Intact quantitation of cysteine-conjugated antibody-drug conjugates using native mass spectrometry.
Rapid Commun Mass Spectrom
; 38(15): e9774, 2024 Aug 15.
Article
em En
| MEDLINE
| ID: mdl-38812280
ABSTRACT
RATIONALE A common strategy for antibody-drug conjugate (ADC) quantitation from in vivo study samples involves measurement of total antibody, conjugated ADC, and free payload concentrations using multiple reaction monitoring (MRM) mass spectrometry. This not only provides a limited picture of biotransformation but can also involve lengthy method development. Quantitation of ADCs directly at the intact protein level in native conditions using high-resolution mass spectrometers presents the advantage of measuring exposure readout as well as monitoring the change in average drug-to-antibody ratio (DAR) and in vivo stability of new linker payloads with minimal method development. Furthermore, site-specific cysteine-conjugated ADCs often rely on non-covalent association to retain their quaternary structure, which highlights the unique capabilities of native mass spectrometry (nMS) for intact ADC quantitation. METHODS:
We developed an intact quantitation workflow involving three stages automated affinity purification, nMS analysis, and data processing in batch fashion. The sample preparation method was modified to include only volatile ion-pairing reagents in the buffer systems. A capillary size-exclusion chromatography (SEC) column was coupled to a quadrupole time-of-flight high-resolution mass spectrometer for high-throughput nMS analysis. Samples from two mouse pharmacokinetic (PK) studies were analyzed using both intact quantitation workflow and the conventional MRM-based approach.RESULTS:
A linear dynamic range of 5-100 µg/mL was achieved using 20 µL of serum sample volume. The results of mouse in vivo PK measurement using the intact quantitation workflow and the MRM-based approach were compared, revealing excellent method agreement.CONCLUSIONS:
We demonstrated the feasibility of utilizing nMS for the quantitation of ADCs at the intact protein level in preclinical PK studies. Our results indicate that this intact quantitation workflow can serve as an alternative generic method for high-throughput analysis, enabling an in-depth understanding of ADC stability and safety in vivo.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Massas
/
Imunoconjugados
/
Cisteína
Limite:
Animals
Idioma:
En
Revista:
Rapid Commun Mass Spectrom
/
Rapid commun. mass spectrom
/
Rapid communications in mass spectrometry
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Estados Unidos