Cleaning-in-place of immunoaffinity resins monitored by in situ ATR-FTIR spectroscopy.
Cleaning-in-place of immunoaffinity resins monitored by in situ ATR-FTIR spectroscopy.
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DOI：
10.1007/s00216-015-8871-3
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发表时间：
2015-09
影响因子：
4.3
通讯作者：
Kazarian SG
中科院分区：
文献类型：
--
作者：
Boulet-Audet M;Byrne B;Kazarian SG
制药业预计，在未来10年内，生物疗法的收入将超过小药物分子产生的收入。尽管有效地治疗了一系列慢性和危及生命的疾病，但生物疗法的高昂成本限制了它们的使用。对于生物治疗性单抗，一个重要的生产成本是用于捕获蛋白质的亲和树脂。现场清洗(CIP)协议旨在通过减缓结合能力衰退来优化树脂的寿命。结合分析可以确定流动相的树脂容量，但不能揭示蛋白A配体降解的根本原因。需要将重点放在固定相上，以检查CIP对树脂的影响。为了直接测定蛋白A在两种蛋白A树脂上的局部配体浓度和构象，我们建立了一种基于衰减全反射(ATR)傅里叶变换红外光谱的方法。ATR-FTIR光谱成像显示，对琼脂糖珠施加精心控制的负载可与内反射元件产生均匀且可重复的接触。这使得能够检测和定量固定相的结合能力。ATR-FTIR光谱还表明，在典型的CIP条件下(低于1M NaOH)，蛋白质A似乎不发生蛋白质分解。然而，我们的数据显示，高于0.1M的NaOH浓度会导致蛋白A构象的显著变化。在CIP过程中添加>；0.4%M海藻糖显著减少了所测试的两种蛋白A树脂中的一种的NaOH诱导的配体展开。这些见解有助于优化CIP方案，以延长树脂寿命并降低单抗生产成本。衰减全反射结合典型傅里叶变换红外光谱分析树脂珠的实验方法本文的在线版本(DOI：10.1007/s002160158871-3)包含补充材料，可供授权用户使用。
In the next 10 years, the pharmaceutical industry anticipates that revenue from biotherapeutics will overtake those generated from small drug molecules. Despite effectively treating a range of chronic and life-threatening diseases, the high cost of biotherapeutics limits their use. For biotherapeutic monoclonal antibodies (mAbs), an important production cost is the affinity resin used for protein capture. Cleaning-in-place (CIP) protocols aim to optimise the lifespan of the resin by slowing binding capacity decay. Binding assays can determine resin capacity from the mobile phase, but do not reveal the underlying causes of Protein A ligand degradation. The focus needs to be on the stationary phase to examine the effect of CIP on the resin. To directly determine both the local Protein A ligand concentration and conformation on two Protein A resins, we developed a method based on attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy. ATR-FTIR spectroscopic imaging revealed that applying a carefully controlled load to agarose beads produces an even and reproducible contact with the internal reflection element. This allowed detection and quantification of the binding capacity of the stationary phase. ATR-FTIR spectroscopy also showed that Protein A proteolysis does not seem to occur under typical CIP conditions (below 1 M NaOH). However, our data revealed that concentrations of NaOH above 0.1 M cause significant changes in Protein A conformation. The addition of >0.4 M trehalose during CIP significantly reduced NaOH-induced ligand unfolding observed for one of the two Protein A resins tested. Such insights could help to optimise CIP protocols in order to extend resin lifetime and reduce mAb production costs. Experimental approach for analysing resin beads by attenduated total reflection alongside typical FTIR spectra The online version of this article (doi:10.1007/s00216-015-8871-3) contains supplementary material, which is available to authorized users.