Waters Corporation, located in CMU-Rue Michel Servet 1, Geneva 1211, Switzerland.
Waters Corporation, 34 Maple Street, Milford, MA 01757, United States.
J Chromatogr A. 2022 Aug 2;1676:463262. doi: 10.1016/j.chroma.2022.463262. Epub 2022 Jun 17.
Size exclusion chromatography has become an essential tool for the protein therapeutics industry. Conceptually, it is a simple form of chromatography that is driven by entropy and sieving effects. An ideal size exclusion column would exhibit no adsorptive interactions between its internal surfaces and the solutes being analysed, but that is not easily achieved. To this end, we have studied the utility of three unique packing materials in pursuit of additional column chemistries that might be less prone to interacting with proteins. These packing materials were each prepared from bridged ethylene hybrid organic/inorganic particles but uniquely derivatized into either hydroxy terminated PEO bonded, methoxy terminated PEO bonded, or diol bonded packing materials. All three materials were packed into column hardware modified with hydrophilic hybrid surface technology (h-HST) so that packing material effects could be more clearly observed without any influence from the secondary interactions that can originate from metal hardware. Non-specific interactions were compared for various challenging protein samples in the presence of ammonium acetate (volatile) and phosphate buffered saline (non-volatile) buffers. It was reconfirmed that the h-HST column hardware mitigates a majority of non-desired secondary interactions. However, during studies on hydrophobic interactions, the new hydroxy terminated PEO packing material showed clear benefit to obtaining higher apparent recoveries to better ensure accurate aggregate quantitation. Further experiments were explored to show that a hydroxy terminated PEO column could be effectively paired with a mobile phase comprised of standard strength phosphate buffered saline to make a fast platform method capable of baseline resolving monoclonal antibody monomer and aggregate peaks within a 3 min analysis time.
尺寸排阻色谱法已成为蛋白质治疗药物行业的重要工具。从概念上讲，它是一种简单的色谱形式，由熵和筛分效应驱动。理想的尺寸排阻柱在其内部表面和被分析的溶质之间不应表现出任何吸附相互作用，但这不容易实现。为此，我们研究了三种独特的填充材料的用途，以期获得可能不易与蛋白质相互作用的额外柱化学。这三种填充材料均由桥接的乙烯基混合有机/无机颗粒制备而成，但独特地衍生为羟基封端的 PEO 键合、甲氧基封端的 PEO 键合或二醇键合的填充材料。所有三种材料都填充到经过亲水混合表面技术 (h-HST) 修饰的柱硬件中，以便更清楚地观察填充材料的作用，而不受可能源自金属硬件的二次相互作用的影响。在存在乙酸铵（挥发性）和磷酸盐缓冲盐水（非挥发性）缓冲液的情况下，对各种具有挑战性的蛋白质样品进行了非特异性相互作用的比较。再次证实 h-HST 柱硬件减轻了大多数不需要的二次相互作用。然而，在研究疏水性相互作用时，新的羟基封端的 PEO 填充材料显示出明显的优势，可以获得更高的表观回收率，以更好地确保准确的聚集体定量。进一步的实验探索表明，羟基封端的 PEO 柱可以有效地与由标准强度磷酸盐缓冲盐水组成的流动相配对，以形成快速平台方法，能够在 3 分钟的分析时间内基线分离单克隆抗体单体和聚集体峰。
