Department of Chemistry and Faculty of Pharmacy, Université de Montréal, CP 6128 Succursale Centre Ville, Montréal, H3C 3J7, QC, Canada.
Anal Bioanal Chem. 2014 Nov;406(29):7539-47. doi: 10.1007/s00216-014-8200-2. Epub 2014 Oct 17.
Complexes formed between poly(acrylates) and polyclonal immunoglobulin G (IgG) in its native conformation and after heat stress were characterized using asymmetric flow field-flow fractionation (AF4) coupled with on-line UV-Vis spectroscopy and multi-angle light-scattering detection (MALS). Mixtures of IgG and poly(acrylates) of increasing structural complexity, sodium poly(acrylate) (PAA), a sodium poly(acrylate) bearing at random 3 mol % n-octadecyl groups, and a random copolymer of sodium acrylate (35 mol%), N-n-octylacrylamide (25 mol%) and N-isopropylacrylamide (40 mol%), were fractionated in a sodium phosphate buffer (0.02 M, pH 6.8) in the presence, or not, of 0.1 M NaCl. The AF4 protocol developed allowed the fractionation of solutions containing free poly(acrylates), native IgG monomer and dimer, poly(acrylates)/IgG complexes made up of one IgG molecule and a few polymer chains, and/or larger poly(acrylates)/IgG aggregates. The molar mass and recovery of the soluble analytes were obtained for mixed solutions of poly(acrylates) and native IgG and for the same solutions incubated at 65 °C for 10 min. From the combined AF4 results, we concluded that in solutions of low ionic strength, the presence of PAA increased the recovery ratio of IgG after thermal stress because of the formation of electrostatically-driven PAA/IgG complexes, but PAA had no protective effect in the presence of 0.1 M NaCl. Poly(acrylates) bearing hydrophobic groups significantly increased IgG recovery after stress, independently of NaCl concentration, because of the synergistic effect of hydrophobic and electrostatic interactions. The AF4 results corroborate conclusions drawn from a previous study combining four analytical techniques. This study demonstrates that AF4 is an efficient tool for the analysis of protein formulations subjected to stress, an important achievement given the anticipated important role of proteins in near-future human therapies.
采用不对称流场流分离（AF4）结合在线紫外-可见光谱和多角度光散射检测（MALS），研究了天然构象和热应激后的聚（丙烯酸酯）与多克隆免疫球蛋白 G（IgG）形成的复合物。用 AF4 在磷酸钠缓冲液（0.02 M，pH6.8）中对结构复杂度逐渐增加的 IgG 和聚（丙烯酸酯）混合物、随机带有 3 mol%正十八烷基的聚（丙烯酸酯）钠盐、丙烯酸钠（35 mol%）、N-正辛基丙烯酰胺（25 mol%）和 N-异丙基丙烯酰胺（40 mol%）的无规共聚物进行了分离，存在或不存在 0.1 M NaCl。所开发的 AF4 方案允许对含有游离聚（丙烯酸酯）、天然 IgG 单体和二聚体、由一个 IgG 分子和几个聚合物链组成的聚（丙烯酸酯）/IgG 复合物以及/或更大的聚（丙烯酸酯）/IgG 聚集体的溶液进行分级分离。对聚（丙烯酸酯）和天然 IgG 的混合溶液以及在 65°C 下孵育 10 分钟的相同溶液进行了混合溶液中可溶性分析物的摩尔质量和回收率的测定。根据 AF4 的综合结果，我们得出结论，在低离子强度溶液中，由于形成静电驱动的 PAA/IgG 复合物，PAA 的存在增加了热应激后 IgG 的回收比，但在存在 0.1 M NaCl 时，PAA 没有保护作用。带有疏水性基团的聚（丙烯酸酯）大大提高了应激后 IgG 的回收率，而与 NaCl 浓度无关，这是由于疏水和静电相互作用的协同效应。AF4 的结果证实了结合四种分析技术的先前研究得出的结论。这项研究表明，AF4 是分析受应激的蛋白质制剂的有效工具，这是一个重要的成就，因为蛋白质在不久的将来的人类治疗中预期会发挥重要作用。
