An Integrated Experimental and Modeling Approach for Crystallization of Complex Biotherapeutics

TL;DR

This paper develops a coupled modeling approach to understand the crystallization kinetics of complex biotherapeutics like rAAV capsids, revealing how slow vapor diffusion and high molecular weight influence nucleation and growth.

Contribution

It introduces a combined population and species balance model for rAAV capsid crystallization, providing insights into nucleation and growth mechanisms specific to high-molecular-weight biotherapeutics.

Findings

Vapor diffusion rate controls initial nucleation and growth.

Capsid nucleation occurs via heterogeneous nucleation.

Growth rate is limited by slow Brownian motion due to high molecular weight.

Abstract

Crystallization of proteins, specifically proteins of medical relevance, is performed for various reasons such as to understand the protein structure and to design therapies. Obtaining kinetic constants in rate laws for nucleation and growth of advanced biotherapeutics such as capsids, an assembly of macromolecules, is challenging and essential to the design of the crystallization processes. In this work, coupled population balance and species balance equations are developed to extract nucleation and growth kinetics for crystallization of recombinant adeno-associated virus (rAAV) capsids. A comparison of model results with that of experimental data for capsid crystallization in hanging-drop vapor diffusion system shows that slow rate of vapor diffusion from the droplet controls the initial nucleation and growth processes, and the capsid nucleation occurs via heterogeneous nucleation in the microdroplet. Results also show that the capsids, which are of very high molecular weight (~3.6 MDa), have a similar tendency to nucleate as small organic molecules such as glycine (75 Da), low-molecular-weight proteins, and small-molecule active pharmaceutical ingredients due to its ball-shaped outer structure/shape. Capids also show a prolonged nucleation period as for proteins and other macromolecules, but has a slow growth rate with a growth rate pre-factor seven orders of magnitude smaller than that of lysozyme. The capsid crystal growth rate is weakly sensitive to the supersaturation compared to lysozyme and is limited by the transport of capsids due to slow Brownian motion resulting from the very high molecular weight.