Phase Separation of an IgG1 Antibody Solution under a Low Ionic Strength Condition
Phase Separation of an IgG1 Antibody Solution under a Low Ionic Strength Condition
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DOI：
10.1007/s11095-010-0125-7
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发表时间：
2010-07-01
影响因子：
3.7
通讯作者：
Fukui, Kiichi
中科院分区：
文献类型：
--
作者：
Nishi, Hirotaka;Miyajima, Makoto;Fukui, Kiichi
研究了单克隆抗体A（MAb A）溶液的相分离及其与蛋白质自缔合的关系，绘制了MAb A的相图，并研究了其对离子强度和pH的依赖性。通过动态光散射（DLS）、分析超离心分析（AUC）和粘度测量来表征蛋白质的自缔合。MAb A溶液在等渗离子强度条件下具有清澈的外观，在低离子强度条件下变成乳白色，随后液-液相分离（LLPS）成轻相和重相。两相的蛋白质浓度依赖于离子强度和pH值。当离子强度或温度增加时，两相变得可逆混溶。DLS和AUC表明，在低离子强度条件下，MAb A在高于临界浓度16.5 mg/mL的蛋白浓度下自缔合。重相的粘度高，并且取决于剪切速率。这些结果表明，在重相中的蛋白质-蛋白质相互作用的吸引诱导LLPS。LLPS诱导在MAb A溶液中在低离子强度条件下，由于可逆的蛋白质自缔合主要介导的MAb A分子之间的吸引静电相互作用在重相中。
Phase separation of monoclonal antibody A (MAb A) solution and its relation to protein self-association are studied.A phase diagram of MAb A and its dependence on ionic strength and pH were investigated. The protein self-associations were characterized by dynamic light scattering (DLS), analytical ultracentrifugation analysis (AUC) and viscosity measurement.MAb A solution with a clear appearance in an isotonic ionic strength condition turned opalescent in a low ionic strength condition, followed by liquid-liquid phase separation (LLPS) into light and heavy phases. The protein concentrations of the two phases were dependent on the ionic strength and pH. The two phases became reversibly miscible when the ionic strength or temperature was increased. DLS and AUC showed that MAb A under a low ionic strength condition self-associates at a protein concentration above the critical concentration of 16.5 mg/mL. The viscosity of the heavy phase was high and dependent on the shear rate. These results indicate that attractive protein-protein interaction in the heavy phase induces LLPS.LLPS was induced in MAb A solution in a low ionic strength condition due to reversible protein self-association mediated mainly by attractive electrostatic interaction among the MAb A molecules in the heavy phase.