DETERMINING PROTEIN STRUCTURE

Understanding the three-dimensional structure of proteins is critical to life science and biopharmaceutical researchers. This information is used for understanding disease mechanisms, identifying drug targets and optimizing pharmaceutical drug design. Transformative drugs developed using this approach include HIV protease inhibitors and the leukemia drug Gleevec. This structural information is most often obtained by modeling proteins using X-ray crystallography. During X-ray crystallography, structural biologists use synchrotrons to blast X-rays through protein crystals, which are regular arrays of individual protein molecules stabilized by crystal contacts. This creates a “diffraction pattern” – essentially, the angle at which the X-rays reflect when passing through the protein crystals. This diffraction pattern is interpreted and used to create a model of the protein’s three-dimensional structure. Obtaining a useful diffraction pattern requires high-quality protein crystals.

OPTIMIZING FORMULATION

Another potential application of protein crystallization is in biologics formulation. Protein-based therapeutics such as monoclonal antibodies are formulated as a fluid suspension and are typically administered via intravenous (i.v.) infusion or subcutaneous injection.

In order to have a therapeutic effect, high doses of these proteins are required. However, a large quantity of therapeutic protein can potentially yield a high-viscosity solution, which is difficult to inject and can lead to protein aggregation which may reduce drug effectiveness or trigger an immune response. Accordingly, most biologics are formulated as high-volume, low-concentration suspensions that require intravenous infusion—a time-consuming approach that requires administration by medical professionals and a trip to the clinic for the patient.

Subcutaneous injections can be administered at home by the patient, making them the preferred delivery method. However, there is an upper limit on the volume that can be delivered this way. This means that in many cases, in order to achieve the required dose, formulation scientists must use a higher concentration of therapeutic protein – possibly leading to increased viscosity as discussed above.

A 2014 study suggests that there is an exponential relationship between protein concentration and solution viscosity. A 2016 study exemplifies this relationship for the soluble formulation of the therapeutic antibody infliximab, but not for the crystalline form. Namely, viscosity increases at a much lower rate for the crystalline form, thereby allowing formulation scientists to create higher concentration formulations of the crystalline protein with a reduced probability of high viscosity.

THE CHALLENGE OF PROTEIN CRYSTALLIZATION

Both X-ray crystallography and formulation development are dependent on obtaining high-quality protein crystals – a complicated process that starts with a pure, highly concentrated protein sample in solution. In an idealized version of the process, the liquid portion of the solution is slowly evaporated, leaving behind protein crystals. For some proteins, this process reliably produces high quality crystals. For example, a crystalline form of insulin is used as a long-acting version of the drug. For other proteins, it is much more difficult to achieve high quality crystal production. To some extent, it is simply a trial-and-error process, which is why protein crystallization is sometimes referred to as an art as well as a science. However, there are some protein characteristics that are known to increase the difficulty in obtaining a crystal. Membrane proteins, for example, are notoriously difficult to purify and crystalize. This class includes many proteins of great interest from a drug discovery perspective, such as transmembrane receptors and ion channels.

THE MICROGRAVITY ADVANTAGE

As the space station orbits around the earth in free fall, so too does everything inside. The result is a microgravity environment (10-6 g) that can be maintained for significant periods of time, making certain technical feats possible that are difficult to accomplish on Earth.

One such process is protein crystallization. Multiple studies over the past three decades have demonstrated that protein crystals produced in a microgravity environment grow larger and produce higher quality crystals for diffraction than their earth-bound counterparts. This is likely because in microgravity, proteins do not sediment out of solution before they are able to crystallize. A microgravity environment also results in reduced buoyancy-driven convective current—fluid movement that disrupts crystallization. These more uniform and slower-growing conditions lead to fewer but larger and higher-quality protein crystals. In addition, difficult-to-crystalize proteins such as the membrane protein 5 photosystem I and the pharmaceutically important monoclonal antibody pembrolizumab have been successfully crystalized in microgravity.

COMMERCIAL MOTIVATION

Pharmaceutical companies such as Eli Lilly and Company + Merck & Co., Inc. are investing in microgravity research to enhance protein crystallization. They are using these studies to better understand structure-function relationships of drugs and their targets as well as to aid in drug formulation. Microgravity protein crystallization studies have the potential to substantially impact drug discovery and development in a range of therapeutic areas including arthritis, cardiovascular disease, multiple sclerosis, osteoporosis, cystic fibrosis, and oncology.

Making an investment in microgravity research to obtain high-quality crystal structures of proteins can pay dividends for drug discovery and development companies by helping them to identify and move forward with drug candidates most likely to succeed, thereby potentially saving considerable time and money during the clinical phases of drug development. Space-grown protein crystals may also make possible more commercially attractive formulations such as small volume subcutaneous injections and lyophilized proteins.