CFI Inactivation of Human Plasma

描述（由申请人提供）：世界范围内的艾滋病流行，埃博拉和SARS的周期性出现，以及最近爆发的H5N1等潜在的大流行流感毒株，突出了卫生保健界持续关注的一个问题——对人类血浆和血浆衍生产品有效消毒技术的需求。还有一些新出现的病毒，如西尼罗河病毒和一些潜在的生物恐怖主义病原体，如天花，对人类血浆供应的安全构成关切。这些“杀手”病毒迅速出现和传播的原因尚不完全清楚，但据认为是由森林砍伐与野生病毒栖息地城市化、进化突变以及全球各国之间的快速传播等因素共同造成的，这些因素可能促进自然和生物恐怖主义病原体的传播。用于灭活或去除人血浆中的病毒和源自人血浆的治疗性蛋白质的方法有许多，包括：加热或巴氏灭菌；solvent-detergent技巧;紫外线照射；利用2-本体内酯和臭氧等可水解化合物进行化学灭活；用合成补骨脂素进行光化学净化。因此，目前的方法并不总是对广泛的人类和动物病毒有效，有时受到工艺特定缺陷的阻碍，并且往往导致它们旨在保护的生物制品变性。一些商业上可用的方法对灭活包膜病毒（如HIV）是有效的，但对非包膜病毒（如甲型肝炎（HAV）和细小病毒B19）不是很有效。我们建议开发一种安全且具有成本效益的通用方法和设备，用于灭活人类血浆中的非包膜和包膜病毒以及单个单位的包膜病毒。我们建议通过进一步推进一种基于超临界和近临界流体（superfluid或SFS）技术的独特、快速和普遍适用的病毒灭活技术来实现这一点。“超流体”通常是气体，当被压缩时，表现出增强的溶剂化、渗透和膨胀特性。这些气体被用来渗透和膨胀病毒颗粒。然后，过度填充的颗粒被减压，由于快速的相变，在它们最薄弱的地方破裂。该过程是纯物理的，不涉及使用热、化学和/或辐射，每一种方法在人血浆病毒灭活方面都有明显的缺点。我们迄今为止的研究表明，superfluid“CFI（临界流体失活）工艺通过脂质溶解机制灭活胎牛血清中的包膜病毒，如MuLV， VSV， TGE， BVD， Sindbis和HIV，类似于溶剂洗涤剂方法。我们的研究还表明，SFS-CFI通过快速膨胀的液体，同时对病毒颗粒进行物理破坏，使被坚硬蛋白质衣壳包围的非包膜病毒失活。已证明SFS-CFI能够灭活胎牛血清中的脊髓灰质炎、腺病毒、Reo和EMC等非包膜病毒，同时保留处理后产品的生物活性。在与英国伦敦国家生物标准与控制研究所（NIBSC）合作的一项研究中，我们还证明了superfluid“CFI”可以在不到一分钟的时间内在两阶段CFI装置中灭活人血清中的4个以上的人细小病毒B19。拟议的第一阶段研究计划的修订具体目标是：“具体目标1：数学模型和评估对层流CFI单元和SFS-CFI过程的放大至关重要的关键过程参数。将进行数学建模和输运现象计算，以确定最佳的SFS和等离子体参数，如密度、流体类型和液滴大小，以便通过SFS有效接触病毒颗粒、渗透、饱和、膨胀、破坏和失活。”具体目标2：基于该建模的结果，设计和测试注射喷嘴和等压室，并定义操作条件，以在单个层流CFI单元中实现原型包膜病毒和原型非包膜病毒的> 3次病毒灭活日志，并保留> 90%的蛋白质完整性。在第二阶段，我们将按比例放大500倍，然后按比例缩小10倍至100倍，这是一个单级层流SFS-CFI原型。我们还计划在cGMP条件下验证大型superfluid“CFI”人体血浆原型。在III期，我们计划与百特国际等制药/生物制剂合作伙伴和/或美国红十字会、国防部或国家心脏、血液和肺研究所等机构合作伙伴进行临床前毒理学和药理学研究，向FDA提交IND并进行I期临床试验。公共卫生相关性：有一些新出现的病毒，如西尼罗河病毒、埃博拉病毒、SARS潜在的流感大流行毒株（H5N1）和一些潜在的生物恐怖主义病原体，如天花，对人类血浆供应的安全构成关切。目前的方法并不总是对广泛的人类和动物病毒有效，有时受到工艺特定缺陷的阻碍，并且往往导致它们旨在保护的生物制品变性。我们建议开发一种纯粹的物理技术，使病毒“弯曲”，在不破坏医学上重要的液体（如人体血浆）中的蛋白质和酶的情况下灭活它们。该技术不涉及使用热、化学和/或辐照，这些方法在人血浆病毒灭活方面都有明显的缺点。Aphios公司将重点开发这种物理病毒灭活技术，用于人类使用的领域，在这些领域，病毒灭活步骤是必不可少的，并且将继续是生产安全产品所必需的。一种普遍适用和有效的病毒灭活工艺，可以提高血浆产量并降低处理成本，将对市场产生重大影响。我们计划将这项技术商业化，作为一种正交病毒灭活技术，与对非包膜病毒无效的溶剂-洗涤剂（S/D）和纳滤等被动病毒去除技术相结合。这种方法与欧洲和美国的监管机构一致，要求至少有两种病毒灭活技术，这两种技术通过不同的作用机制起作用。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.