Revealing charge heterogeneity of stressed trastuzumab at the subunit level.
Anal Bioanal Chem
; 415(8): 1505-1513, 2023 Mar.
Article
em En
| MEDLINE
| ID: mdl-36693954
ABSTRACT
Trastuzumab is known to be heterogeneous in terms of charge. Stressing trastuzumab under physiological conditions (pH 7.4 and 37 °C) increases charge heterogeneity further. Separation of charge variants of stressed trastuzumab at the intact protein level is challenging due to increasing complexity making it difficult to obtain pure charge variants for further characterization. Here we report an approach for revealing charge heterogeneity of stressed trastuzumab at the subunit level by pH gradient cation-exchange chromatography. Trastuzumab subunits were generated after limited proteolytic cleavage with papain, IdeS, and GingisKHAN®. The basic pI of Fab and F(ab)2 fragments allowed to use the same pH gradient for intact protein and subunit level analysis. Baseline separation of Fab subunits was obtained after GingisKHAN® and papain digestion and the corresponding modifications were determined by LC-MS/MS peptide mapping and middle-down MALDI-ISD FT-ICR MS. The described approach allows a comprehensive charge variant analysis of therapeutic antibodies that have two or more modification sites in the Fab region.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Papaína
/
Anticorpos Monoclonais
Idioma:
En
Revista:
Anal Bioanal Chem
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Holanda