Unravelling the reason for different binding behaviors exhibited by antibody aggregates towards preparative and analytical Protein A columns.
Protein Expr Purif
; 218: 106449, 2024 Jun.
Article
en En
| MEDLINE
| ID: mdl-38423157
ABSTRACT
We previously showed that the root cause of low Protein A step yield observed for certain antibodies/Fc-fusions is the presence of non-binding aggregates in cell culture harvest. A pre-assumption for the above conclusion is that the aggregates, while do not bind to the preparative Protein A column, can bind to the analytical Protein A-high performance liquid chromatography (HPLC) column used for titer measurement. In the current work, using materials from a previous case with the low yield issue, we confirmed that non-binding aggregates in preparative Protein A flow-through can indeed bind to the analytical Protein A column. In addition, we showed that this discrepancy is mainly due to the different loading densities applied under these two circumstances. We also demonstrated that aggregate bound to the analytical Protein A column slightly stronger than the monomer, as it exhibited a longer retention time. In summary, the current study not only confirmed that non-binding aggregates detected in the preparative Protein A flow-through bind to the Protein A-HPLC column and contribute to the measured titer of culture harvest but also unravelled the reason for different binding behaviors exhibited by antibody aggregates towards preparative and analytical Protein A columns.
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Bases de datos:
MEDLINE
Asunto principal:
Fragmentos Fc de Inmunoglobulinas
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Anticuerpos
Idioma:
En
Revista:
Protein Expr Purif
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2024
Tipo del documento:
Article
País de afiliación:
China