Localizing the Conjugation Sites of Cysteine-Conjugated Antibody Drug Conjugates by Improved LC-MS Subunit Analysis for ADC Positional Isomer Identification

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Henry Shion, Robert Birdsall, Liuxi Chen, Ying Qing Yu, Frank W. Kotch, April Xu, Thomas J. Porter, Weibin Chen
Waters & Pfizer Bioprocess Research & Development
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Xevo G2-S QTof

Novel Aspect:

An improved antibody-drug conjugate (ADC) subunit analysis by LC-MS methods for cysteine-conjugated ADC conjugation site localization and positional isomer identification


Subunit analysis of antibody drug conjugates (ADCs) with LC-MS methods provides insightful structural information, such as conjugation isoforms and their modifications (e.g. oxidation, or water losses).  This information is not readily accessible by either intact mass analysis or peptide mapping. Cysteine proteases (e.g. IdeS or SpeB) are often used to cleave the hinge region to generate the LC, Fc/2 and Fd subunits because of their high specificity for mAb sequences.

However, we discovered that the cleavage for ADCs was highly depended upon the degree of conjugation, presumably due to steric hindrance. Here, we report a study to address this cleavage efficiency issue by evaluating IdeS, SpecB, and Lys-C for the localization of conjugation sites and confirmation of positional isomers.


IdeS and SpeB (Genovis, Switzeland) and Lys-C (Roche, Indianapolis, IN) were used for enzymatic digestion of the ADCs. For IdeS or SpeB (100 units each) digestions, 50 µL of an ADC sample (1 mg/mL) was incubated at 37 ⁰C for 30 min before partial DTT reduction. Limited Lys-C digestion was achieved after optimization of enzyme Lys-C to ADC ratio and digestion time. Neat acetic acid was added to terminate all digestions. The digests were diluted to 0.5 µg/µL prior to LC-MS analysis. LC gradients with 5% to 85% organic solvent (60/40, ACN/IPA, 0.1% TFA) in 35 to 50 min were used for the separation of positional isomers. Multiple batches of cysteine-conjugated ADCs (with increasing DAR) were analyzed in this study.

Preliminary Results/Abstract:

This study aims to investigate different enzymatic digestion processes for the generation of ADC subunits and to localize the conjugation sites at the subunit level. Proteases IdeS and SpeB were first used to cleave the ADCs hinge region with subsequent DTT reduction to produce three subunits, LC, Fc/2 and Fd, each with a molecular mass of roughly 25 kDa. Limited Lys-C digestion was also evaluated to cleave at the Lysine residue of the ADC hinge region to generate similar LC, Fc/2 and Fd subunits.

The studies focused on the optimization of digestion processes, the LC separation conditions and the MS parameters. Preliminary results show that IdeS generated expected fragments when the mAb sequence contains the suitable cleavage sites. In comparison, the efficiency of SpeB was inhibited by the presence of conjugated payloads. It was observed that the highly conjugated ADCs  (high DAR values) were only partially cleaved by SpeB. The limited Lys-C digestion was the most efficient in producing expected subunits with minimal miscleavage in the hinge region for highly conjugated ADCs. The major conjugated species were confirmed to be LC+payload, Fc/2+payload, Fc/2+2payloads and Fd+payload.

The intensity of the subunits was used to calculate the DARs for the ADC batches. They were found to be in good agreement with the values from the native intact LC/SEC MS and HIC analyses. Observed peaks for positional isomers of the conjugated ADCs were identified by a middle-down fragmentation approach. The subunit analysis required minimal sample preparation and shorter LC run time when compared to peptide mapping experiments. It also provided more detailed structural information, such as positional isomer locations, which could not be directly obtained from intact mass analysis.

The analysis of ADC subunits from protease digestion has become an enabling tool for the elucidation of ADC structures during the product development processes.

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