School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Bd d'Yvoy 20, 1211 Geneva 4, Switzerland.
J Chromatogr A. 2012 Aug 24;1252:90-103. doi: 10.1016/j.chroma.2012.06.066. Epub 2012 Jun 28.
In the present contribution, columns packed with fully porous widepore 1.7μm particles (Acquity BEH300) and widepore core-shell 3.6μm 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 (∼0.2μm) 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-α-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.
在本研究中，我们评估了填充有全多孔大孔 1.7μm 颗粒（Acquity BEH300）和大孔核壳 3.6μm 颗粒（Aeris WP）的柱子，用于分离大小、疏水性和等电点不同的模型蛋白和治疗蛋白。我们系统地比较了两种键合类型，即 C4 和 C18。这些固定相的动力学性能已在之前的一篇论文中进行了评估，因此这项新工作侧重于它们的保留行为、负载能力和选择性。通过 Tanaka 测试、模型蛋白和其他验证实验，极有可能的是，对于蛋白来说，强相互作用机制在 Aeris WP 上占主导地位，而疏水性相互作用是保留在 Acquity BEH300 材料上的驱动力。这解释了为什么尽管 Aeris WP 材料的孔体积较低，但具有疏水性和带电氨基酸残基的蛋白的表观保留因子在四种研究的柱子上非常接近。就峰宽而言，对于所有测试的固定相，蛋白的值都相似，尽管 Aeris WP 柱子可能存在强离子交换机制。这可能是由于颗粒表面的薄多孔层（约 0.2μm）提供了极好的传质特性，从而弥补了缓慢的二次离子相互作用动力学。我们还使用模型蛋白评估了所有四种大孔柱的负载能力。平均而言，与核壳 Aeris WP 柱相比，在避免峰宽或拖尾变化 10%的情况下，大约可以将 2-4 倍的蛋白量注入全多孔 BEH300。然而，这一结果可能受到蛋白的性质和形状、疏水性、折叠、大小和电荷数量的强烈影响。最后，所有这些柱子都被用于高效分离一种治疗蛋白（干扰素-α-2A）和一些密切相关的蛋白，并表现出优异的性能和选择性。这一结果证实，RPLC 在生物制药领域受到关注，因为它提供了比尺寸排阻或离子交换显著更好的峰宽，并且与 MS 具有内在的兼容性。
