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The Center for Biophysical Sciences and Engineering (CBSE) at the University of Alabama at Birmingham has performed protein crystal growth experiments on more than 39 US space shuttle missions. Results from these experiments have clearly demonstrated that the microgravity environment is beneficial in that a number of proteins crystallized were larger and of higher quality than their Earth-grown counterparts. Improvement in crystal quality is judged by analysis of ultimate diffraction resolution, individual peak mosaicity, and electron density maps. There are now a number of protein crystals that exhibited resolution improvements of 0.5–1.5 A. Mosaicity studies revealed dramatic decreases in peak widths for the microgravity-grown crystals. These microgravity results plus data from a variety of other investigators have stimulated various space agencies to support fundamental studies in macromolecular crystal growth processes. The CBSE has devoted substantial effort toward the development of dynamically controlled crystal growth systems which allow scientists to optimize crystallization parameters on Earth or in space. These systems enable monitoring and control of the approach to nucleation and post-nucleation growth phases, thereby dramatically improving the crystal size and X-ray diffraction characteristics. The CBSE is currently designing a complete crystallographic laboratory for the International Space Station including: a crystal growth rack, which will support a variety of crystallization hardware systems; an X-ray diffraction rack for crystal characterization or a complete X-ray data set collection; and robotically controlled crystal harvesting/cryopreservation systems that can be operated with minimal crew time via telerobotic and/or robotic procedures. Key elements of the X-ray system include unique X-ray focusing technology combined with a lightweight, low-power source. The X-ray detection system is based on commercial CCD-based technology. This paper will describe the X-ray facility envisioned for the International Space Station.

The ability to grow quality protein crystals is necessary to analyze protein structure by X-ray diffraction and related techniques. As such it plays a key role in enzymology, structure-based drug design, molecular biology, and other biomedical areas. It is also required for macromolecule purification by crystallization. Protein crystal growth (PCG) may be negatively influenced by various factors related to nonspecific adsorption and adherence at growth chamber surfaces. Such factors include nucleation and growth of flawed crystals at chamber walls, or wall growth blockage of optical monitoring paths. Surface localized poly(ethylene glycol) (PEG) and other neutral, hydrophilic polymers are known to significantly reduce nonspecific adsorption of biological macromolecules and particles. Preliminary studies, involving various PCG methods (temperature induction, vapor diffusion), apparatii (test tubes, cuvettes, and specialized PCG hardware), growth chamber materials (glass, polystyrene, polysulfone), chamber volumes (0.1–10 ml) and protein samples (lysozyme, thaumatin, insulin) indicate the potential of PEG coatings to significantly reduce problems related to adsorption in PCG. The results, which match the ability of such coatings to reduce protein adsorption as evaluated by both ellipsometry and enzyme linked immunoassay, are discussed in relation to colloidal stabilization theory and properties of PEG coated surfaces.

The protein crystal growth facility (PCF) is space-flight hardware that accommodates large scale protein crystal growth experiments using temperature change as the inductive step. Recent modifications include specialized instrumentation for monitoring crystal nucleation with laser light scattering. This paper reviews results from the PCF's first seven flights on the Space Shuttle, the last with laser light scattering instrumentation. The PCF's objective is twofold: (1) production of high quality protein crystals for X-ray analysis and subsequent structure based drug design and (2) preparation of a large quantity of relatively contaminant free crystals for use as time-release protein pharmaceuticals. The first three Shuttle flights with bovine insulin constituted the PCF's proof of concept, demonstrating that the space-grown crystals were larger and diffracted to higher resolution than their earth-grown counterparts. The later four PCF missions were used to grow recombinant human insulin crystals for X-ray analysis and to continue productions trials aimed at the development of a processing facility for crystalline recombinant alpha interferon.

Protein crystal growth experiments have been performed on 16 space shuttle missions since April, 1985. The initial experiments utilized vapor diffusion crystallization techniques similar to those used in laboratories for earth-based experiments. More recent experiments have utilized temperature induced crystallization as an alternative method for growing high quality protein crystals in microgravity. Results from both vapor diffusion and temperature induced crystallization experiments indicate that proteins grown in microgravity may be larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth.

The Center for Biophysical Sciences and Engineering (CBSE) at the University of Alabama at Birmingham has performed protein crystal growth experiments on more than 37 U.S. space shuttle missions. Results from these experiments have clearly demonstrated that the microgravity environment is beneficial in that a number of proteins crystallized were larger and of higher quality than their earth-grown counterparts. Improvement in crystal quality is judged by analysis of ultimate diffraction resolution, individual peak mosaicity, and electron density maps. There are now a number of protein crystals that exhibited resolution improvements of 0.5Å to 1.5Å. Mosaicity studies revealed dramatic decreases in peak widths for the microgravity-grown crystals. These microgravity results plus data from a variety of other investigators have stimulated various space agencies to support fundamental studies in macromolecular crystal growth processes. The CBSE has devoted substantial effort toward the development of dynamically-controlled crystal growth systems which allow scientists to optimize crystallization parameters on Earth or in space. These systems enable monitoring and control of the approach to nucleation and post-nucleation growth phases, thereby dramatically improving the crystal size and x-ray diffraction characteristics. The CBSE is currently designing a complete crystallographic laboratory for the International Space Station including: a crystal growth rack, which will support a variety of crystallization hardware systems; an x-ray diffraction rack for crystal characterization or a complete x-ray data set collection; and robotically-controlled crystal harvesting/cryopreservation systems that can be operated with minimal crew time via telerobotic and/or robotic procedures. Key elements of the x-ray system include unique x-ray focusing technology combined with a lightweight, low power source. The x-ray detection system is based on commercial CCD-based technology. This paper will describe the x-ray facility envisioned for the International Space Station.

The Center for Macromolecular Crystallography (CMC) has been involved in fundamental studies of protein crystal growth (PCG) in microgravity and in our earth-based laboratories. A large group of co-investigators from academia and industry participated in these experiments by providing protein samples and by performing the x-ray crystallographic analysis. These studies have clearly demonstrated the usefulness of a microgravity environment for enhancing the quality and size of protein crystals. Review of the vapor diffusion (VDA) PCG results from nineteen space shuttle missions is given in this paper.

The Protein Crystal Growth Facility (PCF) is space-flight hardware that accommodates large scale protein crystal growth experiments using temperature change as the inductive step. Recent modifications include specialized instrumentation for monitoring crystal nucleation with laser light scattering. This paper reviews results from its first seven flights on the Space Shuttle, the last with laser light scattering instrumentation in place. The PCF’s objective is twofold: (1) the production of high quality protein crystals for x-ray analysis and subsequent structure-based drug design and (2) preparation of a large quantity of relatively contaminant free crystals for use as time-release protein pharmaceuticals. The first three Shuttle flights with bovine insulin constituted the PCF’s proof of concept, demonstrating that the space-grown crystals were larger and diffracted to higher resolution than their earth-grown counterparts. The later four PCF missions were used to grow recombinant human insulin crystals for x-ray analysis and continue productions trials aimed at the development of a processing facility for crystalline recombinant a-interferon.

The development and production of a zinc‐interferon alpha‐2b crystalline suspension on STS‐52 has accelerated our ability to prepare novel high quality pharmaceutical preparations. Crystalline suspensions of protein therapeutics have applications in drug delivery, formulation, and manufacturing. These applications require crystalline suspensions of relatively small particles (

Macroscale crystallization of zinc interferon alfa‐2b was achieved on STS‐52 in October 1992 in the Protein Crystallization Facility. Conditions for crystallization were established by adapting a microscale vapor diffusion method to a macroscale temperature induction method. A series of earth based pilot experiments established conditions to reproducibly crystallize zinc interferon alfa‐2b in high yield and under cleanroom conditions. As a control for the STS‐52 mission, a ground experiment was run simultaneously and in the same configuration as the flight experiment. Greater than 95% of the available protein crystallized in both the ground and flight experiments. Using a battery of physical, biochemical and biological characterization assays, demonstrated that sample processing, polysulfone bottle confinement and the conditions used for crystallization did not have a negative effect on protein integrity. Redissolved crystals from the flight and ground experiments showed full biological activity in a cytopathic effect inhibition assay as compared to an interferon control standard. Morphometric analysis comparing the overall length and width of the derived crystals showed a 2.4 fold increase in the length and width of the space grown crystals as compared to earth grown crystals. Space grown crystals have remained a stable free flowing suspension for over 2 years. Based on these results, further experiments are envisioned to investigate macroscale crystallization of biologically active macromolecules in microgravity.