Staby A, Jensen I H, Mollerup I
Novo Nordisk A/S, Protein Purification, Gentofte, Denmark.
J Chromatogr A. 2000 Nov 3;897(1-2):99-111. doi: 10.1016/s0021-9673(00)00780-9.
A comparative study has been undertaken on various strong anion-exchangers to investigate the pH dependence, titration curves, efficiency, binding strength, and dynamic capacity of the chromatographic resins. The resins tested included: Macro-Prep 25Q, TSK-Gel Q-5PW-HR, Poros QE/M, Q Sepharose FF, Q HyperD 20, Q Zirconia, Source 30Q, Fractogel EMD TMAE 650s, and Express-Ion Q. Testing was performed with five different proteins: Anti-FVII Mab (IgG), aprotinin, BSA, lipolase, and myoglobin. The dependence of pH on retention varies from generally low to very high for proteins with low pI. No direct link between pH dependence on retention and titration curves of the different resins was observed. Efficiency results show the expected trend of lower dependence of the plate height with increasing flow-rate of resins for medium and high pressure operation compared to the soft resins. Binding to the anion-exchange resins as a function of ionic strength may vary depending on the specific protein. Generally, binding and elution at a high salt concentration may be performed with Poros QE/M or Macro-Prep 25Q, while binding and elution at low salt concentration may be done with TSK-Gel Q-5PW. Dynamic capacities are strongly dependent on the specific protein employed and for some resins dependent on the flow-rate. A general good agreement was obtained between this study and data obtained by suppliers for the dynamic capacity. The results of this study may be used for selection of resins for testing in process development, however, the data does not tell anything about specific selectivity differences or resolution between a target protein and a given impurity. None of the resins studied here should be regarded as good or bad, but more or less suitable for a specific purpose, and only testing for the specific application will determine which one is the optimal resin.
已对各种强阴离子交换剂进行了一项比较研究,以研究色谱树脂的pH依赖性、滴定曲线、效率、结合强度和动态容量。测试的树脂包括:Macro-Prep 25Q、TSK-Gel Q-5PW-HR、Poros QE/M、Q Sepharose FF、Q HyperD 20、Q氧化锆、Source 30Q、Fractogel EMD TMAE 650s和Express-Ion Q。使用五种不同的蛋白质进行了测试:抗FVII单克隆抗体(IgG)、抑肽酶、牛血清白蛋白、脂肪酶和肌红蛋白。对于低pI的蛋白质,pH对保留的依赖性从普遍较低到非常高不等。未观察到pH对保留的依赖性与不同树脂的滴定曲线之间有直接联系。效率结果显示,与软质树脂相比,对于中高压操作,随着树脂流速增加,板高依赖性降低的预期趋势。作为离子强度函数的与阴离子交换树脂的结合可能因特定蛋白质而异。一般来说,在高盐浓度下的结合和洗脱可以用Poros QE/M或Macro-Prep 25Q进行,而在低盐浓度下的结合和洗脱可以用TSK-Gel Q-5PW进行。动态容量强烈依赖于所使用的特定蛋白质,对于某些树脂还依赖于流速。本研究与供应商获得的动态容量数据之间总体上达成了良好的一致性。本研究结果可用于在工艺开发中选择用于测试的树脂,然而,这些数据并未说明目标蛋白质与给定杂质之间的特定选择性差异或分辨率。这里研究的树脂都不应被视为好或坏,而只是或多或少适用于特定目的,只有针对特定应用进行测试才能确定哪种树脂是最佳的。