生物制药的发酵、纯化、冷藏和运输成本高，保质期短，无菌运输方式也是生产者和消费者共同关心的问题。由于 FDA 现已批准植物细胞用于大规模、具有成本效益的生物制药生产，转质体叶绿体的分离和冻干可以消除对局限性的担忧。 DARPins 是经过改造的小型单结构域蛋白，被选择以高亲和力和特异性结合 HER2。 HER2 是一种癌基因，参与某些癌症中的异常细胞生长，也是癌症免疫治疗的靶分子。在这项研究中，我们报道了 DARPin G3 在冻干转质体烟草叶和叶绿体中的长期稳定性和功能性。对室温下保存长达九个月的冻干叶子和叶绿体进行的蛋白质印迹分析表明，DARPin G3 蛋白是稳定的并保持正确的折叠。叶子和分离叶绿体的冻干使 DARPin G3 蛋白浓度分别增加 16 倍和 32 倍。 HER2 结合测定表明，叶绿体制造的 DARPin G3 在整个研究过程中，在室温下超过 9 个月的时间里，在冻干叶子材料中可以保持其稳定性和结合活性，而不会出现任何亲和力下降。表达 DARPin G3 的叶绿体的冻干将进一步降低成本并简化下游加工、纯化和储存。考虑到九个月后 DARPin G3 占用的空间以及下游提取和纯化，冻干叶绿体的压缩包比冻干转质体叶更有效。这些方法有利于这些化合物的任何相关配制实践，以满足任何以需求为导向的需求。© 2023。BioMed Central Ltd.，Springer Nature 的一部分。

The high cost of fermentation, purification, cold storage and transportation, short shelf life, and sterile delivery methods of biopharmaceuticals, is a matter for producers and consumers as well. Since the FDA has now approved plant cells for large-scale, cost-effective biopharmaceutical production, the isolation and lyophilization of transplastomic chloroplasts can cover concerns about limitations. DARPins are engineered small single-domain proteins that have been selected to bind to HER2 with high affinity and specificity. HER2 is an oncogene involved in abnormal cell growth in some cancers and the target molecule for cancer immunotherapy.In this study, we reported the prolonged stability and functionality of DARPin G3 in lyophilized transplastomic tobacco leaves and chloroplasts. Western blot analysis of lyophilized leaves and chloroplasts stored at room temperature for up to nine months showed that the DARPin G3 protein was stable and preserved proper folding. Lyophilization of leaves and isolated chloroplasts increased DARPin G3 protein concentrations by 16 and 32-fold, respectively. The HER2-binding assay demonstrated that the chloroplast-made DARPin G3 can maintain its stability and binding activity without any affinity drop in lyophilized leaf materials throughout this study for more than nine months at room temperature.Lyophilization of chloroplasts expressing DARPin G3 would further reduce costs and simplify downstream processing, purification, and storage. Compressed packages of lyophilized chloroplasts were much more effective than lyophilized transplastomic leaves considering occupied space and downstream extraction and purification of DARPin G3 after nine months. These methods facilitate any relevant formulation practices for these compounds to meet any demand-oriented needs.© 2023. BioMed Central Ltd., part of Springer Nature.