Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates

Beck, Alain; Terral, Guillaume; Debaene, François; Wagner‐Rousset, Elsa; Marcoux, Julien; Janin-Bussat, Marie-Claire; Colas, Olivier; Dorsselaer, Alain Van; Cianférani, Sarah

doi:10.1586/14789450.2016.1132167

Cited by 94 publications

(71 citation statements)

References 217 publications

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“…3A, second panel), but a lower recovery of the LC and the Fd glycovariants to 39% and 62% was observed at 40 • C. Brentuximab vedotin has several highly hydrophobic loaded fragments due to cytotoxic drugs attached to them (LC I, Fd I, Fd II and Fd III fragments, with I, II and III corresponding to the number of bound payloads). The proper recovery of these loaded subunits is important for the estimation of the average drug-to-antibody ratio (DAR), which is a critical quality attribute (CQA) of the antibody drug conjugates [31,[42][43][44]. Average DAR can be calculated based on the formula reported by Wagner-Rousset et al [45] and it is generally close to 4.0 [46,47].…”

Section: Recovery Of Mab Species In Hilic Conditionsmentioning

confidence: 99%

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

Bobály

D’Atri

Beck³

et al. 2017

Journal of Pharmaceutical and Biomedical Analysis

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a b s t r a c tHydrophilic interaction liquid chromatography (HILIC) is a well-established technique for the separation and analysis of small polar compounds. A recently introduced widepore stationary phase expanded HILIC applications to larger molecules, such as therapeutic proteins. In this paper, we present some generic HILIC conditions adapted for a wide range of FDA and EMA approved recombinant monoclonal antibody (mAb) species and for an antibody-drug conjugate (ADC). Seven approved mAbs possessing various isoelectric point (pI) and hydrophobicity as well as a cysteine conjugated ADC were used in this study. Samples were digested by IdeS enzyme and digests were further fragmented by chemical reduction. The resulting fragments were separated by HILIC. The main benefit of HILIC was the separation of polar variants (glycovariants) in a reasonable analysis time at the protein level, which is not feasible with other chromatographic modes. Three samples were selected and chromatographic conditions were further optimized to maximize resolution. A commercial software was used to build up retention models. Experimental and predicted chromatograms showed good agreement and the average error of retention time prediction was less than 2%. Recovery of various species and sample stability under the applied conditions were also discussed.

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Section: Recovery Of Mab Species In Hilic Conditionsmentioning

confidence: 99%

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

Bobály

D’Atri

Beck³

et al. 2017

Journal of Pharmaceutical and Biomedical Analysis

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“…State-of-the-art MS-based approaches, including top-down, middle-down and bottom-up, combined with the power of chromatographic or electrophoretic techniques, have been successfully used for identification of CQAs such as drug distribution, DAR value, site of conjugation and chemical liability in ADCs. 37,38 Here, similar analytical methods were used to evaluate the CQAs of a lysine-conjugated ADC.…”

Section: Discussionmentioning

confidence: 99%

The impact of trisulfide modification of antibodies on the properties of antibody-drug conjugates manufactured using thiol chemistry

Liu

Chen

Dushime

et al. 2017

mAbs

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Antibody-drug conjugates (ADCs) are promising biotherapeutic agents for the treatment of cancer. The careful monitoring of critical quality attributes is important for ADCs' development, manufacturing and production. In this work, the effect of the presence of a trisulfide bond in the monoclonal antibody (mAb) conjugated to DM4 cytotoxic payload through a disulfide-bond linker sulfo-SPDB (sSPDB) was investigated. Three lots of antibody containing variable levels of trisulfide bonds were used. The identity and levels of trisulfide bonds were determined by liquid chromatography/ mass spectrometry (MS)/MS analysis. The antibodies were conjugated to sSPDB-DM4 to generate ADCs. Further analysis indicated that the drug-to-antibody ratio (DAR) value, a critical quality attribute, slightly increased for the conjugates made from antibody containing higher levels of trisulfide bond. Also, higher fragmentation levels were observed in the conjugates with more trisulfide bond. Detailed characterization by MS revealed that a small amount of DM4 payload was directly attached to inter-chain cysteine residues by disulfide or trisulfide bonds. Overall, our investigation indicated that the trisulfide bond present in the mAb could react with DM4 during the conjugation process. Therefore, the presence of trisulfide bonds in the antibody moiety should be carefully monitored and well controlled during the development of a maytansinoid ADC.

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“…ADC bioanalysis was also outlined in a recent industry white paper on ADME (Absorption, Distribution, Metabolism, Excretion) characterization of ADCs [4] and a review of ADC analytical characterization by mass spectrometry [5]. It is fair to say that all literature and conference discussions and evolution on ADC bioanalysis in recent years have been around the topics of selection of analytes, DAR characteristics of the assay, and choice of assay platform (LBA or hybrid).…”

Section: Affinity Capture Lc-ms Measurement Of Intactmentioning

confidence: 99%

Current status of antibody-drug conjugate bioanalysis

Wang¹

2017

J Appl Bioanal

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Keywords: antibody-drug conjugate bioanalysis, ligand-binding assay, hybrid ligand-binding/LC-MS assay, drug-toantibody ratio Antibody-drug conjugate (ADC) consists of a cytotoxic drug covalently bound to a monoclonal antibody via a linker. Because of the complexity of ADC structure unique bioanalytical strategies are needed to identify, characterize and quantify the ADC species most relevant to safety and efficacy. ADC bioanalysis need an integrated bioanalytical approach including ligand-binding assay (LBA) and LC-MS based assays to provide comprehensive characterization of the exposure/response relationship. This mini-review will summarize recent publications on ADC bioanalytical strategies and will focus on the publications within last three years on the advancement of hybrid ligand-binding/LC-MS methods for ADC PK and stability evaluation and for intact ADC-DAR distribution measurement to profile ADC biotransformation and catabolism. Special attention is paid to the publications on selection of ADC analytes, assay platforms and DAR characteristics of LBA as well as on the transition of the bioanalytical testing strategy at different phases of clinical development.

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Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates

Cited by 94 publications

References 217 publications

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

The impact of trisulfide modification of antibodies on the properties of antibody-drug conjugates manufactured using thiol chemistry

Current status of antibody-drug conjugate bioanalysis

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