Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization.
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.
Reliance Life Sciences, Dhirubhai Ambani Life Sciences Center, Navi Mumbai 400701, India. sanjukta_chakrabarti@relbio.com.
Sanjukta
S
Chakrabarti
School of Life and Environmental Sciences, Deakin University, Waurn Ponds Campus, Geelong 3216, Australia. colin.barrow@deakin.edu.au.
Colin J
CJ
Barrow
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. rupinder.kanwar@deakin.edu.au.
Rupinder K
RK
Kanwar
Reliance Life Sciences, Dhirubhai Ambani Life Sciences Center, Navi Mumbai 400701, India. venkata.ramana@relbio.com.
Venkata
V
Ramana
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. jagat.kanwar@deakin.edu.au.
Jagat R
JR
Kanwar
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D057809:HEK293 Cells; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000378:metabolism*
D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins; D040281:Vascular Endothelial Growth Factor Receptor-1
CHOK1SV GS-KO; Fc fusion protein; VEGFR1(D1–D3)-Fc; biological activity; clipping; folded; inhibition; proteolysis; stable