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

ABSTRACT [ 14 C]2‐Iminothiolane ([ 14 C]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 [ 14 C]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 [ 14 C]2‐IT‐to‐antibody ratio ([ 14 C]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 [ 14 C]ITAR and DAR measurements of the isolated [ 14 C]mAb. These measurements combined to demonstrate that significant amounts of the cyclized products were shed from the [ 14 C]ADC as [ 14 C]thiobutyrolactone.