The worldwide AIDS epidemic, the periodic emergence of Ebola and SARS, and the recent outbreaks of potentially pandemic strains of influenza such as H5N1 have highlighted a persistent concern in the health- care community -- the need for effective sterilization techniques for human blood plasma and plasma-derived products. There are also a number of emerging viruses such as West Nile and a number of potential bioterrorism pathogens such as smallpox that are of concern to the safety of the human plasma supply. The causes of the more rapid emergence and spread of these """"""""killer"""""""" viruses are not entirely known, but are thought to be caused by some combination of deforestation with urbanization of wild virus habitats, evolutionary mutations, and rapid travel between countries around the globe that can facilitate the spread of both natural and bioterrorism pathogens. A number of approaches have been employed for the inactivation or removal of viruses in human plasma and therapeutic proteins derived from human plasma, including: heating or pasteurization;solvent-detergent technique;Ultra Violet (UV) irradiation;chemical inactivation utilizing hydrolyzable compounds such as 2- proprionolactone and ozone;and photochemical decontamination using synthetic psoralens. Thus, current approaches are not always effective against a wide spectrum of human and animal viruses, are sometimes encumbered by process-specific deficiencies, and often result in denaturation of the biologicals that they are designed to protect. Some of the commercially available methods are effective in inactivating enveloped viruses, such as HIV, but are not very effective against nonenveloped viruses such as Hepatitis A (HAV) and parvovirus B19. We propose to develop a safe and cost-effective universal process and equipment for the inactivation of non- enveloped and enveloped viruses in pooled as well as single units of human plasma. We propose to do so by further advancing a unique, rapid and generally applicable virus inactivation technique based on supercritical and near-critical fluids (SuperFluids"""""""" or SFS) technology. SuperFluids"""""""" are normally gases which, when compressed, exhibit enhanced solvation, penetration and expansion properties. These gases are used to permeate and inflate the virus particles. The overfilled particles are then decompressed and, as a result of rapid phase conversion, rupture at their weakest points. The process is purely physical and does not involve the use of heat, chemicals and/or irradiation, each of which has significant drawbacks in the viral inactivation of human plasma. Our research to date indicates that the SuperFluids"""""""" CFI (critical fluid inactivation) process inactivates enveloped viruses such as MuLV, VSV, TGE, BVD, Sindbis and HIV in fetal bovine serum by a lipid solubilization mechanism, similar to the solvent detergent method. Our research also indicates that SFS-CFI inactivates non-enveloped viruses surrounded by a tough protein capsid through rapid expansion of the fluid with concomitant physical disruption of viral particles. SFS-CFI's ability to inactivate non-enveloped viruses such as Polio, Adeno, Reo and EMC in fetal bovine serum, while preserving biological activity of the treated product, has been demonstrated. In a research collaboration with the National Institute of Biological Standards and Control (NIBSC), London, England, we have also demonstrated that SuperFluids"""""""" CFI can inactivate more than 4 logs of human Parvovirus B19 in human serum in a two-stage CFI unit in less than one minute. The revised Specific Aims of the proposed Phase I research program are: """""""" Specific Aim 1: Mathematically model and evaluate key process parameters that are critical for scaling-up of the laminar flow CFI unit and the SFS-CFI process. Mathematical modeling and transport phenomena calculations will be conducted to define optimum SFS and plasma parameters such as density, fluid type and droplet size for effective contact of the viral particles by the SFS, penetration, saturation, expansion, disruption and inactivation. """""""" Specific Aim 2: Based on the results of this modeling, design and test injection nozzles and isobaric chamber as well as define operating conditions for achieving >3 logs of virus inactivation of a prototypical enveloped virus and a prototypical nonenveloped virus in a single laminar flow CFI unit with retention of >90% of protein integrity. In Phase II, we will scale-up by a factor of 500X then scale-down by a factor of 10X to 100X, a single-stage laminar flow SFS-CFI prototype. We also plan to validate a large-scale SuperFluids"""""""" CFI prototype for human plasma under cGMP conditions. In Phase III, with a pharmaceutical/biologics partner such as Baxter International and/or an institutional partner such as the American Red Cross, the Department of Defense or the National Heart, Blood and Lung Institute, we plan to conduct pre-clinical toxicology and pharmacology studies, file an IND with the FDA and conduct Phase I clinical trials.
There are a number of emerging viruses such as West Nile, Ebola, SARS potential pandemic strains of influenza (H5N1) and a number of potential bioterrorism pathogens such as smallpox that are of concern to the safety of the human plasma supply. Current approaches are not always effective against a wide spectrum of human and animal viruses, are sometimes encumbered by process-specific deficiencies, and often result in denaturation of the biologicals that they are designed to protect. We propose to develop a purely physical technique that gives the virus the """"""""bends,"""""""" inactivating them without damaging proteins and enzymes in medically important fluids such as human plasma. This technique does not involve the use of heat, chemicals and/or irradiation, each of which has significant drawbacks in the viral inactivation of human plasma. Aphios Corporation will focus on developing this physical virus inactivation technique for areas of human use where a virus inactivation step is essential and will remain necessary for the manufacture of safe products. A generally applicable and effective virus inactivation process that increases yield from plasma and reduces processing cost will have a significant impact in the marketplace. We plan to commercialize this technology as an orthogonal virus inactivation technology to techniques such as solvent-detergent (S/D) that is not effective against non-enveloped viruses and passive virus removal techniques such as nanofiltration. This approach is consistent with the regulatory authorities in Europe and the US that require a minimum of two virus inactivation technologies, which work by different mechanisms of action.
