Characterization of Interchain Cysteine Linked Antibody Drug Conjugates…

Antibody drug conjugates (ADCs) has become promising therapy for the treatment of cancers. Among all the ADCs under developing, 2/3 of them are interchain cysteine linked ADCs. The ADCs are manufactured by partially reduce the 4 pairs of interchain disulfide bond followed by conjugate cytotoxic payloads to the thiols, as a consequence, the antibodies are linked with 0, 2, 4, 6, 8 drugs. The drug to antibody ratio (DAR) and the drug linking position are important parameters that affect the therapeutic effects and need to be well characterized.
Sample preparation:
The ADCs in the mouse plasma were purified by affinity capture with anti-human IgG beads followed wash with PBS supplied with 0.1% tween-20. The ADC was eluted with 0.1% TFA and neutralized with 1M tris (PH=8).
1. Heavy chain positional isomers separation and Identification.
2. Heavy chain positional isomers abundance determination.
1. Reduced ADC HRMS analysis. The Purified ADC was deglycosylated and reduced and then analyzed with HRMS.
2. Bottom up analysis. Purified ADC was treated with IdeS to remove Fc part followed by denaturation, reduction, alkylation, chymotrpsin digestion and then subject to LC/MS analysis.
3. LC-MS/MS analysis. Isomer fraction were collected and digested with trypsin, the digest was analyzed by LC/MS/MS for drug linking position identification.