Construction and Expression of Fc-FGF21 by Different Expression Systems and Comparison of Their Similarity and Difference with Efruxifermin by In Vitro and In Vivo Studies.

Non-alcoholic steatohepatitis (NASH) is a potential serious disease, which almost has no available medicine for effective treatment today. Efruxifermin is a bivalent Fc-FGF21 candidate drug developed by Akero Therapeutics that has shown promising results in preclinical and clinical trials for NASH and may be approved in future. However, it is produced by Escherichia coli (E. coli) expressing system, which has no glycosylation modifications and is hard to purify for inclusion body. Suspension mammalian cell expression systems, human embryonic kidney 293 (HEK293), and Chinese hamster ovary (CHO) are good choice for protein expression of biopharmaceutical use. In this report, the objective was to produce Fc-FGF21 by mammalian cell expression systems, which enabled necessary glycosylation modifications to occur on the Fc-FGF21 protein and was relatively easy for future large-scale production. We observed that the target protein Fc-FGF21 could be easily degraded in CHO system, such as CHOK1SV or CHOZN, and it was hard to purify, whereas it was more stable in the HEK293 expressing system. Then, similarity between Fc-FGF21 from E. coli and Fc-FGF21 from HEK293 was focused by in vitro and in vivo studies, and we observed no significant difference between the proteins expressed from the two different expressing systems, indicating that a biosimilar of Efruxifermin has been developed successfully. Proteomics analysis from in vivo study samples further identified some potential biomarkers or FGF21 related signaling pathways. Taken together, this study demonstrates a good example of how to develop and validate a biosimilar for therapeutic purposes. In future, more efforts, such as mutation to FGF21 or linking FGF21 with effective antibody to form dual targets, could be done to obtain more effective FGF21 analogs.