Intact Protein Mass Spectrometry of Cell Culture Harvest Guides Cell Line Development for Trispecific Antibodies

Tousi, Fateme; Jiang, Yan; Sivendran, Sharmila; Song, Yvonne; Elliott, Susan; Paiva, Anthony; Lund, Anders H.; Albee, Karen; Lee, Karen

doi:10.1021/acs.analchem.9b05018

Cited by 8 publications

(16 citation statements)

References 17 publications

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“…Intact Mw measurements were also achieved. Tousi elegantly demonstrated coupling denaturing UPLC SEC-MS to analyze both ProA purified material and clarified harvest samples to profile mis-paired and other product-related impurities, including half antibodies, of the expected trispecific antibody construct.…”

Section: Denaturing Ms and Its Use In Biopharmaceutical Researchmentioning

confidence: 99%

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

Campuzano

Sandoval²

2021

J. Am. Soc. Mass Spectrom.

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Mass spectrometry (MS) plays a key role throughout all stages of drug development and is now as ubiquitous as other analytical techniques such as surface plasmon resonance, nuclear magnetic resonance, and supercritical fluid chromatography, among others. Herein, we aim to discuss the history of MS, both electrospray and matrix-assisted laser desorption ionization, specifically for the analysis of antibodies, evolving through to denaturing and native-MS analysis of newer biologic moieties such as antibody–drug conjugates, multispecific antibodies, and interfering nucleic acid–based therapies. We discuss challenging therapeutic target characterization such as membrane protein receptors. Importantly, we compare and contrast the MS and hyphenated analytical chromatographic methods used to characterize these therapeutic modalities and targets within biopharmaceutical research and highlight the importance of appropriate MS deconvolution software and its essential contribution to project progression. Finally, we describe emerging applications and MS technologies that are still predominantly within either a development or academic stage of use but are poised to have significant impact on future drug development within the biopharmaceutic industry once matured. The views reflected herein are personal and are not meant to be an exhaustive list of all relevant MS performed within biopharmaceutical research but are what we feel have been historically, are currently, and will be in the future the most impactful for the drug development process.

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Section: Denaturing Ms and Its Use In Biopharmaceutical Researchmentioning

confidence: 99%

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

Campuzano

Sandoval²

2021

J. Am. Soc. Mass Spectrom.

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“…For example, an intact mass spectrometry assay using denaturing SEC-MS has been reported with strong acceleration potential for cell line development. 39 Implementation of the automated analysis described here could further reduce timelines of the denaturing SEC-MS assay.…”

Section: ■ Discussion and Conclusionmentioning

confidence: 99%

“…21 In addition to the typical challenges of biotherapeutics such as appropriate physical and chemical stability, the molecular integrity of msAbs can be impaired by chain mispairing. 21,37,39 Chain mispairing of msAbs leads to non-functional proteins, low purity of the desired product and hence insufficient production yield, and high cost of goods. In theory, combinatorial chain pairing in a classical IgG-like msAb format, where two heavy chains (HCs) are combined with two light chains (LCs), can result in ten different complexes due to random chain pairing (Figure 1).…”

Section: ■ Introductionmentioning

confidence: 99%

“…Advances have been reported in the literature such as a solution for rapid sample injection, absolute quantification of correct and mispaired species via 2D-LC HIC-RP-MS (two-dimensional hydrophobic interaction chromatography-reversed-phase chromatography-mass spectrometry), and direct injection of cell supernatant. 39,48,49 These approaches have been shown to greatly accelerate the respective applications, but they usually require individual assay development and thus lack the ability for generic use over a broad spectrum of sequences and formats.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Especially in research where candidate numbers are high and generic assays are needed, MS is considered the ultimate choice for the identification of mispaired variants. , Consistently, significant effort has been spent to adapt conventional LC-MS, a labor- and time-intensive method, to a higher throughput. Advances have been reported in the literature such as a solution for rapid sample injection, absolute quantification of correct and mispaired species via 2D-LC HIC-RP-MS (two-dimensional hydrophobic interaction chromatography-reversed-phase chromatography-mass spectrometry), and direct injection of cell supernatant. ,, These approaches have been shown to greatly accelerate the respective applications, but they usually require individual assay development and thus lack the ability for generic use over a broad spectrum of sequences and formats.…”

Section: Introductionmentioning

confidence: 99%

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High-Throughput and Format-Agnostic Mispairing Assay for Multispecific Antibodies Using Intact Mass Spectrometry

Ziegengeist,

Orth,

Kroll

et al. 2023

Anal. Chem.

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Multispecific antibodies have gained significant importance in a broad indication space due to their ability to engage multiple epitopes simultaneously and to thereby overcome therapeutic barriers. With growing therapeutic potential, however, the molecular complexity increases, thus intensifying the demand for innovative protein engineering and analytical strategies. A major challenge for multispecific antibodies is the correct assembly of light and heavy chains. Engineering strategies exist to stabilize the correct pairing, but typically individual engineering campaigns are required to arrive at the anticipated format. Mass spectrometry has proven to be a versatile tool to identify mispaired species. However, due to manual data analysis procedures, mass spectrometry is limited to lower throughputs. To keep pace with increasing sample numbers, we developed a high-throughput-capable mispairing workflow based on intact mass spectrometry with automated data analysis, peak detection, and relative quantification using Genedata Expressionist. This workflow is capable of detecting mispaired species of ∼1000 multispecific antibodies in three weeks and thus is applicable to complex screening campaigns. As a proof of concept, the assay was applied to engineering a trispecific antibody. Strikingly, the new setup has not only proved successful in mispairing analysis but has also revealed its potential to automatically annotate other product-related impurities. Furthermore, we could confirm the assay to be format-agnostic, as shown by analyzing several different multispecific formats in one run. With these comprehensive capabilities, the new automated intact mass workflow can be applied as a universal tool to detect and annotate peaks in a format-agnostic approach and in high-throughput, thus enabling complex discovery campaigns.

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Protein analysis by desorption electrospray ionization mass spectrometry

Venter

2024

Mass Spectrometry Reviews

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This review presents progress made in the ambient analysis of proteins, in particular by desorption electrospray ionization‐mass spectrometry (DESI‐MS). Related ambient ionization techniques are discussed in comparison to DESI‐MS only to illustrate the larger context of protein analysis by ambient ionization mass spectrometry. The review describes early and current approaches for the analysis of undigested proteins, native proteins, tryptic digests, and indirect protein determination through reporter molecules. Applications to mass spectrometry imaging for protein spatial distributions, the identification of posttranslational modifications, determination of binding stoichiometries, and enzymatic transformations are discussed. The analytical capabilities of other ambient ionization techniques such as LESA and nano‐DESI currently exceed those of DESI‐MS for in situ surface sampling of intact proteins from tissues. This review shows, however, that despite its many limitations, DESI‐MS is making valuable contributions to protein analysis. The challenges in sensitivity, spatial resolution, and mass range are surmountable obstacles and further development and improvements to DESI‐MS is justified.

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Intact Protein Mass Spectrometry of Cell Culture Harvest Guides Cell Line Development for Trispecific Antibodies

Abstract: Figure 1. Illustration of subunit mispairing in trispecific antibodies with CODV (Crossover Dual Variable) IgG-like design. Article pubs.acs.org/ac

Cited by 8 publications

References 17 publications

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

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

High-Throughput and Format-Agnostic Mispairing Assay for Multispecific Antibodies Using Intact Mass Spectrometry

Protein analysis by desorption electrospray ionization mass spectrometry

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