Continuous purification of antibodies from cell culture supernatant with aqueous two-phase systems: From concept to process
Continuous purification of antibodies from cell culture supernatant with aqueous two-phase systems: From concept to process
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DOI：
10.1002/biot.201200031
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发表时间：
2013-03-01
影响因子：
4.7
通讯作者：
Aires-Barros, M. Raquel
中科院分区：
文献类型：
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作者：
Rosa, Paula A. J.;Azevedo, Ana M.;Aires-Barros, M. Raquel
开发了一种基于PEG/磷酸盐系统的双水相萃取（ATPE）工艺，用于捕获人免疫球蛋白G，并成功应用于中国仓鼠卵巢和PER. C6（R）细胞上清液。在泵混合沉降器组中建立并验证了包含三个不同步骤（萃取、反萃取和洗涤）的连续ATPE工艺。大多数较高分子量的细胞上清液杂质在提取步骤中被去除，而大多数较低分子量的杂质在后续步骤中被去除。从CHO细胞上清液中纯化IgG获得了80%的总体回收率和99%以上的最终蛋白纯度，这代表蛋白/IgG比率降低了155倍。对于从PER. C6（R）细胞上清液中纯化IgG，获得了100%的总体回收率和宿主细胞蛋白纯度，这表示宿主细胞蛋白/IgG比率降低了22倍。因此，这些结果为开发的ATPE过程作为捕获抗体的平台的应用开辟了有希望的前景。事实上，这种新方法已经显示出成功地从不同的细胞培养上清液中回收和纯化不同抗体的能力。除了可扩展性、过程集成、连续操作能力和经济可行性的固有优势之外，该技术还可以克服使用典型色谱过程遇到的一些限制。
An aqueous two-phase extraction (ATPE) process based on a PEG/phosphate system was developed for the capture of human immunoglobulin G and successfully applied to a Chinese hamster ovary and a PER.C6(R) cell supernatant. A continuous ATPE process incorporating three different steps (extraction, back-extraction, and washing) was set up and validated in a pump mixer-settler battery. Most of the higher molecular weight cell supernatant impurities were removed during the extraction step, while most of the lower molecular weight impurities were removed during the subsequent steps. A global recovery yield of 80% and a final protein purity of more than 99% were obtained for the IgG purification from a CHO cell supernatant, representing a 155-fold reduction in the protein/IgG ratio. For the purification of IgG from a PER.C6(R) cell supernatant, a global recovery yield of 100%, and a host cell protein purity were attained, representing a 22-fold reduction in the host cell proteins/IgG ratio. These results, thus, open promising perspectives for the application of the developed ATPE process as a platform for the capture of antibodies. In fact, this new process has shown the ability to successfully recover and purify different antibodies from distinct cell culture supernatants. This technology can also overcome some of the limitations encountered using the typical chromatographic processes, besides inherent advantages of scalability, process integration, capability of continuous operation, and economic feasibility.