Conjugations to Endogenous Cysteine Residues.

Area of Science:

• Bioconjugation Chemistry
• Antibody Drug Conjugates (ADCs)
• Protein Chemistry

Background:

• Antibodies possess interchain disulfide bonds that can be reduced to form reactive cysteine residues.
• These endogenous cysteines are utilized for conjugating antibodies to cytotoxic drugs, forming antibody drug conjugates (ADCs).
• ADCs are under investigation as potential cancer therapeutics.

Purpose of the Study:

• To characterize the production of an anti-Notch 3 antibody drug conjugate (ADC).
• To investigate the impact of TCEP reduction temperature on ADC formation and purity.
• To optimize the manufacturing process for ADCs targeting various cancers.

Main Methods:

• Partial reduction of IgG1 monoclonal antibody (mAb) using TCEP reducing agent.
• Conjugation of the reduced mAb to a cytotoxic microtubulin inhibitor via a maleimide linker.
• Purification of the ADC using column chromatography and ultrafiltration-diafiltration.
• Analysis of ADC species, including drug loading and aggregates.

Main Results:

• The conjugation process yielded a mixture of 2, 4, 6, and 8 loaded ADC species.
• Product- and process-related impurities, including aggregates, were generated during conjugation.
• TCEP reduction temperature significantly influenced the level of aggregates produced.
• Reduction temperature also affected the isomeric composition of the 4-loaded ADC species.

Conclusions:

• Controlling the TCEP reduction temperature is critical for minimizing aggregate formation during ADC manufacturing.
• Optimizing reduction conditions can improve the purity and isomeric composition of ADCs.
• This optimized ADC production method supports the clinical development of novel cancer therapies.