Precise quantification of mixtures of bispecific IgG produced in single host cells by liquid chromatography-Orbitrap high-resolution mass spectrometry

Yin, Yiyuan; Han, Guanghui; Zhou, Jian; Dillon, Michael A.; McCarty, Luke; Gavino, Lou; Ellerman, Diego; Spiess, Christoph; Sandoval, Wendy; Carter, Paul J.

doi:10.1080/19420862.2016.1232217

Cited by 35 publications

(76 citation statements)

References 37 publications

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“…RP chromatography requires significant concentrations of organic solvent often adjusted to very low pH (<3.0) using elevated column temperatures. 1 These conditions result in denaturation of most proteins and a broad distribution of relatively high charge states at low m/z ranges. Denaturing LC-MS can present analytical challenges for moderately heterogeneous samples.…”

Section: Introductionmentioning

confidence: 99%

“…2,9,10 Modern mass analyzers, such as Orbitrap MS systems, have enabled native MS to deliver increasingly higher resolution data. 1 , 2,11–15 Achieving baseline resolution of intact protein isoforms, such as monoclonal antibody (mAb) glycoforms, allows heterogeneous intact protein samples to be efficiently measured without sample pre-treatment (e.g., reduction or deglycosylation), and offers a true intact mass measurement of complex biologics.…”

Section: Introductionmentioning

confidence: 99%

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Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis

Bailey

Han²,

Phung³

et al. 2018

mAbs

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The preponderance and diversity of charge variants in therapeutic monoclonal antibodies has implications for antibody efficacy and degradation. Understanding the extent and impact of minor antibody variants is of great interest, and it is also a critical regulatory requirement. Traditionally, a combination of approaches is used to characterize antibody charge heterogeneity, including ion exchange chromatography and independent mass spectrometric variant site mapping after proteolytic digestion. Here, we describe charge variant native mass spectrometry (CVMS), an integrated native ion exchange mass spectrometry-based charge variant analytical approach that delivers detailed molecular information in a single, semi-automated analysis. We utilized pure volatile salt mobile phases over a pH gradient that effectively separated variants based on minimal differences in isoelectric point. Characterization of variants such as deamidation, which are traditionally unattainable by intact mass due to their minimal molecular weight differences, were measured unambiguously by mass and retention time to allow confident MS1 identification. We demonstrate that efficient chromatographic separation allows introduction of the purified forms of the charge variant isoforms into the Orbitrap mass spectrometer. Our CVMS method allows confident assignment of intact monoclonal antibody isoforms of similar mass and relative abundance measurements across three orders of magnitude dynamic range.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis

Bailey

Han²,

Phung³

et al. 2018

mAbs

Self Cite

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“…Generally, LC mispairing is driven by chain expression stoichiometry mismatches, so the levels of double mispaired LC is significantly lower than the single mispaired LC. The levels of double mispaired LC can be estimated by the abundance of single mispaired species through a validated probability-based mathematical model to differentiate the two isobaric species (26). In addition, for differentiating the isobaric species on our lead clones, we also perform MS characterization using middle down digestion to analyze the individual Fab arms (23).…”

mentioning

confidence: 99%

High-throughput antibody screening from complex matrices using intact protein electrospray mass spectrometry

Sawyer¹,

Srikumar²,

Carver³

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Toward the goal of increasing the throughput of high-resolution mass characterization of intact antibodies, we developed a RapidFire-mass spectrometry (MS) assay using electrospray ionization. We achieved unprecedented screening throughput as fast as 15 s/sample, which is an order of magnitude improvement over conventional liquid chromatography (LC)-MS approaches. The screening enabled intact mass determination as accurate as 7 ppm with baseline resolution at the glycoform level for intact antibodies. We utilized this assay to characterize and perform relative quantitation of antibody species from 248 samples of 62 different cell line clones at four time points in 2 h using RapidFiretime-of-flight MS screening. The screening enabled selection of clones with the highest purity of bispecific antibody production and the results significantly correlated with conventional LC-MS results. In addition, analyzing antibodies from a complex plasma sample using affinity-RapidFire-MS was also demonstrated and qualified. In summary, the platform affords high-throughput analyses of antibodies, including bispecific antibodies and potential mispaired side products, in cell culture media, or other complex matrices.antibody screening | high throughput | intact protein mass spectrometry | electrospray U nambiguous characterization of analytes from complex matrices with high content information in a label-free format continues to expand the application of mass spectrometry (MS) in drug discovery (1-3). With the need for high accuracy, sensitivity, and selectivity, the rapidly improving MS instrumentations are emerging at the forefront of analyzing analytes with limited alternative assays for biologics developability (4-6), biotransformation, and high-throughput screening (HTS). For ultra-high-throughput screening, matrix-assisted laser desorption/ionization (MALDI) is amenable to speeds >100,000 samples per day (7-11). Through incorporating self-assembled monolayers for MALDI-time-of-flight (TOF) (SAMDI) technology (12-14), it is also possible to infer small molecule noncovalent hit identification from MALDI-MS detection under native conditions. While such MALDI-MS approaches have the capability for screening small molecule and peptide analytes, larger molecular weight proteins suffer from limited mass resolution and quantitative challenges. Alternatively, electrospray ionization MS (ESI-MS) can provide isotopic resolution for molecules as large as antibodies (15). Although modern mass spectrometers can scan as rapidly as tens of microseconds, highthroughput antibody analyses using ESI-MS is challenged by the relatively low ion sampling rate from chromatographic elution coupled to MS. For rapid sampling, Agilent RapidFire MS (RF-MS) utilizes a rapid trap and elute strategy to enable desalting and ion sampling coupled to a MS ion source. RF-MS has been shown to afford HTS triage for small molecules and proteins <30 kDa from biochemical buffers as fast as 15 s as opposed to minutes observed with conventional chromatography (16). Altern...

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“…With the improvement in the resolution power of mass spectrometry, mispaired species can be characterized with better accuracy and sensitivity even when mass differences are small. 33 …”

Section: Discussionmentioning

confidence: 99%

A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture

Wang¹,

Vemulapalli²,

Cao³

et al. 2018

mAbs

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Immunoglobulin G–like bispecific antibodies with asymmetric architecture are among the most widely used bispecific antibody formats for diagnostic and therapeutic applications. The primary technical challenge for this format is how to achieve correctly paired assembly of four unique polypeptide chains. Advances in protein engineering and process development are being used to overcome these challenges and are driving a corresponding demand for sensitive analytical tools to monitor and control mispaired species. Here, we report a systematic approach for analysis and characterization of mispairing in asymmetric bispecific antibodies. This approach consists of three orthogonal components, the first of which is a liquid chromatography (LC)-mass spectrometry (MS)–based method to measure the mass of intact antibodies. This method is used for fast analysis of mispairing and requires minimal method development, which makes it an ideal choice for early-stage development. The second component is a hydrophobic interaction chromatography (HIC)–based mispairing method that is suitable for lot release testing. The HIC method is robust and quality control friendly, and offers great linearity, precision, and accuracy. The third component is a two-dimensional LC-MS method for on-line chromatographic peak identification, which not only expedites this task but also reduces the risk of undesirable modifications during conventional fraction collection. These three methods dovetail to form the foundation of a complementary toolbox for analysis and characterization of mispairing in asymmetric bispecific antibodies and provide guidance and support for process development throughout the drug development life cycle.

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Precise quantification of mixtures of bispecific IgG produced in single host cells by liquid chromatography-Orbitrap high-resolution mass spectrometry

Cited by 35 publications

References 37 publications

Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis

Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis

High-throughput antibody screening from complex matrices using intact protein electrospray mass spectrometry

A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture

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