Insight into purification of monoclonal antibodies in industrial columns via studies of Protein A binding capacity by in situ ATR-FTIR spectroscopy.
Insight into purification of monoclonal antibodies in industrial columns via studies of Protein A binding capacity by in situ ATR-FTIR spectroscopy.
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通过原位ATR-FTIR光谱研究蛋白A结合能力，深入了解工业柱中单克隆抗体的纯化。
DOI：
10.1039/d1an00985k
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发表时间：
2021-08-09
期刊：
影响因子：
--
通讯作者：
Byrne B
中科院分区：
文献类型：
--
作者：
Beattie JW;Rowland-Jones RC;Farys M;Tran R;Kazarian SG;Byrne B
治疗性单克隆抗体（mAb）是一系列癌症和其他严重疾病的有效治疗方法，但mAb治疗平均每位患者每年花费10万美元，限制了其使用。目前，工业上倾向于蛋白A亲和层析（PrAc）作为mAb下游加工的关键步骤。该步骤虽然高效，但代表了显著的mAb生产成本。蛋白A柱的污垢和蛋白A配体浸出会缩短树脂的寿命，从而增加mAb生产的成本。在这项研究中，我们评估了从中间入口，中心和出口以及侧入口的中试规模的工业柱回收的用过的PrAc树脂的性能。我们使用静态结合能力（SBC）分析和衰减全反射-傅里叶变换红外（ATR-FTIR）光谱的组合来探索所使用的树脂样品。SBC分析表明，来自柱入口的树脂具有比来自柱出口的树脂更低的结合能力。ATR-FTIR光谱与PLS（偏最小二乘）分析证实了从SBC分析获得的结果。重要的是，原位ATR-FTIR光谱还允许测量结合蛋白A的浓度和评估结合蛋白A的构象状态。我们的研究结果表明，使用后的PrAc树脂降解取决于柱的位置，蛋白A配体浸出和变性都不是结合能力损失的原因。静态结合能力分析和ATR-FTIR光谱的组合显示，通过使用的蛋白A柱，结合能力的损失是不均匀的，并且不是由于蛋白A配体的损失。
Therapeutic monoclonal antibodies (mAbs) are effective treatments for a range of cancers and other serious diseases, however mAb treatments cost on average ∼$100 000 per year per patient, limiting their use. Currently, industry favours Protein A affinity chromatography (PrAc) as the key step in downstream processing of mAbs. This step, although highly efficient, represents a significant mAb production cost. Fouling of the Protein A column and Protein A ligand leaching contribute to the cost of mAb production by shortening the life span of the resin. In this study, we assessed the performance of used PrAc resin recovered from the middle inlet, center and outlet as well as the side inlet of a pilot-scale industrial column. We used a combination of static binding capacity (SBC) analysis and Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy to explore the used resin samples. SBC analysis demonstrated that resin from the inlet of the column had lower binding capacity than resin from the column outlet. ATR-FTIR spectroscopy with PLS (partial least square) analysis confirmed the results obtained from SBC analysis. Importantly, in situ ATR-FTIR spectroscopy also allowed both measurement of the concentration and assessment of the conformational state of the bound Protein A. Our results reveal that PrAc resin degradation after use is dependent on column location and that neither Protein A ligand leaching nor denaturation are responsible for binding capacity loss. A combination of static binding capacity analysis and ATR-FTIR spectroscopy reveals that loss of binding capacity is not uniform through a used Protein A column and is not due to loss of Protein A ligand.
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DOI：
10.1016/s1570-0232(03)00092-8
发表时间：
2003-06-25
影响因子：
3
作者：
Hahn, R;Schlegel, R;Jungbauer, A
通讯作者：
Jungbauer, A
影响因子：
5.3
作者：
Kaplon H;Reichert JM
通讯作者：
Reichert JM
影响因子：
2.9
作者：
deJongh, HHJ;Goormaghtigh, E;Ruysschaert, JM
通讯作者：
Ruysschaert, JM
影响因子：
2.9
作者：
DEISENHOFER, J
通讯作者：
DEISENHOFER, J
影响因子：
5.3
作者：
Kaplon, Helene;Muralidharan, Mrinalini;Reichert, Janice M.
通讯作者：
Reichert, Janice M.