Denaturing and Native Mass Spectrometric Analytics for Biotherapeutic Drug Discovery Research: Historical, Current, and Future Personal Perspectives

Campuzano, Iain D. G.; Sandoval, Wendy

doi:10.1021/jasms.1c00036

Cited by 34 publications

(49 citation statements)

References 307 publications

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“…Deconvolution is the process by which pressures are assigned for each gas considered to be present in the MS. It is one of the most widely used techniques in the reconstruction and analysis of samples obtained by spectrometry and spectroscopy in many different areas of study, including Raman spectrometry for pediatric diagnoses [26], time-resolved fluorescence spectroscopy [27], nuclear instruments MS [28] and spectrometry analytics [29,30], among others.…”

Section: Iterative Deconvolutionmentioning

confidence: 99%

Reconstruction of Ultra-High Vacuum Mass Spectra Using Genetic Algorithms

et al. 2021

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In ultra-high vacuum systems, obtaining the composition of a mass spectrum is often a challenging task due to the highly overlapping nature of the individual profiles of the gas species that contribute to that spectrum, as well as the high differences in terms of degree of contribution (several orders of magnitude). This problem is even more complex when not only the presence but also a quantitative estimation of the contribution (partial pressure) of each species is required. This paper aims at estimating the relative contribution of each species in a target mass spectrum by combining a state-of-the-art machine learning method (multilabel classifier) to obtain a pool of candidate species based on a threshold applied to the probability scores given by the classifier with a genetic algorithm that aims at finding the partial pressure at which each one of the species contributes to the target mass spectrum. For this purpose, we use a dataset of synthetically generated samples. We explore different acceptance thresholds for the generation of initial populations, and we establish comparative metrics against the most novel method to date for automatically obtaining partial pressure contributions. Our results show a clear advantage in terms of the integral error metric (up to 112 times lower for simpler spectra) and computational times (up to 4 times lower for complex spectra) in favor of the proposed method, which is considered a substantial improvement for this task.

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Section: Iterative Deconvolutionmentioning

confidence: 99%

Reconstruction of Ultra-High Vacuum Mass Spectra Using Genetic Algorithms

et al. 2021

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“…1,4,16−18 The native MS approach has been shown to have advantages for the analysis of both noncovalently and covalently bound ADC samples compared to analysis performed under denaturing conditions. 1,4,16,17 Lower-resolution levels, ∼5000−15,000 at the target m/z peak, are common for mass measurements of proteins in the native MS approach. 16,17,19−21 Analysis of ADCs/AOCs of a low-to-medium sample complexity could be efficiently conducted with Orbitrap Fourier transform MS (FTMS), 8,9,20−23 time-of-flight (TOF) MS, 4,18,20,24 and triple quadrupole MS platforms.…”

Section: ■ Introductionmentioning

confidence: 99%

“…High levels of selectivity and specificity of mass spectrometry (MS), typically coupled to liquid chromatography (LC), render it an analytical technique of choice to provide the required accuracy needed in deducing the DAR values. ,,, The currently employed MS methods differ by the resolution performance (low vs high resolution) and the degree of protein unfolding (denaturing vs native MS conditions). ,,− The native MS approach has been shown to have advantages for the analysis of both noncovalently and covalently bound ADC samples compared to analysis performed under denaturing conditions. ,,, Lower-resolution levels, ∼5000–15,000 at the target m / z peak, are common for mass measurements of proteins in the native MS approach. ,,− Analysis of ADCs/AOCs of a low-to-medium sample complexity could be efficiently conducted with Orbitrap Fourier transform MS (FTMS), ,,− time-of-flight (TOF) MS, ,,, and triple quadrupole MS platforms . The increased complexity of ADC samples has paved the way to a broader application of high-resolution MS instruments, , which may still prove to be inadequate at resolving highly congested mass spectra derived from complex ADC/AOC samples.…”

Section: Introductionmentioning

confidence: 99%

Drug-to-Antibody Ratio Estimation via Proteoform Peak Integration in the Analysis of Antibody–Oligonucleotide Conjugates with Orbitrap Fourier Transform Mass Spectrometry

et al. 2021

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The therapeutic efficacy and pharmacokinetics of antibody−drug conjugates (ADCs) in general, and antibody−oligonucleotide conjugates (AOCs) in particular, depend on the drug-to-antibody ratio (DAR) distribution and average value. The DAR is considered a critical quality attribute, and information pertaining to it needs to be gathered during ADC/AOC development, production, and storage. However, because of the high structural complexity of ADC/AOC samples, particularly in the initial drug-development stages, the application of the current state-of-the-art mass spectrometric approaches can be limited for DAR analysis. Here, we demonstrate a novel approach for the analysis of complex ADC/AOC samples, following native sizeexclusion chromatography Orbitrap Fourier transform mass spectrometry (FTMS). The approach is based on the integration of the proteoform-level mass spectral peaks in order to provide an estimate of the DAR distribution and its average value with less than 10% error. The peak integration is performed via a truncation of the Orbitrap's unreduced time-domain ion signals (transients) before mass spectra generation via FT processing. Transient recording and processing are undertaken using an external data acquisition system, FTMS Booster X2, coupled to a Q Exactive HF Orbitrap FTMS instrument. This approach has been applied to the analysis of whole and subunit-level trastuzumab conjugates with oligonucleotides. The obtained results indicate that ADC/AOC sample purification or simplification procedures, for example, deglycosylation, could be omitted or minimized prior to the DAR analysis, streamlining the drug-development process.

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confidence: 99%

“…15 By combining alanine scanning with CIU, it is possible to quantify the contributions of individual residues on the conformational stabilities of protein ions. 16 IM-MS is furthermore employed in the pharmaceutical industry, 17 for example to characterize drug conjugates. 18 Common to all of these applications is a reliance on the generation of native-like protein ions through electrospray ionization (ESI), where the protein is desolvated and charges are added to facilitate its detection in the mass spectrometer.…”

mentioning

confidence: 99%

Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry

Abramsson

Sahin

Hopper

et al. 2021

JACS Au

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In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.

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Denaturing and Native Mass Spectrometric Analytics for Biotherapeutic Drug Discovery Research: Historical, Current, and Future Personal Perspectives

Cited by 34 publications

References 307 publications

Reconstruction of Ultra-High Vacuum Mass Spectra Using Genetic Algorithms

Reconstruction of Ultra-High Vacuum Mass Spectra Using Genetic Algorithms

Drug-to-Antibody Ratio Estimation via Proteoform Peak Integration in the Analysis of Antibody–Oligonucleotide Conjugates with Orbitrap Fourier Transform Mass Spectrometry

Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry

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