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Int. J. Mol. Sci. 2016, 17(6), 913; doi:10.3390/ijms17060913

Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization

1
Reliance Life Sciences, Dhirubhai Ambani Life Sciences Center, Navi Mumbai 400701, India
2
School of Life and Environmental Sciences, Deakin University, Waurn Ponds Campus, Geelong 3216, Australia
3
Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Centre for Molecular and Medical Research (C-MMR), Deakin University, Waurn Ponds Campus, Geelong 3216, Australia
These authors contributed equally to this work.
*
Authors to whom correspondence should be addressed.
Academic Editor: Salvador Ventura
Received: 5 January 2016 / Revised: 24 May 2016 / Accepted: 27 May 2016 / Published: 9 June 2016
(This article belongs to the Section Bioinorganic Chemistry)
View Full-Text   |   Download PDF [6516 KB, uploaded 9 June 2016]   |  

Abstract

Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1–D3)-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities. View Full-Text
Keywords: Fc fusion protein; VEGFR1(D1–D3)-Fc; CHOK1SV GS-KO; clipping; proteolysis; inhibition; stable; folded; biological activity Fc fusion protein; VEGFR1(D1–D3)-Fc; CHOK1SV GS-KO; clipping; proteolysis; inhibition; stable; folded; biological activity
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Chakrabarti, S.; Barrow, C.J.; Kanwar, R.K.; Ramana, V.; Kanwar, J.R. Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization. Int. J. Mol. Sci. 2016, 17, 913.

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