Simultaneous Protein Quantitation and Glycosylation Profiling of Antigen-Specific Immunoglobulin G1 in Large Clinical Studies DOI Creative Commons
Steinar Gijze,

Anna M Wasynczuk,

Leanne P. M. van Leeuwen

et al.

Journal of Proteome Research, Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 13, 2024

Antibodies have a key role in the immune system, making their characterization essential to biomedical, biopharmaceutical, and clinical research questions. Antibody effector functions are mainly controlled by quantity, subclass, Fc glycosylation. We describe an integrated method measure these three critical dimensions simultaneously. The subclass-specific immunoglobulin G (IgG) glycosylation analysis combines immunosorbance with glycopeptide-centered LC-MS detection. For IgG1-specific quantitation, commercial, stable isotope labeled IgG1 protein standard was spiked into immunosorbent eluates. Robust quantitation achieved, relying on combination of proteotypic peptide most abundant glycopeptides, generated through proteolytic cleavage from mixture natural recombinant standard. Method performance demonstrated large coronavirus vaccination cohort at throughput 100 samples/day. LC-MS-derived, anti-SARS-CoV-2 spike concentrations ranged 10000 ng/mL correlated well clinically relevant immunoassay. Technical variation 200 times lower than biological variation; intermediate precision 44%. In conclusion, we present capable robustly simultaneously assessing antigen-specific IgG studies. This will facilitate broader understanding responses, especially important interplay among dimensions.

Language: Английский

Stellabody: A novel hexamer‐promoting mutation for improved IgG potency DOI Creative Commons
Clarissa A. Whitehead, Bruce D. Wines, Anna M. Davies

et al.

Immunological Reviews, Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 4, 2024

Summary Advances in antibody engineering are being directed at the development of next generation immunotherapeutics with improved potency. Hexamerisation IgG is a normal physiological aspect biology and recently described mutations that facilitate this process have substantial impact upon monoclonal behavior resulting elicitation dramatically enhanced complement‐dependent cytotoxicity, Fc receptor function, antigen binding effects, such as targeted agonism or microbe neutralization. Whereas discovery hexamerisation enhancing has largely focused on residues exposure surface Fc‐Fc CH2‐CH3 interfaces, our unique approach mostly buried residue H429 CH3 domain. Selective substitution position 429 forms basis Stellabody technology, where choice amino acid results distinct outcomes. H429F monomeric hexamerises after target binding, so called “on‐target” hexamerisation, while H429Y mutant pH‐sensitive hexamers in‐solution prior to binding. Moreover, technologies broadly applicable across family antibody‐based biologic therapeutics, including conventional mAbs, bispecific Ig‐like biologics Fc‐fusions, applications diverse diseases.

Language: Английский

Citations

1
High-throughput N-glycan analysis in aging and inflammaging: State of the art and future directions DOI
Ana Cindrić, Tea Pribić, Gordan Lauc

et al.

Seminars in Immunology, Journal Year: 2024, Volume and Issue: 73, P. 101890 - 101890

Published: May 1, 2024

Language: Английский

Citations

1
Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation DOI Creative Commons

Tamiris Azamor,

Débora Familiar-Macedo, Gielenny M. Salem

et al.

The EMBO Journal, Journal Year: 2024, Volume and Issue: 43(22), P. 5494 - 5529

Published: Oct. 10, 2024

Language: Английский

Citations

1
2D-CEX–FcRn–MS to Study Structure/Function Relation of mAb Charge Variants DOI
Liesa Verscheure,

Isabel Vandenheede,

Eline De Rore

et al.

Analytical Chemistry, Journal Year: 2024, Volume and Issue: 96(45), P. 18122 - 18131

Published: Oct. 29, 2024

The automated elucidation of the interplay between monoclonal antibody (mAb) structure and function using two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS) is reported. Charge variants, induced through forced degradation, are resolved by first-dimension (

Language: Английский

Citations

1
Simultaneous Protein Quantitation and Glycosylation Profiling of Antigen-Specific Immunoglobulin G1 in Large Clinical Studies DOI Creative Commons
Steinar Gijze,

Anna M Wasynczuk,

Leanne P. M. van Leeuwen

et al.

Journal of Proteome Research, Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 13, 2024

Antibodies have a key role in the immune system, making their characterization essential to biomedical, biopharmaceutical, and clinical research questions. Antibody effector functions are mainly controlled by quantity, subclass, Fc glycosylation. We describe an integrated method measure these three critical dimensions simultaneously. The subclass-specific immunoglobulin G (IgG) glycosylation analysis combines immunosorbance with glycopeptide-centered LC-MS detection. For IgG1-specific quantitation, commercial, stable isotope labeled IgG1 protein standard was spiked into immunosorbent eluates. Robust quantitation achieved, relying on combination of proteotypic peptide most abundant glycopeptides, generated through proteolytic cleavage from mixture natural recombinant standard. Method performance demonstrated large coronavirus vaccination cohort at throughput 100 samples/day. LC-MS-derived, anti-SARS-CoV-2 spike concentrations ranged 10000 ng/mL correlated well clinically relevant immunoassay. Technical variation 200 times lower than biological variation; intermediate precision 44%. In conclusion, we present capable robustly simultaneously assessing antigen-specific IgG studies. This will facilitate broader understanding responses, especially important interplay among dimensions.

Language: Английский

Citations

1