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生产中最大的行业之一。美国在这一行业中发挥着重要作用，因为它是最大的血浆收集和制造基地，占世界血浆供应量的50%以上。尽管每年收集2200多万升血浆用于分离，但这一数量仍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公斤）和IgG（40吨）。因此，最佳的分离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量是重要的。尽管基于蛋白质的血液制品在临床领域持续流行，目前用于获得它们的方法被称为分离，是过时的和低效的，一个很好的例子是乙醇分离。近年来，用于工业规模血浆分离的色谱方法已成为一种标准方法，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血剂）方面发挥了核心作用。然而，由于人类血浆的稀缺性，迫切需要利用当前的分离技术来提高纯化血浆蛋白的产量。FDA通过生物制品评估中心（CDER）很早就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，如果美国要在血浆衍生疗法的开发中保持领先地位，就需要卓越的产品开发科学来应对更高效率和更低成本的挑战，这是必不可少的目标。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载体上的特定亲和力配体，从根本上改善血浆衍生蛋白治疗药物的生物处理，这种方法应该提高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），这是一种最近开发的基于甲基丙烯酸酯单体的革命性色谱载体。在拟议的研究中，将设计在几种亲和CIM支架上对人血浆进行顺序色谱分离，以简化现有血浆衍生物（如免疫球蛋白和凝血因子）以及新型血浆衍生产品（如蛋白酶抑制剂）的分离过程。在Specific Aim 1中，我们将通过在环氧活化的CIM柱上固定抗体、肽或合成染料来开发针对各种血浆蛋白的亲和支持物。一个主要的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、稳定性和可重用性来优化固定条件。在具体目标2中，我们将开发基于串联亲和CIM圆盘的纯化方案，目标是在单步纯化中获得高水平的纯度和活性。我们还将评估CIM分离协议扩大后的产量和纯度，这是迈向工业水平生产的必要步骤，将通过独立于其大小或形状（圆盘、柱或管）的短单体具有相同的性能和纯化特性，从而促进生产。这些调查的结果将为CIM技术在人类血浆工业分离中的使用提供“概念证明”。

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based blood products in the clinical arena, the methods currently used to obtain them known as fractionation, are outdated and inefficient, a case in point being ethanol fractionation, a purification process developed during World War II that still remains the backbone of most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.