Evaluating the efficacy of immobilized metal affinity chromatography (IMAC) for host cell protein (HCP) removal from anti-HER2 scFv expressed in Escherichia coli.
Protein Expr Purif
; 190: 106004, 2022 02.
Article
en En
| MEDLINE
| ID: mdl-34688918
ABSTRACT
Host cell proteins (HCPs) are process-related impurities that have influence on product safety and efficacy. HCPs should effectively be removed by chromatographic steps in downstream purification process. In this study, we aimed to evaluate the efficacy of immobilized-metal affinity chromatography (IMAC) for separation of HCPs from anti-HER2 single chain fragment variable (scFv) expressed in E. coli. This study explored how different purification conditions including native, denaturing and hybrid affect HCP level in purified anti-HER2 scFv. Furthermore, the effects of NaCl concentration in wash buffer as well as imidazole concentration in wash and elution buffer on purification yield and HCP level in purified anti-HER2 scFv were evaluated. It was found that increasing imidazole concentration in wash and elution buffers in native conditions reduced the yield of anti-HER2 scFv purification. However, enhancing NaCl concentration in wash buffer in purification under native conditions led to significant increase in the amount of anti-HER2 scFv without any change in protein purity. Herein, none of the IMAC purification methods conducted on soluble cytoplasmic proteins under native conditions could reduce the amount of HCP to acceptable level. HCP content was only lowered to Ë 10 ppm when inclusion bodies were purified under hybrid conditions. Furthermore, increasing imidazole concentration in wash buffer in purification under hybrid conditions led to significant increase in eluted anti-HER2 scFv concentration, while HCP content was also increased in this condition. Overall, purification under hybrid conditions using wash buffer containing 40 mM imidazole resulted in the highest yield and acceptable level of HCP.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Contexto en salud:
3_ND
Problema de salud:
3_neglected_diseases
/
3_zoonosis
Asunto principal:
Expresión Génica
/
Cromatografía de Afinidad
/
Receptor ErbB-2
/
Proteínas de Escherichia coli
/
Escherichia coli
/
Anticuerpos de Cadena Única
Tipo de estudio:
Evaluation_studies
Límite:
Humans
Idioma:
En
Revista:
Protein Expr Purif
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2022
Tipo del documento:
Article
País de afiliación:
Irán