Tracing transport of protein aggregates in microgravity versus unit gravity crystallization

Abstract

Microgravity conditions have been used to improve protein crystallization from the early 1980s using advanced crystallization apparatuses and methods. Early microgravity crystallization experiments confirmed that minimal convection and a sedimentation-free environment is beneficial for growth of crystals with higher internal order and in some cases, larger volume. It was however realized that crystal growth in microgravity requires additional time due to slower growth rates. The progress in space research via the International Space Station (ISS) provides a laboratory-like environment to perform convection-free crystallization experiments for an extended time. To obtain detailed insights in macromolecular transport phenomena under microgravity and the assumed reduction of unfavorable impurity incorporation in growing crystals, microgravity and unit gravity control experiments for three different proteins were designed. To determine the quantity of impurity incorporated into crystals, fluorescence-tagged aggregates of the proteins (acting as impurities) were prepared. The recorded fluorescence intensities of the respective crystals reveal reduction in the incorporation of aggregates under microgravity for different aggregate quantities. The experiments and data obtained, provide insights about macromolecular transport in relation to molecular weight of the target proteins, as well as information about associated diffusion behavior and crystal lattice formation. Results suggest one explanation why microgravity-grown protein crystals often exhibit higher quality. Furthermore, results from these experiments can be used to predict which proteins may benefit more from microgravity crystallization.

Microgravity environment grown crystal structure information based engineering of direct electron transfer type glucose dehydrogenase

“…Therefore, we joined the JAXA program to crystallize BcGDH. The effect of suppressing convection in liquid solution in microgravity crystallization has been reported by JAXA and others [19][20][21][22][23][24] . In microgravity environments, high-quality protein crystals without cracks caused by convection can be obtained which result in nice diffraction with low mosaicity for structural analysis.…”

“…The Japan Aerospace Exploration Agency (JAXA) has been conducting High-quality Protein Crystal Growth experiments (JAXA PCG) in the Japanese Kibo module on the International Space Station (ISS). Owing to the unique environment in space where there is no convection to disrupt liquid solution nor precipitation causing heavier molecules to sink, many crystals of various proteins have been successfully created in this way [19][20][21][22][23][24] . To improve the stability of the DET-type FADGDH complex via quaternary structure engineering, we elucidated the partial structure of this enzyme complex by preparing a protein crystal of BcGDH heterotrimer complex in space through JAXA PCG experiments.…”

Verification of the Inverse Scale Effect Hypothesis on Viscosity and Diffusion by Azo-Amino Acid Schiff Base Copper Complexes

“…In addition to chemical engineering and fluid dynamics in viscous media, there is currently insufficient chemical understanding of how to treat molecules using classical mechanics. In addition, by referring to (protein) crystal growth and molecular orientation under the conditions in droplets, it is thought that an analogy of gravity [28] and other "forces acting on objects" to molecules and nanoaggregates may be effective.…”

Monoclonal Antibodies from Space: Improved Crystallization Under Microgravity During Manufacturing in Orbit

“…9 Reports in the literature over the last three decades indicate that the microgravity environment can be used to create novel protein crystalline polymorphs with physicochemical properties significantly different from protein crystals grown on Earth at 1g (one g is the force per unit mass due to gravity at the Earth's surface; this standard gravity is defined as 9.8 meters per second squared), thus enabling the production of low-viscosity, highly concentrated colloidal protein crystal suspensions. [10][11][12] These physiochemical characteristics are essential for developing therapeutic mAb and ADC formulations that are suitable for simple SC injection. These high-concentration crystalline mAb formulations could then ideally be administered at a regular doctor's office or even by patients themselves as home therapy, thereby avoiding the traditional, hospital-based, day-long, IV infusion treatment protocols.…”