Identification and characterization of carbonylation sites in trastuzumab biosimilars

Detection of metal catalyzed carbonylation in proteins is traditionally based on derivatization followed by detection and quantification via spectroscopy or immunodetection. However, these measure only cumulative carbonylation and do not identify the specific sites of modification within the protein. Recently, fluorescein thiosemicarbazide (FTC) based semi-microplate method was adapted for high throughput monitoring of carbonyl content during mAb process development, using size-exclusion chromatography followed by ultraviolet and fluorescence detection. Here, we have examined carbonylation in originators and 4 biosimilars of an IgG1 therapeutic monoclonal antibody, trastuzumab, a first line of therapy for HER2 positive breast cancer. The hyphenated RP-ESI-MS/MS detection was able to identify the location of each of the carbonylated amino acids for all products. The result is a comprehensive map of a total of 27 unique identified carbonylation sites of trastuzumab found across multiple batches of originator as well as marketed biosimilars. Our results demonstrate that although the different carbonylation sites are spread across different domains throughout the mAb sequence, the complementarity determining regions (CDRs) are free of carbonylation and all identified sites lie within the framework region of the variable domain. Moreover, the constant- heavy domain 3 (CH₃) region seems to be particularly resistant to process induced carbonylation.