Interrogating heterogeneity of cysteine-engineered antibody-drug conjugates and antibody-oligonucleotide conjugates by capillary zone electrophoresis-mass spectrometry

Xu, Tian; Zhang, Fan; Chen, Daoyang; Sun, Liangliang; Tomazela, Daniela M.; Fayadat‐Dilman, Laurence

doi:10.1080/19420862.2023.2229102

Cited by 6 publications

(3 citation statements)

References 50 publications

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“…Given the low normal blood levels of ammonia (<30 μM 26 ), this mechanistically undesirable reaction can be considered pharmacologically irrelevant, but may be observed in mass spectrometric analysis of the bioconjugates, which typically utilizes volatile ammoniumbased buffers. 27 The hydrolytic loss of the equatorial chloro ligand observed in 11 is consistent with reactivity patterns reported in other Pt(IV) prodrugs, 28,29 but occurs at a considerably slower rate than in the Pt(II)-based parent PAs (t 1/2(aquation) < 30 min 9 ). Most importantly, reductive degradation to Pt(II) over time, which would result in the loss of axial ligands and formation of compound 1, is not observed for any of the payloads.…”

Section: ■ Results and Discussionsupporting

confidence: 78%

“…In ammonium bicarbonate (ABC) buffer, the chloro ligand is readily replaced by ammonia. Given the low normal blood levels of ammonia (<30 μM), this mechanistically undesirable reaction can be considered pharmacologically irrelevant, but may be observed in mass spectrometric analysis of the bioconjugates, which typically utilizes volatile ammonium-based buffers . The hydrolytic loss of the equatorial chloro ligand observed in 11 is consistent with reactivity patterns reported in other Pt(IV) prodrugs, , but occurs at a considerably slower rate than in the Pt(II)-based parent PAs ( t 1/2(aquation) < 30 min).…”

Section: Resultsmentioning

confidence: 63%

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Development of Prodrug–Payloads for Targeted Therapeutic Applications of Platinum–Acridine Anticancer Agents

Mancera-Ortiz,

Chen,

Slade

et al. 2023

Bioconjugate Chem.

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A synthetic platform has been developed that provides access to platinum(IV) prodrugs of highly cytotoxic platinum−acridine anticancer agents and allows them to be incorporated into conjugation-ready prodrug−payloads (PPLs). The PPLs can be conveniently assembled in highly efficient microscale reactions utilizing strain-promoted azide−alkyne cycloaddition chemistry. Model reactions were performed to study the stability of the PPLs in buffers and media and to assess their compatibility with cysteine−maleimide Michael addition chemistry. Amide coupling was a successful strategy to generate a conjugate containing integrin-targeted cyclo[RGDfK] peptide. Reactions with ascorbate were performed to mimic the reductive activation of the PPLs and the latter conjugate, and a cyanine (Cy5) fluorophore-labeled PPL was used to probe the reduction of platinum(IV) in cancer cells by confocal microscopy. The PPL concept introduced here should be evaluated for treating solid tumors with PAs using cancer-targeting vehicles, such as antibody− drug conjugates.

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Section: ■ Results and Discussionsupporting

confidence: 78%

Section: Resultsmentioning

confidence: 63%

Development of Prodrug–Payloads for Targeted Therapeutic Applications of Platinum–Acridine Anticancer Agents

Mancera-Ortiz,

Chen,

Slade

et al. 2023

Bioconjugate Chem.

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“…4,5,13 With the improvement in HRMS instrumentation and sample preparation strategies, recent literature has demonstrated the feasibility of routine qualitative and quantitative analyses of mAbs and ADCs at the intact protein level from complex biological matrices. 2,[4][5][6][13][14][15][16][17][18][19][20][21] For example,…”

Section: Introductionmentioning

confidence: 99%

Intact quantitation of cysteine‐conjugated antibody‐drug conjugates using native mass spectrometry

Li,

Wang,

Shenoy

et al. 2024

Rapid Comm Mass Spectrometry

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RationaleA 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.MethodsWe 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.ResultsA 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.ConclusionsWe 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.

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Determination of the decapping efficiency of THIOMAB™ antibodies with the engineered cysteine in the Fc region for making antibody–drug conjugates by specific hinge fragmentation-liquid chromatography

Yang,

Patel,

Yang

et al. 2024

Anal Bioanal Chem

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Interrogating heterogeneity of cysteine-engineered antibody-drug conjugates and antibody-oligonucleotide conjugates by capillary zone electrophoresis-mass spectrometry

Cited by 6 publications

References 50 publications

Development of Prodrug–Payloads for Targeted Therapeutic Applications of Platinum–Acridine Anticancer Agents

Development of Prodrug–Payloads for Targeted Therapeutic Applications of Platinum–Acridine Anticancer Agents

Intact quantitation of cysteine‐conjugated antibody‐drug conjugates using native mass spectrometry

Determination of the decapping efficiency of THIOMAB™ antibodies with the engineered cysteine in the Fc region for making antibody–drug conjugates by specific hinge fragmentation-liquid chromatography

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