Process development for the controlled expansion and differentiation of human primary B lymphocytes outside the human body. Part 2: Use in bioprocess technology

第二个项目阶段的目的是使用第一个项目阶段开发的工具，控制原代人B淋巴细胞的离体增殖和分化，以制备用于生物工艺技术中示例性应用的细胞。利用B淋巴细胞的自然能力，如：快速繁殖，然后产生大量蛋白质的能力（抗体）（生产类型A：“浆细胞”），在体内长时间持续存在并保持生物活性的能力（生产类型B：“活性药物”），以及以靶向方式激活T淋巴细胞的能力（生产类型C：“抗原呈递细胞”），我们打算证明这种细胞在生物生产和基于细胞的治疗中的应用潜力。每种设想的应用/生产类型都需要不同类型的定制B细胞亚群，有时量更大。实现A型生产的挑战是使记忆B细胞的增殖和它们分化成抗体生产细胞解耦。就A型生产而言，该项目的“演示应用”目的是产生抗血清，作为常见血源性血浆制剂的替代品。在生产类型B的情况下，B细胞在人体中以代谢活性形式持续存在的能力决定了这些细胞用作“活性药物”，例如用于蛋白质替代疗法。在这种情况下，B细胞的遗传修饰提出了巨大的挑战。基于第一个项目阶段的结果，此处使用非病毒转染，使用认为在工艺条件下更适合的B细胞亚型。在C型生产的情况下，目的是提供定制的B淋巴细胞作为抗原呈递细胞（APC）。因此，活化的T淋巴细胞在这种情况下代表实际的“产物”。在生产C型的情况下，主要挑战是需要以受控的方式一起培养两种类型的淋巴细胞。虽然生产类型A主要用于生物生产，但生产类型B和C也与未来的医学应用相关。然而，在这个项目的范围内，只有细胞生物学和工艺工程的基础知识，直到第一个“示范应用”。

The aim of the second project phase applied here, is to use the tools developed in the first project phase for the controlled ex vivo proliferation and differentiation of primary human B lymphocytes to prepare such cells for exemplary applications in bioprocess technology. Using natural abilities of the B lymphocytes such as: the ability to multiply quickly and then to pro-duce large amounts of protein (antibodies) (production type A: 'plasma cell'), the ability to per-sist for extended lengths of time in the body and remain biologically active (production type B: 'Living Drug'), as well as the ability to activate T lymphocytes in a targeted manner (produc-tion type C: 'antigen-presenting cell'), we intend to demonstrate the potential of such cells for applications in bioproduction and cell-based therapies. Each envisioned application/production types require different types of tailor-made B-cell subpopulations, sometimes in larger amounts. The challenge in realizing production type A is to decouple the proliferation of memory B cells and their differentiation into antibody-producing cells. The aim of the project in the sense of a 'demonstrator application' in case of production type A is the generation of an antiserum as a replacement for common blood-derived plasma preparations. In the case of production type B, the ability of B cells to persist in a metabolically active form in the hu-man body predestines such cells for use as 'living drugs', e.g. for protein replacement thera-py. The genetic modification of the B cells presents a great challenge in this case. Building on the results of the first project phase, non-viral transfection is to be used here, using the B cells subtype which is considered more suitable under process conditions. In the case of produc-tion type C, the aim is to provide tailor-made B-lymphocytes as antigen-presenting cells (APC). The activated T-lymphocytes thus represent the actual 'product' in this case. The main challenge in the case of production type C is the need to cultivate both types of lymphocytes together in a controlled manner. While production type A is primarily of interest for bioproduc-tion, production types B and C are also relevant for future medical applications. Within the scope of this project, however, only the basics of cell biology and process engineering are laid, right up to the first ‘demonstrator applications’.