Zinc supplementation protects human endostatin Fc fusion against proteolytic degradation during cell culture.

Abstract

Endostatin is a potent anti-angiogenesis compound with efficacy in treating solid tumors and other diseases. However, its clinical application has been hampered by the susceptibility to proteolytic degradation during cell culture production. Here we describe a simple and effective strategy for stabilizing a CHO cell-derived human endostatin Fc fusion. Mass spectrometry analysis of the prominent clipped species revealed that the cleavage sites are located at the N-terminal zinc binding region, which is known to be critical for the structural stability of the molecule. Accordingly, we tested the effect of zinc supplementation on stabilizing the molecule and found that micromolar concentrations of zinc chloride significantly reduced the level of clipping. The protective effect appeared to be mediated via direct interaction between zinc and endostatin, as zinc protects purified endostatin spiked into conditioned medium. Interestingly, copper which is known to have high affinity to endostatin, also prevents degradation. The method provides a robust process for manufacturing Fc-endostatin.

• Authors: Gang Hao, Jessica Dawson, Gene Lee, Robert Longley
• Journal: Protein expression and purification (Protein Expr Purif) Vol. 93 Pg. 18-22 (Jan 2014) ISSN: 1096-0279 [Electronic] United States
• PMID: 24161563 (Publication Type: Journal Article)
• Copyright: Copyright © 2013 Elsevier Inc. All rights reserved.