Causes of Industrial Protein A Column Degradation, Explored Using Raman Spectroscopy.
Causes of Industrial Protein A Column Degradation, Explored Using Raman Spectroscopy.
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DOI：
10.1021/acs.analchem.2c03063
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发表时间：
2022-11-15
影响因子：
7.4
通讯作者：
Byrne B
中科院分区：
文献类型：
--
作者：
Beattie JW;Istrate A;Lu A;Marshall C;Rowland-Jones RC;Farys M;Kazarian SG;Byrne B
单克隆抗体（mAb）被广泛用作慢性和急性病症的生物治疗剂。单克隆抗体的生产是漫长和昂贵的，蛋白A亲和捕获是最昂贵的步骤，这是由于树脂的性质和重复使用后结合能力的显著降低。我们先前使用原位ATR-FTIR光谱的研究表明，蛋白A结合能力的损失不是蛋白A配体的浸出或降解的结果，这表明结垢是主要原因。在这里，我们探讨结合行为和树脂容量损失使用拉曼光谱。我们的数据揭示了与MabSelect SuRe的蛋白A配体结合的mAb的独特拉曼光谱指纹。结果表明，通过拉曼光谱结合偏最小二乘回归，可辨别先前观察到的使用过的蛋白A树脂的静态结合容量（SBC）下降。与使用过的出口（40.17 mg mL-1）和未使用的树脂样品（70.35 mg mL-1）相比，使用过的入口树脂的SBC最低（35.76 mg mL-1）。通过拉曼光谱法进行的深度分析表明，在低于饱和浓度（约18 mg mL-1）时，通过使用的树脂珠粒，mAb的结合不均匀，蛋白质优先结合到珠粒的外部区域，与通过未使用的对照MabSelect SuRe树脂珠粒的完全均匀分布相反。拉曼光谱分析表明，一种污垢是不可逆结合的mAb。通过对使用过的树脂珠进行质谱分析，证实了mAb和宿主细胞蛋白不可逆结合的存在。
Monoclonal antibodies (mAbs) are used extensively as biotherapeutics for chronic and acute conditions. Production of mAbs is lengthy and expensive, with protein A affinity capture the most costly step, due both to the nature of the resin and its marked reduction in binding capacity with repeated use. Our previous studies using in situ ATR-FTIR spectroscopy indicated that loss in protein A binding capacity is not the result of leaching or degradation of protein A ligand, suggesting fouling is the principal cause. Here we explore binding behavior and resin capacity loss using Raman spectroscopy. Our data reveal a distinct Raman spectral fingerprint for mAb bound to the protein A ligand of MabSelect SuRe. The results show that the drop in static binding capacity (SBC) previously observed for used protein A resin is discernible by Raman spectroscopy in combination with partial least-squares regression. The SBC is lowest (35.76 mg mL–1) for used inlet resin compared to used outlet (40.17 mg mL–1) and unused resin samples (70.35 mg mL–1). Depth profiling by Raman spectroscopy indicates that at below saturating concentrations (∼18 mg mL–1), binding of mAb is not homogeneous through used resin beads with protein binding preferentially to the outer regions of the bead, in contrast to fully homogeneous distribution through unused control MabSelect SuRe resin beads. Analysis of the Raman spectra indicates that one foulant is irreversibly bound mAb. The presence of irreversibly bound mAb and host cell proteins was confirmed by mass spectrometric analysis of used resin beads.
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DOI：
10.1039/d1an00985k
发表时间：
2021-08-09
期刊：
The Analyst
影响因子：
--
作者：
Beattie JW;Rowland-Jones RC;Farys M;Tran R;Kazarian SG;Byrne B
通讯作者：
Byrne B
影响因子：
5.3
作者：
Lampson LA
通讯作者：
Lampson LA
影响因子：
11.5
作者：
Markham A
通讯作者：
Markham A
影响因子：
5.3
作者：
Kaplon H;Reichert JM
通讯作者：
Reichert JM
影响因子：
3.5
作者：
Everall, NJ
通讯作者：
Everall, NJ