/mL for the “bottom-up” surrogate peptide approach.
The accuracy (% bias) and precision (% CV) of all QCs were within:
• ±25% for LLOQ level and ±20% for the other concentration levels.
Multiplexing methods allow simultaneous quantitation of total mAb & conjugated warhead.
• Very useful for cases like very limited sample volume with need of multiple bioanalysis. For example, sample volume less than 100 uL, need Four PK (free drug, free drug metabolite, total mAb, conjugated drug) plus immunogenicity assay.
Conclusion: LC-HRMS (top-down) and LC-MS/MS (bottom-up) based bioanalytical methods were developed, validated, and successfully used for the quantitation of ADC moieties in biological samples for PK evaluations.
The methods were able to provide comprehensive ADC PK profiling, including not only total antibody, conjugated warhead, free warhead and metabolites, but also DAR characterization from an intact level.
The multiplexing approach generated two concentration measurements from one single sample assay, which significantly reduced the assay cost.