Bioprocessing of Plasma Therapeutic Proteins using Sequential Affinity Monolithic

描述（由申请人提供）：血浆分离是全球治疗性蛋白质生产中最大的行业领域之一。美国在该行业中扮演着重要角色，因为它是最大的血浆采集和制造基地，占全球血浆供应量的50%以上。尽管每年收集超过2200万升血浆用于分离，但这一数量无法满足全球对血浆衍生物的高需求，如白蛋白（约500吨）、FVIII（2 kg）和IgG（40吨）。因此，最佳的分级方法对于增加从血浆中提取的各种治疗性蛋白质和提高纯化产物的产率是重要的。尽管基于蛋白质的血液制品在临床竞技场中持续流行，但目前用于获得它们的称为分馏的方法是过时且低效的，一个恰当的例子是乙醇分馏，在第二次世界大战期间开发的纯化过程，仍然是当今使用的大多数分离策略的支柱。这是过去几年的标准方法，在开发新一代高度纯化的治疗性血浆衍生物（包括凝血因子、蛋白酶抑制剂和抗凝剂）中发挥了核心作用。然而，人血浆的稀缺已经产生了对提高使用当前分级分离技术获得的纯化血浆蛋白的产率的迫切需要。FDA通过生物制品评价中心（CDER）长期以来一直认识到生物制品的开发正变得越来越具有挑战性。该机构建议，需要上级产品开发科学来解决更高效率和更低成本的挑战，如果美国要保持血浆衍生治疗药物开发的领导地位，这是基本目标。认识到这一需求，在本提案中，我们将开发一种策略，通过使用固定在新一代色谱支持物上的特异性亲和配体，从根本上改善血浆衍生蛋白治疗剂的生物加工，这种方法应该增加血浆衍生产品的产量、生产速度和质量。具体来说，我们将利用对流相互作用介质（CIM），一个最近开发的革命性的色谱支持甲基丙烯酸酯的整体。在拟定的研究中，将设计在几种亲和CIM支持物上连续色谱分离人血浆，以简化现有血浆衍生物（如免疫球蛋白和凝血因子）以及新类别血浆衍生产品（如蛋白酶抑制剂）的分馏过程。在特定目标1中，我们将通过将抗体、肽或合成染料固定在环氧活化的CIM柱上来开发对各种血浆蛋白具有特异性的亲和支持物。一个主要目标将是通过评估在不同条件和密度下偶联的配体的性能、稳定性和可重复使用性来优化固定化条件。在特定目标2中，我们将开发基于串联亲和CIM盘的纯化方案，目标是在一步纯化中获得高水平的纯度和活性。我们还将评估CIM分离方案放大后的产率和纯度，这是迈向工业水平生产的必要步骤，这将通过与其尺寸或形状无关的短整料的相同性能和纯化概况来促进（盘、柱或管），这些研究的结果应该为CIM技术在人血浆工业分离中的应用提供“概念验证”。

DESCRIPTION (provided by applicant): Plasma fractionation is one of the largest industry segments in global therapeutic protein manufacture. The United States plays an important role in this industry since it represents the largest plasma collection and manufacturing base that accounts for more than 50% of the world supply of plasma. Even though more than 22 million liters of plasma are collected each year for fractionation, this amount cannot meet the high demand for plasma derivatives worldwide such as albumin (about 500 tons), FVIII (2 kg) and IgG (40 tons). Consequently, optimal fractionation methods are important to increase the various therapeutic proteins extracted from plasma and to improve the yields of the purified products. In spite of the continued prevalence of protein based blood products in the clinical arena, the methods currently used to obtain them known as fractionation, are outdated and inefficient, a case in point being ethanol fractionation, a purification process developed during World War II that still remains the backbone of most fractionation strategies in use today The recent addition of chromatographic methods for industrial scale plasma fractionation has become a standard approach in the last few years that has played a central role in the development of a new generation of highly purified therapeutic plasma derivatives that includes coagulation factors, protease inhibitors and anticoagulants. Nevertheless, the scarcity of human plasma has created a pressing need for improving the yield of purified plasma proteins obtained using current fractionation technologies. The FDA through the Center of Biologics Evaluation (CDER) has long recognized that the development of biological products is becoming increasingly challenging. The agency has suggested that superior product development sciences are needed to address the challenges of greater efficiency and lower costs, essential goals if the United States is going to remain a leader in the develop of plasma derived therapeutics. In recognition of this need, in this proposal, we will develop a strategy to radically improve the bioprocessing of plasma derived protein therapeutics by using specific affinity ligands immobilized on a new generation of chromatographic supports, an approach that should increase the yield, speed of production and quality of plasma derived products. Specifically, we will make use of Convective Interaction Media (CIM), a recently developed revolutionary chromatographic support based on methacrylate monoliths. In the proposed studies, a sequential chromatographic separation of human plasma on several affinity CIM supports will be designed to streamline the fractionation process of existing plasma derivatives such as immunoglobulins and clotting factors but also of newer classes of plasma derived products such as protease inhibitors. In Specific Aim 1, we will develop affinity supports specific for various plasma proteins by immobilizing antibodies, peptides or synthetic dyes on epoxy-activated CIM columns. A major goal will be to optimize the immobilization conditions by assessing performance, stability, and reusability of ligands coupled under different conditions and densities. In Specific Aim 2, we will develop purification protocols based on serially linked-affinity CIM discs with the goal of obtaining a high level of purity and activity in a single step purification. We will also evaluate the yield and purity following scale-up of CIM separation protocols, a necessary step towards industrial level production that will be facilitated by the identical performance and purification profiles of short monoliths independently from their size or shape (disk, column or tube), The results of these investigations should provide the "proof of concept" for the use of CIM technology in industrial fractionation of human plasma.