Identification and characterization of buried unpaired cysteines in a recombinant monoclonal IgG1 antibody.
Anal Chem
; 84(16): 7112-23, 2012 Aug 21.
Article
em En
| MEDLINE
| ID: mdl-22794164
ABSTRACT
The heterogeneity in therapeutic antibodies arising from buried unpaired cysteines has not been well studied. This paper describes the characterization of two unpaired cysteines in a recombinant humanized IgG1 monoclonal antibody (referred to as mAb A). The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis of mAb A samples showed three distinct peaks, indicating the presence of three species. The heterogeneities observed in the RP-HPLC have been determined to arise from unpaired cysteines (Cys-22 and Cys-96) that are buried in the V(H) domain. The Fab containing free thiols (referred to as "free-thiol Fab") and the Fab containing the disulfide (referred to as "intact Fab") of mAb A were generated through limited Lys-C digestion and purified with an ion exchange chromatography method. The binding of free-thiol Fab and intact Fab to its antigen was measured in a cell-based binding assay and an enzyme linked immunosorbent assay. The unpaired cysteines in the Fab of mAb A were found to have no significant impact on the binding to its target. Consistent with these Fab binding data, the enriched intact mAb A containing free thiols was determined to be fully active in a potency assay. The data reported here demonstrate that the redox status of cysteines is potentially a major source of heterogeneity for an antibody.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes
/
Imunoglobulina G
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Cisteína
/
Anticorpos Monoclonais
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article