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Evaluating the efficacy of immobilized metal affinity chromatography (IMAC) for host cell protein (HCP) removal from anti-HER2 scFv expressed in Escherichia coli.

Authors :
Soltaninasab S
Ahmadzadeh M
Shahhosseini S
Mohit E
Source :
Protein expression and purification [Protein Expr Purif] 2022 Feb; Vol. 190, pp. 106004. Date of Electronic Publication: 2021 Oct 21.
Publication Year :
2022

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 ˂ 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.<br /> (Copyright © 2021 Elsevier Inc. All rights reserved.)

Details

Language :
English
ISSN :
1096-0279
Volume :
190
Database :
MEDLINE
Journal :
Protein expression and purification
Publication Type :
Academic Journal
Accession number :
34688918
Full Text :
https://doi.org/10.1016/j.pep.2021.106004