Evaluation of recent very efficient wide-pore stationary phases for the reversed-phase separation of proteins
Evaluation of recent very efficient wide-pore stationary phases for the reversed-phase separation of proteins
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DOI：
10.1016/j.chroma.2012.06.066
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发表时间：
2012-08-24
影响因子：
4.1
通讯作者：
Guillarme, Davy
中科院分区：
文献类型：
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作者：
Fekete, Szabolcs;Berky, Robert;Guillarme, Davy
在目前的贡献中，用全孔宽孔1.7 μ m颗粒（Acquity BEH300）和宽孔核壳3.6 μ m颗粒（Aeris WP）填充的柱对不同大小、疏水性和等电点的模型蛋白和治疗蛋白的分离进行了评估。系统比较了C4和C18两种键合方式。这些固定相的动力学性能在以前的论文中进行了评估，因此这项新的工作侧重于它们的保留行为，负载能力和选择性。通过Tanaka实验、模型蛋白和其他验证性实验，很可能与蛋白质的强相互作用机制在Aeris WP上占主导地位，而疏水相互作用是Acquity BEH300材料上滞留的驱动力。这就解释了为什么尽管Aeris WP材料的孔隙体积较小，但具有疏水性和带电氨基酸残基的蛋白质的表观保留因子在四个所研究的柱上非常接近。就峰宽而言，尽管Aeris WP柱可能具有强离子交换机制，但所有测试的固定相中蛋白质的值相似。这可以用粒子表面的薄多孔层（类似于0.2 μ m）提供的优良传质特性来解释，这可能弥补了缓慢的二次离子相互作用动力学。使用模型蛋白对所有四根宽孔柱的负载能力进行了评估。平均而言，在避免峰宽或尾迹变化10%的情况下，全多孔BEH300上的蛋白质注入量比Aeris核壳WP柱高约2-4倍。然而，这一结果可能受到蛋白质的性质和形状、疏水性、折叠性、大小和电荷数量的强烈影响。最后，所有这些色谱柱用于干扰素- α - 2a和一些密切相关的蛋白的高效分离，并显示出良好的性能和选择性。这一结果证实了RPLC在生物制药领域获得的兴趣，因为它提供了比尺寸排除或离子交换更好的峰宽，并且与ms (C) 2012 Elsevier B.V.具有固有的兼容性。
In the present contribution, columns packed with fully porous widepore 1.7 mu m particles (Acquity BEH300) and widepore core-shell 3.6 mu m particles (Aeris WP) were evaluated for the separation of model and therapeutic proteins of varying sizes, hydrophobicity and isoelectric points. Two types of bonding were compared, namely C4 and C18 in a systematic way. The kinetic performance of these stationary phases was evaluated in a previous paper hence this new work focuses on their retention behaviour, loading capacity and selectivity. Using the Tanaka tests, model proteins, and other confirmatory experiments, it is highly probable that with proteins, strong interaction mechanisms were predominant on the Aeris WP while the hydrophobic interaction was the driving force of the retention on the Acquity BEH300 material. This explained why, despite the lower pore volume of the Aeris WP material, the apparent retention factors of proteins possessing both hydrophobic and charged amino acids residues were very close on the four investigated columns. In terms of peak widths, values for proteins were similar for all the tested stationary phases, despite the probable strong ion exchange mechanisms of Aeris WP column. This could be explained by the excellent mass transfer characteristics afforded by the thin porous layer (similar to 0.2 mu m) at the surface of the particle which probably compensates for the slow secondary ionic interaction kinetics. The loading capacity was also evaluated on all the four widepore columns, using model proteins. On average, approximately 2-4 times higher amount of proteins can be injected on the fully porous BEH300 compared to the core-shell Aeris WP columns when avoiding 10% change in peak width or in tailing. However, this result could be strongly influenced by the nature and shape of the protein, its hydrophobicity, folding, size and number of charges. Finally, all of these columns were employed for the highly efficient separation of a therapeutic protein (interferon-alpha-2A) and some closely related proteins and showed excellent performance and selectivity. This result confirms that RPLC gained interest in the biopharmaceutical field as it provides significantly better peak widths than size-exclusion or ion-exchange and inherent compatibility with MS. (C) 2012 Elsevier B.V. All rights reserved.