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蛋白制造中最大的行业之一。美国在这个行业中扮演着重要的角色，因为它代表着最大的血浆收集和制造基地，占世界血浆供应的50%以上。即使每年收集2200多万升血浆用于分级，这一数量也不能满足世界范围内对白蛋白(约500吨)、FVIII(2公斤)和免疫球蛋白(40吨)等血浆衍生品的高需求。因此，优化的分离方法对于增加从血浆中提取的各种治疗蛋白和提高纯化产品的产率具有重要意义。尽管以蛋白质为基础的血液产品在临床上继续流行，但目前用于获得它们的方法被称为分级，过时且效率低下，乙醇分级就是一个很好的例子，乙醇分级是第二次世界大战期间开发的一种纯化工艺，至今仍是当今使用的大多数分级策略的支柱。最近增加的用于工业规模血浆分级的色谱方法在过去几年已成为标准方法，在开发包括凝血因子、蛋白酶抑制剂和抗凝剂在内的新一代高纯度治疗性血浆衍生物方面发挥了核心作用。然而，人类血浆的稀缺性导致迫切需要提高使用当前分离技术获得的纯化血浆蛋白的产率。FDA通过生物制品评估中心(CDER)早就认识到，生物制品的开发正变得越来越具有挑战性。该机构表示，需要卓越的产品开发科学来应对提高效率和降低成本的挑战，如果美国要保持在血浆衍生疗法开发方面的领先地位，这些目标是必不可少的。认识到这一需要，在这项建议中，我们将开发一种策略，通过使用固定在新一代层析载体上的特定亲和配体，从根本上改进血浆衍生蛋白疗法的生物处理，这一方法应该会提高血浆衍生产品的产量、生产速度和质量。具体地说，我们将利用对流相互作用介质(CIM)，这是最近开发的一种基于甲基丙烯酸酯整体的革命性色谱支持。在拟议的研究中，将设计一种在几种亲和CIM载体上顺序分离人血浆的方法，以简化现有血浆衍生物(如免疫球蛋白和凝血因子)以及较新类型的血浆衍生产品(如蛋白酶抑制剂)的分离过程。在具体目标1中，我们将通过将抗体、多肽或合成染料固定在环氧活化的CIM柱上，开发针对各种血浆蛋白的亲和载体。一个主要的目标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化条件。在具体目标2中，我们将开发基于串联亲和CIM光盘的纯化方案，目标是在一步纯化中获得高水平的纯度和活性。我们还将评估扩大CIM分离方案后的产率和纯度，这是迈向工业级生产的必要步骤，短整体的相同性能和纯化曲线将促进工业化生产，而不受其大小或形状(盘、柱或管)的影响，这些研究结果应为CIM技术在工业分离人血浆中的使用提供“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based blood products in the clinical arena, the methods currently used to obtain them known as fractionation, are outdated and inefficient, a case in point being ethanol fractionation, a purification process developed during World War II that still remains the backbone of most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.