Evaluation of protein disulfide conversion in vitro using a continuous flow dialysis system

Area of Science:

• Biopharmaceutical development
• Protein chemistry
• Analytical chemistry

Background:

• Recombinant therapeutic proteins exhibit heterogeneity due to chemical and physical modifications.
• Understanding these modifications is crucial for drug safety, efficacy, and process development.
• Establishing appropriate specification limits requires knowledge of modification impacts.

Purpose of the Study:

• To develop an in vitro continuous flow dialysis system.
• To evaluate the in vivo behavior of thiol adducted and incorrectly disulfide bonded therapeutic protein species.
• To assess the stability of modified Fc-fusion protein forms under physiological conditions.

Main Methods:

• Development of a continuous flow dialysis system.
• Simulation of low-level cysteine concentrations found in human blood.
• Investigation of cysteamine adducted, incorrectly disulfide bonded, and correctly disulfide bonded Fc-fusion protein species.

Main Results:

• The system maintained physiological cysteine concentrations.
• 90% of cysteamine adduct converted to correctly and incorrectly disulfide bonded forms within 4 hours.
• Approximately 50% of incorrectly disulfide bonded species converted to the correctly bonded form within 2 days.

Conclusions:

• The developed system provides valuable data on the in vivo stability of modified therapeutic proteins.
• Results inform the evaluation of product quality attributes and process optimization.
• Understanding protein modification stability is critical for biopharmaceutical development.