Trisulfide modification impacts the reduction step in antibody-drug conjugation process

Area of Science:

• Bioconjugation Chemistry
• Protein Chemistry
• Pharmaceutical Manufacturing

Background:

• Antibody-drug conjugates (ADCs) link cytotoxic drugs to antibodies via disulfide bonds.
• Cysteine-directed linker chemistry relies on reducing interchain disulfide bonds for drug conjugation.
• The reduction step using tris(2-carboxyethyl)phosphine (TCEP) is critical for ADC manufacturing.

Purpose of the Study:

• To investigate the variability in TCEP reductant requirements during ADC manufacturing.
• To identify the root cause of inconsistent drug-to-antibody ratios in ADC production.
• To understand the impact of antibody quality on the reduction and conjugation steps.

Main Methods:

• Analysis of interchain disulfide and trisulfide bond levels in monoclonal antibody starting materials.
• Titration of TCEP reductant to determine the molar ratio required for achieving target drug-to-antibody ratios.
• Characterization of reaction products between TCEP and antibody trisulfide bonds.

Main Results:

• Inconsistent levels of interchain trisulfide bonds were identified as the cause of TCEP variability.
• Antibody trisulfide bonds react with TCEP, consuming the reductant without generating free thiols for conjugation.
• Higher trisulfide content in antibodies requires increased TCEP:antibody molar ratios for successful conjugation.

Conclusions:

• The presence of interchain trisulfide bonds significantly impacts ADC manufacturing efficiency.
• Accurate quantification of antibody trisulfide levels is crucial for optimizing TCEP usage.
• Controlling antibody quality, specifically minimizing trisulfide bonds, is essential for reproducible ADC production.