Engineering protein A ligands to mitigate antibody loss during high-pH washes in protein A chromatography.
J Chromatogr A
; 1696: 463962, 2023 May 10.
Article
em En
| MEDLINE
| ID: mdl-37043977
ABSTRACT
Protein A chromatography is a workhorse in monoclonal antibody (mAb) manufacture since it provides effective separation of mAbs from impurities such as host-cell proteins (HCPs) in a single capture step. HCP clearance can be aided by the inclusion of a wash step prior to low-pH elution. Although high-pH washes can be effective in removing additional HCPs from the loaded column, they may also contribute to a reduced mAb yield. In this work we show that this yield loss is reflected in a pH-dependent variation of the equilibrium binding capacity of the protein A resin, which is also observed for the capacity of the Fc fragments alone and therefore not a result of steric interactions involving the Fab fragments in the intact mAbs. We therefore hypothesized that the high-pH wash loss was due to protonation or deprotonation of ionizable residues on the protein A ligand. To evaluate this, we applied a rational protein engineering approach to the Z domain (the Fc-binding component of most commercial protein A ligands) and expressed engineered mutants in E. coli. Biolayer interferometry and affinity chromatography experiments showed that some of the Z domain mutants were able to mitigate wash loss at high pH while maintaining similar binding characteristics at neutral pH. These experiments enabled elucidation of the roles of specific interactions in the Z domain - Fc complex, but more importantly offer a route to ameliorating the disadvantages of high-pH washes in protein A chromatography.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteína Estafilocócica A
/
Escherichia coli
Limite:
Animals
Idioma:
En
Revista:
J Chromatogr A
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Estados Unidos