High-throughput Carbonyl Content Method of Therapeutic mAb using size-exclusion chromatography with ultraviolet and fluorescence detection.
Anal Biochem
; 571: 25-36, 2019 04 15.
Article
em En
| MEDLINE
| ID: mdl-30653944
ABSTRACT
Monoclonal antibody (mAb), one of the major types of therapeutic proteins in the pharmaceutical industry, is predominantly manufactured using mammalian cell culture [1]. Oxidative stress, potentially present during cell culture process, may increase the protein carbonyl content in the mAb product, which was reported to positively correlate with aggregate burst rate during storage [2]. In order to monitor carbonyl content during mAb process development, we developed a high-throughput screening method for therapeutic mAbs using size-exclusion chromatography followed by ultraviolet and fluorescence detection (SEC-UV/FL), optimized from a fluorescein thiosemicarbazide (FTC) semi-microplate method. The method demonstrated a good correlation with conventional ELISA assay and FTC-based fluorometric semi-microplate method with improved throughput and precision. The method was successfully applied in three case studies to improve our understanding of mAb carbonylation, including the impact of metal-catalyzed oxidation on an IgG4 mAb, comparison of carbonyl content between several mAbs expressed by CHO cell culture with human serum antibody pool, as well as the surface charge property of carbonylated mAb assessed by ion-exchange chromatography.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Ensaios de Triagem em Larga Escala
/
Anticorpos Monoclonais
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
Estados Unidos