Purification Process Development, Biogen IDEC Corporation, 5200 Research Place, San Diego, CA 92122, USA.
Biotechnol Prog. 2010 Nov-Dec;26(6):1662-70. doi: 10.1002/btpr.477.
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% (w/w) PEG, 8.4% (w/w) citrate, and 7.2% (w/w) 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% (w/w) PEG, 8% (w/w) 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.
采用聚乙二醇（PEG）-柠檬酸钠双水相体系（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对抗体分配有显著影响的因素。使用实验设计（DOE）研究了澄清的细胞培养物上清液中抗体与宿主细胞蛋白（HCP）的分离。抗体的分配在整个操作空间范围内几乎完全完成。生成了 HCP 分配模型，其中 NaCl 和柠檬酸钠浓度均被确定为重要因素。为了获得最高纯度，使用最速下降算法将 HCP 从细胞培养液分配到含产物的相中，从而最小化 HCP 的分配。由 14.0%（w/w）PEG、8.4%（w/w）柠檬酸钠和 7.2%（w/w）NaCl 在 pH 7.2 组成的最佳 ATPS 导致产物收率为 89%，与提取前澄清的细胞培养液相比，HCP 水平降低了约 7.6 倍，总纯度为 70%。由 15%（w/w）PEG、8%（w/w）柠檬酸钠和 15%（w/w）NaCl 在 pH 5.5 组成的系统将产物相关杂质（聚集体和低分子量产物片段）从约 40%降低至小于 0.5%，同时实现了 95%的产物回收率。在分批模式下优化的实验条件下，开发了用于逆流萃取技术的放大模型，以确定在连续操作模式下可以实现的纯度和回收率的潜在提高。
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