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

Jiang, M.-S.; Yang, X.; Esposito, D.; Nelson, E.; Yuan, J.; Hopkins, R.F.; Broadt, T.; Xiao, Z.; Colantonio, S.; Prieto, D.A.; Welch, A.R.; Creekmore, S.P.; Mitra, G.; Zhu, J.

International Immunopharmacology 29(2): 476-487

2015


ISSN/ISBN: 1878-1705
PMID: 26474694
DOI: 10.1016/j.intimp.2015.10.008
Accession: 058260874

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Abstract
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 ~50mg/L was selected for production at 2L 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 ~50mg/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.