Therapeutic proteins provide uniquely effective treatments for numerous diseases and disorders. However, these products can have safety problems which can be due to degradation products of the protein. Aggregates are critical degradation products in therapeutic protein products because even trace amounts can cause adverse effects in patients (e.g., immunogenicity). Protein aggregates are also particularly difficult to control because aggregation can occur at any step from initial fermentation to delivery to the patients including: during purification, filtration and other downstream processes steps; during shipping and storage in the final drug container/closure; and even during administration to the patient. Protein aggregates form from partially unfolded species in the native state ensemble. So even in solution conditions greatly favoring the native state, during pharmaceutical time scales of storage (e.g., two years) significant aggregation can occur. Also, the numerous stress to which therapeutic proteins are subjected (e.g., exposure to light, interfaces, freeze-thawing or agitation) can rapidly cause aggregation. Finally, even without stress and in an optimized formulation there can be formation of particles that are caused by adsorption of protein molecules to microparticles of foreign materials found in therapeutic protein products such as glass, stainless steel, silicone oil and rubber. Recent studies in mice with murine growth hormone showed that one such protein particle (protein adsorbed to microparticles obtained from grinding glass syringes) was particularly immunogenic. Therefore, it is critical to develop formulations and drug container/closure that also control the heterogeneous nucleation of protein particles onto foreign microparticles.

Typically, aggregates are arbitrarily divided in subclasses: 1) soluble aggregates are oligomers that are found in solution and include species ranging in size from dimers to those large enough to elute in the void volume of a size exclusion chromatography (SEC) column; 2) insoluble aggregates are those that can be separated from the native protein and soluble aggregates by centrifugation and filtration; and 3) particles are aggregates that are usually too large to be analyzed by SEC and are at too low of a mass percent to be quantified by mass loss due to filtration or centrifugation. Particles typically are counted for quantification and the size range examined is from about 1 micron to 125 micron. The connections between all of these species in the aggregation pathways of proteins are poorly understood. However, recent studies in which all three types were quantified during pharmaceutically relevant stresses (e.g., freeze-thawing, agitation and agitation in the presence of silicone oil) have shown that the first detectable aggregate type is subvisible particles. These can be quantified at mass percent far below the limit of detection for loss of monomer. Thus, particle counting provides the most sensitive method to date to observe and quantify protein aggregation. Also, it appears that when there is sufficient mass of aggregate such that insoluble aggregates can be quantified due to loss of monomer, these aggregates are composed of agglomerates of subvisible particles. Therefore, subvisible particles are fundamentally important components on the protein aggregation pathway.

Bacterial glycobiology involves a network of diverse proteins that can, for example, metabolize and modify glycans, synthesize oligosaccharides and polysaccharides, transport proteins that can flip lipid-linked oligosaccharides and even transport them to outer membrane. WbgU (Pleisomonas shigelloides) is a protein involved in biosynthetic pathway of a specific kind of surface polysaccharide found in gram-negative bacteria called lipopolysaccharide (LPS). It catalyzes inter-conversion of UDP-GalNAc and UDP-GlcNAc. We present here crystal structure of WbgU and a structure based rational design of mutant enzymes that might carry altered substrate specificity. The implications of this study can lead to a more economic synthesis of UDP-GalNAc and downstream glycans as well as structure based design of antibacterial drugs.

The carotid bodies are small organs located on the carotid arteries. They serve an essential biological function by detecting and relaying information regarding blood gas composition to the breathing centers of the brain. Thus, a deficit in blood oxygen (hypoxia) is 'sensed' by the carotid body, resulting in an increased firing frequency of the carotid sinus nerve and ultimately a change in the pattern of breathing. Recent research suggests that the exact molecular mechanism by which hypoxia is transduced into increased carotid sinus nerve activity is close to being resolved. Evidence indicates that activation of the energy-sensing enzyme AMP-activated protein kinase (AMPK) may be critical for the transduction of an acute hypoxic stimulus by the carotid bodies.