Live-cell imaging to analyze intracellular aggregation of recombinant IgG in CHO cells
Live-cell imaging to analyze intracellular aggregation of recombinant IgG in CHO cells
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DOI：
10.1016/j.chembiol.2021.08.010
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发表时间：
2022-01-20
影响因子：
8.6
通讯作者：
Honda，Shinya
中科院分区：
文献类型：
--
作者：
Senga，Yukako;Doi，Motomichi;Honda，Shinya
重组免疫球蛋白G（IgG）聚集体在其生产过程中形成。然而，在细胞培养条件下的细胞内/细胞外聚集的潜在过程尚未得到很好的理解，并且不存在评估IgG聚集体的有效方法。在这里，我们建立了一种方法来检测细胞内的聚集体使用AF.2A1，一个小的人工蛋白，结合到非天然IgG构象和聚集体。通过偶联制备荧光标记的AF. 2A 1，并转染到产生抗体的中国仓鼠卵巢（CHO）细胞中。显微图像显示CHO细胞中的细胞内IgG聚集体。细胞内聚集体的相对量（相对于总细胞内IgG）根据细胞培养期间使用的添加剂的类型而不同。有趣的是，细胞内聚集体的相对量与体外细胞外IgG聚集体的相对量适度相关，表明它们是分泌的。该方法将允许研究细胞中的抗体聚集，并且可以指导具有高产量/质量的治疗性抗体的生产。
Recombinant immunoglobulin G (IgG) aggregates are formed during their production. However, the process underlying intracellular/extracellular aggregation in cell culture conditions is not well understood, and no effective method exists to assess IgG aggregates. Here, we establish an approach to detect intracellular aggregates using AF.2A1, a small artificial protein that binds to non-native IgG conformers and aggregates. Fluorescent-labeled AF.2A1 is prepared via conjugation and transfected into antibody-producing Chinese hamster ovary (CHO) cells. Micrographic images show intracellular IgG aggregates in CHO cells. The relative amount of intracellular aggregates (versus total intracellular IgG) differed depending on the type of additives used during cell culture. Interestingly, the relative amount of intracellular aggregates moderately correlates with that ofin vitroextracellular IgG aggregates, suggesting they are secreted. This method will allow the investigation of antibody aggregation in cells, and may guide the production of therapeutic antibodies with high yield/quality.