Structural Changes and Aggregation Mechanisms for Anti-Streptavidin IgG1 at Elevated Concentration.

Area of Science:

• Biopharmaceutical manufacturing
• Protein aggregation science
• Drug product stability

Background:

• Non-native protein aggregation in therapeutics poses risks to efficacy and patient safety.
• Monitoring structural changes during aggregation is challenging, especially at high protein concentrations.
• Understanding aggregation mechanisms is crucial for regulatory compliance and drug development.

Purpose of the Study:

• To investigate non-native aggregation of antistreptavidin (AS) IgG1 at high concentration (30 mg/mL).
• To compare aggregation mechanisms and resulting aggregate structures under various solution conditions.
• To assess the impact of sucrose on aggregation kinetics and mechanisms.

Main Methods:

• In situ monitoring using dynamic light scattering, small-angle neutron scattering, and Raman scattering.
• Ex situ analysis with far-UV circular dichroism and second-derivative UV spectroscopy.
• Evaluation of aggregation under different pD, salt concentrations, and with sucrose addition.

Main Results:

• Sucrose addition reduced monomer loss rates but did not alter protein-protein interactions or aggregation mechanisms.
• Altering pD or salt concentration significantly changed the aggregation mechanism.
• Aggregates formed at pD 5.3 without salt were smallest but showed the most significant structural changes.

Conclusions:

• Solution conditions, particularly pD and salt, are key drivers of aggregation mechanisms and aggregate structure.
• Orthogonal techniques provide consistent qualitative assessment of aggregate structure and morphology.
• Electrostatic repulsions may influence both aggregate growth and structural changes within aggregates, a phenomenon potentially applicable to other proteins.