单克隆抗体（mAbs）是常用的生物制剂，用于治疗风湿性关节炎、多发性硬化症、COVID-19和各种癌症等疾病。它们是在中国仓鼠卵巢细胞系中生产的，并通过一系列基于色谱的复杂且昂贵的步骤进行纯化，这些步骤是批量操作的，且严重依赖蛋白A树脂。传统程序的主要缺点是吸附介质成本高，色谱柱再生时需要大量使用化学品，对环境造成了负担。我们已经表明，传统的蛋白A色谱可以用单一结晶步骤替代，并且可以使用模板辅助膜结晶过程，在连续流中实现克级生产。模板嵌入在膜中（例如，多孔聚偏氟乙烯和聚乙烯醇聚合的层），用作结晶的成核剂。mAbs是柔性蛋白质，很难结晶，因此确定结晶的最佳条件可能具有挑战性。本协议的目的是建立一种系统性和灵活的方法，以设计一个强大、经济和可持续的mAb纯化平台，至少能够替代传统色谱纯化平台中的蛋白A亲和阶段。该程序提供了建立分离的最佳参数（结晶条件、模板选择、膜选择）和分析和表征方法的详细信息。© 2023年 Springer Nature Limited.

Monoclonal antibodies (mAbs) are commonly used biologic drugs for the treatment of diseases such as rheumatoid arthritis, multiple sclerosis, COVID-19 and various cancers. They are produced in Chinese hamster ovary cell lines and are purified via a number of complex and expensive chromatography-based steps, operated in batch mode, that rely heavily on protein A resin. The major drawback of conventional procedures is the high cost of the adsorption media and the extensive use of chemicals for the regeneration of the chromatographic columns, with an environmental cost. We have shown that conventional protein A chromatography can be replaced with a single crystallization step and gram-scale production can be achieved in continuous flow using the template-assisted membrane crystallization process. The templates are embedded in a membrane (e.g., porous polyvinylidene fluoride with a layer of polymerized polyvinyl alcohol) and serve as nucleants for crystallization. mAbs are flexible proteins that are difficult to crystallize, so it can be challenging to determine the optimal conditions for crystallization. The objective of this protocol is to establish a systematic and flexible approach for the design of a robust, economic and sustainable mAb purification platform to replace at least the protein A affinity stage in traditional chromatography-based purification platforms. The procedure provides details on how to establish the optimal parameters for separation (crystallization conditions, choice of templates, choice of membrane) and advice on analytical and characterization methods.© 2023. Springer Nature Limited.