Mammalian cell transient expression, non-affinity purification, and characterization of human recombinant IGFBP7, an IGF-1 targeting therapeutic protein

摘要

Targeted inhibiting insulin-like growth factor 1 is an effective approach for cancer therapy. Insulin-like growth factor binding protein 7 (IGFBP7) is considered as a potential therapeutic protein. However, producing high quality of such non-IgG proteins in mammalian cells is still a challenge in biopharmaceutical development Here, we report a rapid production process by using transient gene transfection in HEK 293E cells. A set of constructs combining several expression promoters, leader sequences, and 5' un-translated regions were generated and optimized, from which the best vector with expression level at similar to 50 mg/L was selected for production at 2 L cell culture scale. Comparison study in downstream purification methods led to development of a scalable, non-affinity chromatography strategy through Super Q Fast Flow Q and Heparin columns. The product was characterized in purity (99%), isoelectric point, molecule weight, glycosylation, and stability by using SEC-HPLC, SDS-PAGE, isoelectric focusing and mass spectrometry. The highly purified product shows IGF-1 binding activity and inhibits IGF-1-induced cell proliferation. This process not only provides a remarkable high expression at similar to 50 mg/L and pure glycosylated mammalian rhIGFBP7, also highlights that transient gene expression technology is practical to be used for production and early development of recombinant non-IgG therapeutic proteins. (C) 2015 Elsevier B.V. All rights reserved.