High-yield production of recombinant CLCF1 protein fused with human serum albumin in animal cells and toxicity evaluation in rodents

Recombinant protein based biopharmaceutics have been developed as therapeutics of various diseases, especially cancer, diabetes, infectious diseases, and autoimmune diseases. In this study, we conducted a study for the development of biopharmaceuticals based on the CLCF1 protein. First, we established strategies for producing recombinant human CLCF1 protein by transient gene expression in ExpiCHO-S™ Cells and Expi293F™ Cells. For the secretion of CLCF1 protein, we established strategies that human CRLF1 or sCNTFR with CLCF1 protein were co-expressed. As a result, the CLCF1 protein formed a complex with CRLF1 or sCNTFR, which was successfully secreted. Furthermore, the productivity of CLCF1 protein was significantly increased. The ratio of co-transfected plasmids, temperature, CO₂ level and time of harvest were explored, so that the productivity of CLCF1 was remarkably increased 7-fold from 3 mg/L to 22 mg/L. Next, we generated recombinant CLCF1 fusion protein with HSA (Albumin CLCF1) considering the improvement of pharmacokinetic properties and the proven production method in GMP facilities. We evaluated the biological activity of various CLCF1 proteins. In consideration of protein productivity, physical properties, and efficacy, we conducted a single intravenous administration of 4 types of proteins in Sprague-Dawley rats to evaluate the short-term toxicity. As a result, no toxicity related CLCF1 proteins was observed based on the behavior sign observation and body weight changes. In conclusion, we successfully established the strategies of production and characterization of biologically active recombinant CLCF1 proteins in mammalian cells as potential biotherapeutics.