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.
Highlights
Fusion proteins are generated by combining the genes encoding two or more proteins, where the benefits of both the components are achieved [1]
We have shown different ways of minimizing degradation in VEGFR1(D1–D3)-Fc fusion protein in lab scale experiments
The VEGFR1(D1–D3)-Fc gene was generated by fusing the first three domains (D1, D2 and D3) of VEGFR1 gene from a VEGFR1 clone to the Fc (CH2–CH3) region of IgG1 of a monoclonal antibody clone already existing in our laboratory
Summary
Fusion proteins are generated by combining the genes encoding two or more proteins, where the benefits of both the components are achieved [1]. The Fc region of a Fc-fusion protein confers additional pharmacokinetic (immunoglobulin-like) properties, such as increasing the drug half-life due to interaction with the cellular Fc receptor (FcRn), and serum stability of most biologically active proteins. The sales of approved protein-based drugs in 2010 was approximately USD $108 billion [4], with half of it accounting for monoclonal antibodies and the remaining for Fc fusions, antibody conjugated drugs, bispecific antibodies, antigen-binding antibody fragments, and other engineered therapeutic molecules. The total revenue generated from therapeutic proteins, including monoclonal antibodies and fusion proteins, in the US is about $64 billion [5]. In 2012, US sales for Fc fusions grew at the highest rate for all bio-therapeutics in the USA, at an incredible ~35.3% (USD $5.8 billion) [7]. The global sale for fusion proteins is predicted to rise to $10.1 billion on a risk-adjusted basis by 2016 [8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
