Expression of human coagulation factor VIII in a human hybrid cell line, HKB11

Loss of coagulation factor VIII (FVIII) function results in a bleeding disorder, hemophilia A, which requires FVIII replacement therapy. Owing to its large size and complexity, the expression level of recombinant FVIII is two to three orders of magnitude lower than other recombinant proteins produced in mammalian cell lines. To understand cellular factors limiting FVIII expression, we studied the expression of FVIII in a human cell line, HKB11 (a hybrid cell line of HEK293 and a human B cell line). In comparison with other cell lines, such as HEK293 and BHK-21, HKB11 showed increased FVIII expression levels. With unamplified, pooled stable cells, FVIII expression in HKB11 cells was 8- to 30-fold higher than the other cell lines tested. In this study, HKB11 clones expressing varying levels of FVIII were analyzed and FVIII secreted from these clones had similar specific activity. Characterization of these clones by immunofluorescence staining, Western blotting analysis, and flow cytometry showed that high-producing cells not only secreted more active FVIII but also accumulated more FVIII protein intracellularly. FVIII expression appears to be controlled by the rates of transcription, translation, and secretion, but transcription and translation may play more important roles than secretion in determining expression level in HKB11 cells. FACS analysis of live cells showed that the high-producing clones also had more FVIII on the HKB11 cell surface than low-producing cells, thus opening the possibility of using FACS to select high-producing cell lines. Expression levels of the chaperone protein Hsp70 and antiapoptotic proteins such as Bcl-2 and Bcl-xL were similar among HKB11 clones with different FVIII productivity. In conclusion, HKB11 is an efficient host cell line for expression of FVIII and possibly other recombinant proteins. Systematic approaches, such as gene expression profiling by DNA microarray, will be necessary to understand the global changes in the cells producing recombinant proteins.