Classification and characterization of therapeutic antibody aggregates

Area of Science:

• Biochemistry
• Protein Chemistry
• Pharmaceutical Science

Background:

• Protein aggregation is a critical concern in biopharmaceutical development.
• Understanding aggregate formation is essential for predicting drug stability and immunogenicity.

Purpose of the Study:

• To characterize protein aggregates formed under various stress conditions.
• To establish a classification system for protein aggregates based on their attributes.

Main Methods:

• Monoclonal antibodies (IgG subtypes) were subjected to diverse stress techniques (mechanical, thermal, metal-catalyzed).
• Aggregated solutions were analyzed for aggregation percentage, particle characteristics (count, size, morphology), structural changes (secondary, tertiary), hydrophobicity, metal content, and reversibility.
• A classification system was developed for seven distinct aggregate classes.

Main Results:

• Protein aggregates exhibit significant heterogeneity in attributes and morphology, dependent on the applied stress.
• Mechanical stress produced the most subvisible particles; thermal stress yielded the most visible particles.
• Most aggregate classes showed disrupted higher-order structures, with varying degrees of reversibility (except thermal stress).
• Metal-catalyzed aggregates contained high copper levels, indicating potential immunogenicity.

Conclusions:

• Protein aggregate formation is stress-dependent, leading to diverse populations.
• A unified classification system can categorize aggregates across different IgG molecules.
• Stress conditions dictate aggregate characteristics, including structure, size, and reversibility.