Native size-exclusion chromatography-mass spectrometry: suitability for antibody–drug conjugate drug-to-antibody ratio quantitation across a range of chemotypes and drug-loading levels

Jones, J. Leighton; Pack, Laura; Hunter, Joshua H.; Valliere-Douglass, John

doi:10.1080/19420862.2019.1682895

Cited by 48 publications

(41 citation statements)

References 37 publications

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“…Chen et al described successful use of nanoESI instead of conventional ESI for cysteine-linked ADCs after proteolytic drug removal [ 25 ]. Online coupling of size exclusion chromatography (SEC) to nMS was then implemented by different groups for the analysis of mAbs and ADCs [ 26 , 27 , 28 , 29 , 30 ], paving the way for routine integration of nMS in high throughput analytical workflows of biopharmaceutical companies. An additional level of separation can be achieved through ion mobility spectrometry coupled to nMS (nIM-MS), which provides conformational characterization in the gas phase.…”

Section: Introductionmentioning

confidence: 99%

State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

et al. 2021

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Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines.

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Section: Introductionmentioning

confidence: 99%

State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

et al. 2021

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“…Conventional liquid separation mechanisms (e.g., reversed-phase liquid chromatography; RPLC) are not suitable for this purpose due to denaturing conditions during the separation. In reaction to that, we have been witnessing rapid development of separation techniques maintaining native-like conformations of the protein analytes, including ion-exchange chromatography (IEX), 14 , 15 size exclusion chromatography (SEC), 16 , 17 and hydrophobic interaction chromatography (HIC). 18 , 19 However, one of the primary bottlenecks for broader implementation of these approaches is the mobile phase incompatibility with mass spectrometry (MS) due to the high concentrations of nonvolatile salts required for the separation.…”

Section: Introductionmentioning

confidence: 99%

Native Structural and Functional Proteoform Characterization of the Prolyl-Alanyl-Specific Endoprotease EndoPro from Aspergillus niger

et al. 2021

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The prolyl-alanyl-specific endoprotease (EndoPro) is an industrial enzyme produced in Aspergillus niger . EndoPro is mainly used for food applications but also as a protease in proteomics. In-depth characterization of this enzyme is essential to understand its structural features and functionality. However, there is a lack of analytical methods capable of maintaining both the structural and functional integrity of separated proteoforms. In this study, we developed an anion exchange (AEX) method coupled to native mass spectrometry (MS) for profiling EndoPro proteoforms. Moreover, we investigated purified EndoPro proteoforms with complementary MS-based approaches, including released N-glycan and glycopeptide analysis, to obtain a comprehensive overview of the structural heterogeneity. We showed that EndoPro has at least three sequence variants and seven N-glycosylation sites occupied by high-mannose glycans that can be phosphorylated. Each glycosylation site showed high microheterogeneity with ∼20 glycans per site. The functional characterization of fractionated proteoforms revealed that EndoPro proteoforms remained active after AEX-separation and the specificity of these proteoforms did not depend on N-glycan phosphorylation. Nevertheless, our data confirmed a strong pH dependence of EndoPro cleavage activity. Altogether, our study demonstrates that AEX-MS is an excellent tool to characterize complex industrial enzymes under native conditions.

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“…Native MS (nMS) technology minimizes sample preparation and preserves the protein higher-order structure and noncovalent interactions (23,24). For instance, nMS is commonly used for the characterization of cysteine-linked ADCs, which consist of a broad population of noncovalent assemblies (25)(26)(27)(28)(29). Some of the typical critical quality attributes (CQAs) such as i) the average drug-to-antibody ratio (DAR); ii) the distribution of drug-loaded species; and iii) the amount of unconjugated mAbs can be determined by nMS (30).…”

Section: Intact Mass Analysis 11 Native Mass Spectrometry (Nms)mentioning

confidence: 99%

Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches

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et al. 2021

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Native size-exclusion chromatography-mass spectrometry: suitability for antibody–drug conjugate drug-to-antibody ratio quantitation across a range of chemotypes and drug-loading levels

Abstract: Native size-exclusion chromatography-mass spectrometry: suitability for antibody-drug conjugate drug-to-antibody ratio quantitation across a range of chemotypes and drug-loading levels, mAbs, 12:1, 1682895,

Cited by 48 publications

References 37 publications

State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

Native Structural and Functional Proteoform Characterization of the Prolyl-Alanyl-Specific Endoprotease EndoPro from Aspergillus niger

Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches

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