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首页> 外文期刊>Biotechnology Progress >Downstream Antibody Purification using Aqueous Two-Phase Extraction
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Downstream Antibody Purification using Aqueous Two-Phase Extraction

机译：两相水相萃取纯化下游抗体

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The extraction of antibodies using a polyethylene glycol (PEG)-citrate aqueous two-phase system (ATPS) was investigated. Studies using purified monoclonal antibody (mAb) identified operating ranges for successful phase formation and factors that significantly affected antibody partitioning. The separation of antibody and host cell protein (HCP) from clarified cell culture media was examined using statistical design of experiments (DOE). The partitioning of antibody was nearly complete over the entire range of the operating space examined. A model of the HCP partitioning was generated in which both NaCl and citrate concentrations were identified as significant factors. To achieve the highest purity, the partitioning of HCP from cell culture fluid into the product containing phase was minimized using a Steepest Descent algorithm. An optimal ATPS consisting of 14.0% (wlw) PEG, 8.4% (w/w) citrate, and 7.2% (wlw) NaCl at pH 7.2 resulted in a product yield of 89%, an approximate 7.6-fold reduction in HCP levels relative to the clarified cell culture fluid before extraction and an overall purity of 70%. A system consisting of 15% (wlw) PEG, 8% (wlw) citrate, and 15% (w/w) NaCl at pH 5.5 reduced product-related impurities (aggregates and low molecular product fragments) from ~40%_ to less than 0.5% while achieving 95% product recovery. At the experimental conditions that were optimized in the batch mode, a scale-up model for the use of counter-current extraction technology was developed to identify potential improvements in purity and recovery that could be realized in the continuous operational mode.
机译：研究了使用聚乙二醇（PEG）柠檬酸盐水溶液两相系统（ATPS）提取抗体。使用纯化的单克隆抗体（mAb）进行的研究确定了成功形成相的操作范围以及显着影响抗体分配的因素。使用实验统计设计（DOE）检查了从澄清的细胞培养基中分离抗体和宿主细胞蛋白（HCP）的过程。在所检查的整个操作空间范围内，抗体的分配几乎完全完成。生成了HCP分配模型，其中NaCl和柠檬酸盐的浓度均被确定为重要因素。为了获得最高的纯度，使用最速下降算法将HCP从细胞培养液分配到含有产物的相中的情况降到最低。在pH值为7.2时，由14.0％（wlw）PEG，8.4％（w / w）柠檬酸盐和7.2％（wlw）NaCl组成的最佳ATPS可使产品收率为89％，相对于HCP含量降低约7.6倍提取前先加入澄清的细胞培养液中，总纯度为70％。 pH为5.5时，由15％（wlw）PEG，8％（wlw）柠檬酸盐和15％（w / w）NaCl组成的系统将与产品相关的杂质（聚集体和低分子产物碎片）从约40％减少到更少低于0.5％，同时实现95％的产品回收率。在以批处理模式优化的实验条件下，开发了使用逆流萃取技术的放大模型，以识别在连续操作模式下可以实现的纯度和回收率方面的潜在改进。

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