Purification and determination of antibody drugs in bio-samples by EGFR/cell membrane chromatography method.

With the rapid development of therapeutic monoclonal antibody drugs, it is increasingly difficult to meet clinical needs using traditional antibody purification techniques. In this study, epidermal growth factor receptor (EGFR)-SNAP-tag was expressed in HEK293 cells. Then we captured the EGFR-SNAP-tag from the cell membrane suspension onto a O6-benzylguanine-modified silica gel to prepare a new EGFR stationary phase as a bioactive material, which could specifically recognize its antibody in bio-samples. The EGFR stationary phase was systematically characterized via scanning electron microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, and fourier transform infrared spectroscopy. Then we used EGFR stationary phase to establish a new EGFR cell membrane chromatography (CMC) model. The EGFR/CMC-online-ion exchange chromatography (IEC)/high performance liquid chromatography (HPLC) was established for the efficient purification and specific identification of cetuximab, nituzumab, and panizumab from cell culture medium and human serum. The results show that the EGFR stationary phase prepared by one-step immobilized technique can maintain biological activity and stability like EGFR in cell membrane. The EGFR/CMC-online-IEC/HPLC method has a high specificity, accuracy and sensitivity. Therefore, it will present a valuable method for the purification, identification, and analysis of monoclonal antibody drugs. In this study, HEK293 cells with a high expression of the EGFR carboxy-terminal-labeled SNAP-tag were constructed. The EGFR-SNAP-tag on the cell membrane could interact with BG-modified silica gels (SiO 2 -BG) to obtain a new EGFR stationary phase (EGFR/SP) with EGFR extracellular domain facing outward, which can specifically recognize the monoclonal antibodies acting on the extracellular domain of EGFR. [Display omitted] • A new EGFR/CMC model with EGFR extracellular domain facing outward was prepared. • A highly specific EGFR/CMC model was obtained by SNAP-tag technique. • The EGFR/CMC model was used to purify monoclonal antibody for the first time. [ABSTRACT FROM AUTHOR]