High-Throughput mAb Expression and Purification Platform Based on Transient CHO
High-Throughput mAb Expression and Purification Platform Based on Transient CHO
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DOI：
10.1002/btpr.2012
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发表时间：
2015-01-01
影响因子：
2.9
通讯作者：
Rajendra, Yashas
中科院分区：
文献类型：
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作者：
Barnard, Gavin C.;Hougland, Maria D.;Rajendra, Yashas
我们实验室最近基于CHO-GS-KO细胞系开发了一种高细胞密度瞬时转染系统。该方法通过简单的7天工艺，在2 mL至2 L的体积范围内产生高达350 mg/L的单克隆抗体滴度。通过在24深孔板中进行转染，可以同时表达大量mAb。我们将这种新的高通量转染过程与半自动化的蛋白A纯化过程相结合。使用Biomek FXp液体处理机器人，最多可同时纯化72种独特的mAb。我们的主要目标是获得>0.25 mg浓度>0.5 mg/mL的纯化mAb，而无需任何浓缩或缓冲液交换步骤。我们优化了批次结合和批次洗脱步骤。批次结合步骤的长度对于最小化流过级分中的mAb损失很重要。洗脱步骤被证明是具有挑战性的，以同时最大限度地提高蛋白质回收率和蛋白质浓度。我们设计了一种基于平均上清液滴度的可变体积洗脱策略。最后，我们提出了两个案例研究。在第一项研究中，我们以0.33 +/-0.05 mg的平均产量（平均浓度为0.65 +/-0.10 mg/mL）生产了56种亲和力成熟mAb变体。在第二项研究中，我们从早期发现工作中产生了42种独特的mAb，平均产量为0.79 +/- 0.31 mg（平均浓度为1.59 +/- 0.63 mg/mL）。并行高产瞬时转染和半自动化高通量蛋白A纯化的组合代表了有价值的mAb药物发现工具。(c)2014年美国化学工程师学会生物技术。程序：31：239-247，2015
A high-cell-density transient transfection system was recently developed in our laboratory based on a CHO-GS-KO cell line. This method yields monoclonal antibody titers up to 350 mg/L from a simple 7-day process, in volumes ranging from 2 mL to 2 L. By performing transfections in 24-deep-well plates, a large number of mAbs can be expressed simultaneously. We coupled this new high-throughput transfection process to a semiautomated protein A purification process. Using a Biomek FXp liquid handling robot, up to 72 unique mAbs can be simultaneously purified. Our primary goal was to obtain >0.25 mg of purified mAb at a concentration of >0.5 mg/mL, without any concentration or buffer-exchange steps. We optimized both the batch-binding and the batch elution steps. The length of the batch-binding step was important to minimize mAb losses in the flowthrough fraction. The elution step proved to be challenging to simultaneously maximize protein recovery and protein concentration. We designed a variable volume elution strategy based on the average supernatant titer. Finally, we present two case studies. In the first study, we produced 56 affinity maturation mAb variants at an average yield of 0.33 +/- 0.05 mg (average concentration of 0.65 +/- 0.10 mg/mL). In a second study, we produced 42 unique mAbs, from an early-stage discovery effort, at an average yield of 0.79 +/- 0.31 mg (average concentration of 1.59 +/- 0.63 mg/mL). The combination of parallel high-yielding transient transfection and semiautomated high-throughput protein A purification represents a valuable mAb drug discovery tool. (c) 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:239-247, 2015