Monitoring of the disulfide scrambled species by mixed-mode SEC-HPLC during the purification of a bispecific antibody.
Protein Expr Purif
; 223: 106544, 2024 Nov.
Article
em En
| MEDLINE
| ID: mdl-38972616
ABSTRACT
Size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is an analytical method routinely used for assessing aggregation content in protein samples. As SEC-HPLC separates analytes based on their hydrodynamic radius, it generally lacks the capability of differentiating species that are similar in size. Recently while purifying a bispecific antibody (bsAb), we noticed that SEC-HPLC can provide certain degree of resolution between the target bsAb and a disulfide scrambled form, although these two species were identical in molecular weight. In seeing the unexpected potential of SEC-HPLC at resolving species with similar size, we further tested Zenix SEC-300, a mixed-mode SEC-HPLC column from Sepax, which was reported to be capable of separating protein analytes based on other factors besides size. The Zenix column indeed provided resolution much better than the regular SEC-HPLC column. Upon further optimization, the Zenix column allowed close to baseline separation of the correctly folded and the disulfide scrambled species. The current study, as a complement to the previous reports, further demonstrates that mix-mode SEC-HPLC is capable of separating protein analytes that are close in size but are different in conformation and/or surface characteristics.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cromatografia em Gel
/
Anticorpos Biespecíficos
/
Dissulfetos
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Protein Expr Purif
/
Protein expr. purif
/
Protein expression and purification
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
China