Native Mass Spectrometry, Ion mobility, and Collision-Induced Unfolding Categorize Malaria Antigen/Antibody Binding

Huang, Yining; Salinas, Nichole D.; Chen, Edwin; Tolia, Niraj H.; Gross, Michael L.

doi:10.1007/s13361-017-1782-0

Cited by 18 publications

(23 citation statements)

References 34 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%

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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“…The effects of accelerated storage conditions on the native-like folded protein structure of the biotherapeutics included in this study were examined with gas-phase travelling-wave collision cross sections measured in Nitrogen ( TW CCS N2 ) measurements using native IM-MS. 31,32 With optimized instrumental parameters, native IM-MS methods are generally accepted to provide biologically relevant measurements of the rotationally averaged solution phase structure. 33,34 Several relevant review articles provide comprehensive information on IM-MS. 31,32 Calculated TW CCS N2 values comparing the selected biotherapeutics before and after incubating for six months at the accelerated storage conditions (25 ± 2°C with 60 ± 5% RH) stipulated…”

Section: Native Ion Mobility-mass Spectrometry and Collision-induced mentioning

confidence: 99%

“…In tandem with the above native IM-MS studies, we further leveraged the power of CIU, 34,[38][39][40] which is considered an orthogonal measurement to differential scanning calorimetry, 41,42 to measure differences in the gas phase stability of the chosen biotherapeutics before and after sample incubation. CIU leverages the capability of IM-MS to measure changes in structural conformation as a function of gas-phase collisional activation.…”

Section: Native Ion Mobility-mass Spectrometry and Collision-induced mentioning

confidence: 99%

The impact of standard accelerated stability conditions on antibody higher order structure as assessed by mass spectrometry

Kerr

Keire

2019

mAbs

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Protein therapeutic higher order structure (HOS) is a quality attribute that can be assessed to help predict shelf life. To model product shelf-life values, possible sample-dependent pathways of degradation that may affect drug efficacy or safety need to be evaluated. As changes in drug thermal stability over time can be correlated with an increased risk of HOS perturbations, the effect of long-term storage on the product should be measured as a function of temperature. Here, complementary high-resolution mass spectrometry methods for HOS analysis were used to identify storage-dependent changes of biotherapeutics (bevacizumab (Avastin), trastuzumab (Herceptin), rituximab (Rituxan), and the NIST reference material 8671 (NISTmAb)) under accelerated or manufacturer-recommended storage conditions. Collision-induced unfolding ion mobility-mass spectrometry data showed changes in monoclonal antibody folded stability profiles that were consistent with the appearance of a characteristic unfolded population. Orthogonal hydrogendeuterium exchange-mass spectrometry data revealed that the observed changes in unfolding occurred in parallel to changes in HOS localized to the periphery of the hinge region. Using intact reverse-phase liquid chromatography-mass spectrometry, we identified several mass species indicative of peptide backbone hydrolysis, located between the variable and constant domains of the heavy chain of bevacizumab. Taken together, our data highlighted the capability of these approaches to identify age-or temperature-dependent changes in biotherapeutic HOS.

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Native Mass Spectrometry, Ion mobility, and Collision-Induced Unfolding Categorize Malaria Antigen/Antibody Binding

Cited by 18 publications

References 34 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

The impact of standard accelerated stability conditions on antibody higher order structure as assessed by mass spectrometry

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