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Trastuzumab emtansine (T-DM1) is a target-specific anticancer antibody-drug conjugate (ADC). In the present study, critical quality attributes for different manufactured products, such as the drug to antibody ratio (DAR), conjugation site, and site conjugation ratio, are similar, which is contrary to the traditional view that conjugation at lysine sites is randomly assigned. To investigate this result, a series samples with different DARs were prepared. Site conjugation ratios of the 27 different conjugation sites (corresponding to 54 potential sites) were analyzed. We found that the correlation coefficients of the 26 site conjugation ratio and the DAR were R 2 > 0.9, and the remaining one was R 2 > 0.7. By comparing three batches of samples with a DAR value of ∼3.3 in a stability study, we found that degradation rates of conjugation sites of the samples incubated at 40 °C were basically the same. These data show that the conjugation ratio and the conjugation stability of each site may remain consistent if the process parameters are stable. LC/MS/MS was used to study the unconjugated linker of the crosslink byproducts produced by a two-step method. We determined four forms of unconjugated linkers: ( N -maleimidomethyl) cyclohexane-1-carboxylate (MCC) unconjugated to DM1, hydrolyzed MCC unconjugated to DM1, lys-MCC-lys, and lys-MCC-cys. We believe that the current study can provide an effective guide for the processing of ADCs, control of product quality, and reduction of side reaction products., Competing Interests: The authors declare no competing financial interest.