An Integrated Experimental and Modeling Approach for Crystallization of Complex Biotherapeutics.

Area of Science:

• Biophysical Chemistry
• Macromolecular Crystallization
• Biopharmaceutical Development

Background:

• Protein crystallization is crucial for understanding structure and designing therapies, especially for medical proteins.
• Determining nucleation and growth kinetics is essential for advanced biotherapeutic crystallization, like viral capsids.
• Recombinant adeno-associated virus (rAAV) capsids are complex macromolecules requiring specific crystallization process design.

Purpose of the Study:

• To develop and apply coupled population and species balance equations to extract nucleation and growth kinetics for rAAV capsid crystallization.
• To compare model predictions with experimental data from a hanging-drop vapor diffusion system.
• To elucidate the factors controlling nucleation and growth rates of these large biotherapeutic molecules.

Main Methods:

• Development of coupled population balance and species balance equations.
• Modeling of rAAV capsid crystallization in a hanging-drop vapor diffusion system.
• Comparison of model-derived kinetics with experimental observations.

Main Results:

• Slow vapor diffusion from the droplet governs initial nucleation and growth of rAAV capsids.
• Capsid nucleation occurs via heterogeneous mechanisms within the microdroplet.
• Despite high molecular weight (3.6 MDa), capsids exhibit nucleation tendencies similar to small molecules and proteins, but with significantly slower growth rates (prefactor seven orders of magnitude smaller than lysozyme) limited by slow Brownian motion.

Conclusions:

• The study successfully extracted nucleation and growth kinetics for rAAV capsid crystallization.
• Heterogeneous nucleation and slow, supersaturation-insensitive growth, limited by capsid transport, characterize rAAV capsid crystallization.
• Findings provide critical insights for designing and optimizing crystallization processes for large biotherapeutics.