Antibody Drug Conjugate Formation Using 2‐ ¹⁴ C‐Iminothiolane: A Radiochemical Analysis of Sequential Lysine Conjugation, Uncloaking of a Phantom Drug‐to‐Antibody Ratio, and Stability of the ADC

[¹⁴C]2-Iminothiolane ([¹⁴C]2-IT) served as a highly informative analytical tool to determine the rate of lysine thiolation and total addition of 2-IT to a monoclonal antibody. Specific activity measurements on [¹⁴C]2-IT modified mAbs found that lysine thiolation with 2-IT is linear regarding time and dependent on buffer strength. Conjugation with a fluorescent maleimide directly linked the free thiol-to-antibody ratio (FTAR) to the [¹⁴C]2-IT-to-antibody ratio ([¹⁴C]ITAR), providing accurate predictive information on the true number of sites proficient for payload conjugation and, by comparison with conjugation with the cytotoxic payload, quantified the absorbance from the nonconjugated cyclized N-substituted 2-iminothiolane adduct that created "phantom" drug-to-antibody ratios (PhDAR). Hydrolytic loss of the nonconjugated cyclized N-substituted 2-iminothiolane adduct was investigated using carbon-14 based radioquantitative HPLC, mass spectral analysis, and orthogonal [¹⁴C]ITAR and DAR measurements of the isolated [¹⁴C]mAb. These measurements combined to demonstrate that significant amounts of the cyclized products were shed from the [¹⁴C]ADC as [¹⁴C]thiobutyrolactone.