Evaluation of Immobilized- Metal Affinity Chromatography (IMAC) for host cell proteins (HCPs) removal from anti-HER2 scFv expressed in E.coli

Evaluation of Immobilized- Metal Affinity Chromatography (IMAC) for host cell proteins (HCPs) removal from anti-HER2 scFv expressed in E.coli
Elham mohit,^1,* Maryam ahmadzadeh,² Saba soltaninasab,³
1. Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences
2. Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences
3. Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences
Introduction: Host cell proteins (HCPs) are process-related impurities derived from the host organisms and are unrelated to the intended recombinant product. HCPs have potential effects on biological activity, immunogenicity, and consequently product safety and efficacy. To produce safe and efficient protein biopharmaceuticals, HCPs should effectively be removed by chromatographic steps in downstream purification process. Therefore, monitoring the removal of HCPs in final drug product to acceptable amounts (<100 ppm) is a regulatory requirement. In this study, we aimed to evaluate the efficacy of purification process using Immobilized- Metal Affinity Chromatography (IMAC) for separation of HCPs from the recombinant anti- HER2 single chain fragment variable (scFv) expressed in Escherichia coli. In scFv, the variable regions of heavy (VH) and light (VL) chains of an antibody are connected with each other by a flexible peptide linker. scFvs display improved pharmacokinetic properties, such as better tissue penetration and rapid blood clearance while having specific affinity and low immunogenicity. Anti-HER2 scFv can be fused to the marker proteins, toxins, radioactive agents and drugs and can be used for treatment and diagnosis of HER2+ breast cancer which is the most common cancer and the first cause of cancer death in women, worldwide
Methods: Anti- HER2 scFv was expressed in BL21 (DE3). Anti- HER2 scFv protein was purified by IMAC using Ni-NTA resin under native, denaturing and hybrid conditions. Purity of samples was investigated by SDS-PAGE. Total protein concentration of samples was measured by BCA assay. Herein, a commercial sandwich Enzyme-Linked Immunosorbent Assay (ELISA) (E.coli HCP ELISA kit) was used to determine HCP concentration of different samples.
Results: The yield and purity of anti-HER2 scFv purified under hybrid condition was higher than those purified under native and denaturing methods. The ELISA result showed that all of three methods caused reduction in HCP concentration of elute samples compared with wash and flow through samples. Furthermore, the lowest concentration of HCP in elute samples was obtained by IMAC purification under hybrid condition (2-3 ng/mL).
Conclusion: : IMAC under hybrid condition was a suitable method for separation of HCPs from the recombinant anti-HER2 scFv expressed in BL21 (DE3).
Keywords: Anti-HER2 scFv, HCP, Purification, IMAC