Fragmentation of recombinant human interleukin-12 by matriptase in CHO cell culture

During the development of a recombinant CHO cell line expressing human Interleukin-12 fused to human IgG1 Fc (rhIL-12), we observed a prominent proteolytic cleavage of the rhIL-12 in its p40 subunit between Lys260 and Arg261. Using class-specific protease inhibitors, we concluded that the serine hydrolase family was responsible for the clipping. To identify the specific serine proteases involved, we conducted transcriptomic and proteomic analyses and identified several potential candidates. By performing in-vitro enzyme digestion experiments with these proteases, we determined that matriptase was responsible for the observed p40 clipping. Further confirmation was obtained through the development of matriptase (St14) knockout cell lines in which rhIL-12 clipping was almost completely abolished. Armed with this knowledge, we devised several strategies including increasing culture pH to reduce matriptase activity and rhIL-12 clipping during the manufacturing process.