Field flow fractionation for assessing neonatal Fc receptor and Fcγ receptor binding to monoclonal antibodies in solution.
Anal Biochem
; 414(1): 88-98, 2011 Jul 01.
Article
em En
| MEDLINE
| ID: mdl-21385563
ABSTRACT
Analysis of the strength and stoichiometry of immunoglobulin G (IgG) binding to neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR) is important for evaluating the pharmacokinetics and effector functions of therapeutic monoclonal antibody (mAb) products, respectively. The current standard for assessing FcγR and FcRn binding is composed of cell-based and surface plasmon resonance (SPR) assays. In this work, asymmetrical flow field flow fractionation (AF4) was evaluated to establish the true stoichiometry of IgG binding in solution. AF4 and liquid chromatography-mass spectrometry (LC-MS) were applied to directly observe IgG/FcγR and IgG/FcRn complexes, which were not observed using nonequilibrium size exclusion chromatography (SEC) analysis. Human serum albumin (HSA), an abundant component of human blood and capable of binding FcRn, was studied in combination with FcRn and IgG. AF4 demonstrated that the majority of large complexes of IgG/FcRn/HSA were at an approximate 121 molar ratio. In addition, affinity measurements of the complex were performed in the sub-micromolar affinity range. A significant decrease in binding was detected for IgG molecules with increased oxidation in the Fc region. AF4 was useful in detecting weak binding between full-length IgG/Fc fragments and Fc receptors and the effect of chemical modifications on binding. AF4 is a useful technique in the assessment of mAb product quality attributes.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Imunoglobulina G
/
Receptores Fc
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Antígenos de Histocompatibilidade Classe I
/
Receptores de IgG
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Mapeamento de Interação de Proteínas
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Fracionamento por Campo e Fluxo
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Anticorpos Monoclonais
Limite:
Animals
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Humans
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Estados Unidos