Identification and characterization of the Fd+ 16 Da fragment impurity in therapeutic CD47 monoclonal antibodies expressed in CHO-K1 cell lines

A range of biotherapeutics are currently manufactured in large-scale production facilities via cell culture techniques to support both commercial applications and clinical research. Among these biotherapeutics, monoclonal antibodies (mAbs) represent the dominant class, and they are typically produced using mammalian expression systems—with Chinese hamster ovary (CHO) cells being the most widely employed. To guarantee the efficacy, safety, and consistency of these mAbs, comprehensive multi-dimensional molecular characterization is indispensable. In our study, following the successful construction of cell lines for a CD47-targeting monoclonal antibody, an impurity with a molecular weight shift of Fd+16 Da was detected in the expression product of CHO-K1 cells, whereas it was absent in the earlier expression system using human HEK293 cells. To trace the origin of this Fd+16 Da impurity (designated for the CD47 mAb WBP2131), we conducted a series of investigations focusing on the potential effects of oxidation on disulfide bond integrity, glycosylation patterns, and other post-translational modifications, as well as the stability of the Fd+16 Da impurity under controlled degradation conditions. Peptide mapping analysis provided definitive evidence that the formation of this Fd+16 Da impurity was attributed to the oxidation of a tryptophan residue within the complementarity-determining region 3 (CDR3) region, specifically in the TWWR peptide segment. Further degradation analysis demonstrated its stability. Collectively, our findings provide valuable data for the development, formulation, storage, and transportation for this CD47 mAb candidate, thereby supporting its translational progression.