Behavior of host-cell-protein-rich aggregates in antibody capture and polishing chromatography.
J Chromatogr A
; 1702: 464081, 2023 Aug 02.
Article
em En
| MEDLINE
| ID: mdl-37244165
ABSTRACT
Recent work has shown that aggregates in monoclonal antibody (mAb) solutions may be made up not just of mAb oligomers but can also harbor hundreds of host-cell proteins (HCPs), suggesting that aggregate persistence through downstream purification operations may be related to HCP clearance. We have examined this in a primary analysis of aggregate persistence through processing steps that are typically implemented for HCP reduction, demonstrating that the phenomenon is relevant to depth filtration, protein A chromatography and flow-through anion-exchange (AEX) polishing. Confocal laser scanning microscopy observations show that aggregates compete with the mAb to adsorb specifically in protein A chromatography and that this competitive interaction is integral to the efficacy of protein A washes. Column chromatography reveals that the protein A elution tail can have a relatively high concentration of aggregates, which corroborates analogous observations from recent HCP studies. Similar measurements in flow-through AEX chromatography show that relatively large aggregates that harbor HCPs and that persist into the protein A eluate can be retained to an extent that appears to depend primarily on the resin surface chemistry. The total aggregate mass fraction of both protein A eluate pools (â¼ 2.4 - 3.6%) and AEX flow-through fractions (â¼ 1.5 - 3.2%) correlates generally with HCP concentrations measured using enzyme-linked immunosorbent assay (ELISA) as well as the number of HCPs that may be identified in proteomic analysis. This suggests that quantification of the aggregate mass fraction may serve as a convenient albeit imperfect surrogate for informing early process development decisions regarding HCP clearance strategies.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cromatografia
/
Proteômica
Limite:
Animals
Idioma:
En
Revista:
J Chromatogr A
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Estados Unidos