title: Quantitative bioanalysis of antibody-conjugated payload in monkey plasma using a hybrid immuno-capture LC-MS/MS approach: Assay development, validation, and a case study.
abstract: Antibody drug conjugates (ADCs) are complex molecules composed of two pharmacologically distinct components, the cytotoxic payload and the antibody. The measurement of the payload molecules that are attached to the antibody in vivo is important for the evaluation of the safety and efficacy of ADCs, and can also provide distinct information compared to the antibody-related analytes. However, analyzing the antibody-conjugated payload is challenging and in some cases may not be feasible. The in vivo change in drug antibody ratio (DAR), due to deconjugation, biotransformation or other clearance phenomena, generates unique and additional challenges for ADC analysis in biological samples. Here, we report a novel hybrid approach with immuno-capture of the ADC, payload cleavage by specific enzyme, and LC-MS/MS of the cleaved payload to quantitatively measure the concentration of payload molecules still attached to the antibody via linker in plasma. The ADC reference material used for the calibration curve is not likely to be identical to the ADC measured in study samples due to the change in DAR distribution over the PK time course. The assay clearly demonstrated that there was no bias in the measurement of antibody-conjugated payload for ADC with varying DAR, which thus allowed accurate quantification even when the DAR distribution dynamically changes in vivo. This hybrid assay was fully validated based on a combination of requirements for both chromatographic and ligand binding methods, and was successfully applied to support a GLP safety study in monkeys.
authors: Analytical and Bioanalytical Development, Bristol-Myers Squibb, Princeton, NJ 08543, USA. Electronic address: ang.liu@bms.com.
authors: Ang
authors: A
authors: Liu
authors: Analytical and Bioanalytical Development, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors: Alexander
authors: A
authors: Kozhich
authors: Biologics Discovery California, Bristol-Myers Squibb, Redwood City, CA 94063, USA.
authors: David
authors: D
authors: Passmore
authors: Analytical and Bioanalytical Development, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors: Huidong
authors: H
authors: Gu
authors: Bioanalytical and Discovery Analytical Sciences, Bristol-Myers Squibb, Pennington, NJ 08534, USA.
authors: Richard
authors: R
authors: Wong
authors: Analytical and Bioanalytical Development, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors: Frank
authors: F
authors: Zambito
authors: Biologics Discovery California, Bristol-Myers Squibb, Redwood City, CA 94063, USA.
authors: Vangipuram S
authors: VS
authors: Rangan
authors: Analytical and Bioanalytical Development, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors: Heather
authors: H
authors: Myler
authors: Analytical and Bioanalytical Development, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors: Anne-Françoise
authors: AF
authors: Aubry
authors: Analytical and Bioanalytical Development, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors: Mark E
authors: ME
authors: Arnold
authors: Analytical and Bioanalytical Development, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
authors: Jian
authors: J
authors: Wang
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D000882:Haplorhini / Q000097:blood*; D018796:Immunoconjugates / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D018796:Immunoconjugates
keywords: Antibody-conjugated payload; Hybrid approach; Immuno-capture; LC–MS/MS; Plasma; Validation