High-Throughput Kinetic Characterization of Irreversible Covalent Inhibitors of KRASG12C by Intact Protein MS and Targeted MRM

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 RapidFire–time-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.

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Automated high-throughput process for site-directed mutagenesis, production, purification, and kinetic characterization of enzymes

Analytical Biochemistry ◽

10.1016/j.ab.2006.04.047 ◽

2006 ◽

Vol 355 (1) ◽

pp. 110-116 ◽

Cited By ~ 11

Author(s):

Raphaël Carapito ◽

Benoit Gallet ◽

André Zapun ◽

Thierry Vernet

Keyword(s):

High Throughput ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Kinetic Characterization

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Kinetic Characterization of Fragment Binding in AmpC β-Lactamase by High-Throughput Molecular Simulations

Journal of Chemical Information and Modeling ◽

10.1021/ci4006063 ◽

2014 ◽

Vol 54 (2) ◽

pp. 362-366 ◽

Cited By ~ 15

Author(s):

P. Bisignano ◽

S. Doerr ◽

M. J. Harvey ◽

A. D. Favia ◽

A. Cavalli ◽

...

Keyword(s):

High Throughput ◽

Molecular Simulations ◽

Kinetic Characterization

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Serum proteomics expands on high-affinity antibodies in immunized rabbits than deep B-cell repertoire sequencing alone

10.1101/833871 ◽

2019 ◽

Author(s):

Stefano R. Bonissone ◽

Thiago Lima ◽

Katherine Harris ◽

Laura Davison ◽

Brian Avanzino ◽

...

Keyword(s):

B Cell ◽

High Throughput ◽

Proteomic Analysis ◽

Intact Protein ◽

Serum Antibodies ◽

Cell Repertoire ◽

Serum Proteomics ◽

B Cell Repertoire ◽

Repertoire Sequencing

AbstractRabbits are a model for immunology studies, and monoclonal antibodies developed from rabbits have been sought after to empower immunoassays in a variety of applications. High-throughput characterization of circulating serum antibodies in response to specific antigens is highly impactful for both humoral immunology studies and antibody development. A combination of high throughput sequencing of antibody transcripts from B cells and proteomic analysis of serum antibodies, an approach referred to as immunoproteogenomics, is applied to profile the immune response of rabbits to β-galactosidase (Beta-gal) in both recombinant antigen and peptide antigen immunization formats. The use of intact protein antigen resulted in observing 56.3% more heavy chains CDR3s in serum than immunization with peptide antigens. Additionally, sampling peripheral blood mononuclear cells (PBMCs) for B-cell repertoire sequencing at different time points throughout the immunization was found to capture 47.8%-72.8% of total proteomically observed heavy chain CDR3s, and would serve well in replacing sequencing the B cell rich, but more difficult to access spleen or bone marrow compartments. Despite B-cell repertoire sequencing to depths of 2M to 10M reads, we found proteomic evidence supporting at least 10% of serum antibodies are still missed. Further improvements to proteomic analysis techniques would enable more precise characterization of antibodies circulating in serum and determine antibody protein sequences missed by repertoire sequencing.

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