Collision induced unfolding detects subtle differences in intact antibody glycoforms and associated fragments

Tian, Yuwei; Ruotolo, Brandon T.

doi:10.1016/j.ijms.2017.12.005

Cited by 49 publications

(63 citation statements)

References 52 publications

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“…This technology involves the collisional activation of protein ions prior to IM‐MS separation in order to initiate protein unfolding events in the gas‐phase. CIU is capable of differentiating IgG subclasses, detecting minor alterations in mAb glycoforms, assessing stability shifts associated with site‐specific ADCs, and evaluating the comparability of biosimilars …”

Section: Introductionmentioning

confidence: 99%

“…CIU is capable of differentiating IgG subclasses, detecting minor alterations in mAb glycoforms, assessing stability shifts associated with site-specific ADCs, and evaluating the comparability of biosimilars. [30][31][32][33][34][35][36][37] In this report, we present a CIU based workflow for the rapid characterization of a human IgG1 mAb conjugated with biotin via its native lysine residues, which we treat as a model ADC system. Despite the high degree of structural similarity across conjugation states revealed by native IM-MS, our CIU results indicate the presence of subtle structural changes in the mAbs upon biotin conjugation, as revealed by shifts in their overall stabilities and ground state CCSs.…”

Section: Introductionmentioning

confidence: 99%

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Quantitative collision‐induced unfolding differentiates model antibody–drug conjugates

et al. 2018

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Antibody–drug conjugates (ADCs) are antibody‐based therapeutics that have proven to be highly effective cancer treatment platforms. They are composed of monoclonal antibodies conjugated with highly potent drugs via chemical linkers. Compared to cysteine‐targeted chemistries, conjugation at native lysine residues can lead to a higher degree of structural heterogeneity, and thus it is important to evaluate the impact of conjugation on antibody conformation. Here, we present a workflow involving native ion mobility (IM)‐MS and gas‐phase unfolding for the structural characterization of lysine‐linked monoclonal antibody (mAb)–biotin conjugates. Following the determination of conjugation states via denaturing Liquid Chromatography‐Mass Spectrometry (LC–MS) measurements, we performed both size exclusion chromatography (SEC) and native IM‐MS measurements in order to compare the structures of biotinylated and unmodified IgG1 molecules. Hydrodynamic radii (Rh) and collision cross‐sectional (CCS) values were insufficient to distinguish the conformational changes in these antibody–biotin conjugates owing to their flexible structures and limited instrument resolution. In contrast, collision induced unfolding (CIU) analyses were able to detect subtle structural and stability differences in the mAb upon biotin conjugation, exhibiting a sensitivity to mAb conjugation that exceeds native MS analysis alone. Destabilization of mAb–biotin conjugates was detected by both CIU and differential scanning calorimetry (DSC) data, suggesting a previously unknown correlation between the two measurement tools. We conclude by discussing the impact of IM‐MS and CIU technologies on the future of ADC development pipelines.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Quantitative collision‐induced unfolding differentiates model antibody–drug conjugates

et al. 2018

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“…Finally, collision induced unfolding (CIU) experiments (Figure ) were performed to compare the gas‐phase unfolding behavior of the three VHHs. This new approach has already been reported for the rapid characterization of intact monoclonal antibodies, fragments and antibody drug conjugates . Briefly, in CIU experiments, ions are progressively accelerated in the trap T‐wave of the mass spectrometer before IM separation in the IM cell.…”

Section: Resultsmentioning

confidence: 99%

“…Ions were activated in the trap cell by increasing progressively the trap collision voltage in 5 V steps from 0 to 120 V prior to IM separation during 1 min run. Generation and data interpretation of CIU plots were performed using the open‐source CIU_Suite software, allowing the extraction of the arrival time distribution (ATD) of ions of interest at each trap collision voltage as described previously …”

Section: Methodsmentioning

confidence: 99%

“…Generation and data interpretation of CIU plots were performed using the open-source CIU_Suite software, allowing the extraction of the arrival time distribution (ATD) of ions of interest at each trap collision voltage as described previously. 22,27 4.16 | VHH-"antigen" interaction measurements SPR experiments were performed to measure the interactions between antibodies and HER2 protein were investigated using a BIAcore T200 at 25 C. Sensor surfaces CM5 sensorchips (BR100530), amine coupling kit and other Biacore consumables were purchased from GE Healthcare. HER2 protein was immobilized on the sensor surface and the VHH antibodies were injected over the surface in a continuous flow.…”

Section: Collision Induced Unfolding Experiments (Ciu)mentioning

confidence: 99%

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VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

Hartmann

Botzanowski

Galibert³

et al. 2019

Protein Science

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In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we investigated two different production strategies of this small antibody‐like protein, using an anti‐HER2 VHH as a model. The total chemical synthesis of the 125 amino‐acid peptide was performed with reasonable yield, even if optimization will be necessary to upgrade this kind of production. In parallel, we expressed the same sequence in two different hosts: Escherichia coli and Pichia pastoris. Both productions were successful and led to a fair amount of VHHs. The integrity and conformation of the VHH were characterized by complementary mass spectrometry approaches, while surface plasmon resonance experiments were used to assess the VHH recognition capacity and affinity toward its “antigen.” Using this combination of orthogonal techniques, it was possible to show that the three VHHs—whether synthetic or recombinant ones—were properly and similarly folded and recognized the “antigen” HER2 with similar affinities, in the nanomolar range. This opens a route toward further exploration of modified VHH with unnatural amino acids and subsequently, VHH‐drug conjugates.

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Native Mass Spectrometry in Drug Discovery and Development

Jia

Wen

Mozziconacci

et al. 2023

High‐Throughput Mass Spectrometry in Drug Discovery

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Collision induced unfolding detects subtle differences in intact antibody glycoforms and associated fragments

Cited by 49 publications

References 52 publications

Quantitative collision‐induced unfolding differentiates model antibody–drug conjugates

Quantitative collision‐induced unfolding differentiates model antibody–drug conjugates

VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

Native Mass Spectrometry in Drug Discovery and Development

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