Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody
Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody
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DOI：
10.1080/19420862.2015.1029217
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发表时间：
2015-05-04
期刊：
影响因子：
5.3
通讯作者：
Volkin, David B.
中科院分区：
文献类型：
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作者：
Arora, Jayant;Hickey, John M.;Volkin, David B.
需要新的分析方法来更好地表征浓度依赖的可逆自结合(RSA)的性质，当这些蛋白被配制为高浓度溶液时，直接和高分辨率地描述这些单抗(MAbs)。在这项工作中，氢交换质谱仪(HX-MS)被用来定义浓度依赖的RSA界面，并表征缔合对IgG1mAb(mAb-C)骨架动力学的影响。采用动态光散射、化学交联法和溶液粘度测定等方法确定引起单抗-C RSA的条件。一种新的HX-MS实验方法被用来直接监测由于RSA在不同蛋白质浓度的氚缓冲液中对mAb-C局部柔韧性的影响。首先，确定了一种含有冻干保护剂的稳定配方，该配方允许mAb-C在5和60 mg/mL的浓度下冷冻干燥。在与RSA促进的氚溶液重组时，低蛋白质浓度样品和高蛋白质浓度样品显示不同水平的溶液粘度(即约。每秒1至75英里)。用HX-MS对重组mAb-C样品进行分析。覆盖互补决定区CDR2H和CDR2L的两个特定序列(分别位于可变的重链和轻链上)显示出显著的保护作用，以防氢摄取(即氢交换减少)。这些结果确定了与mAb-C浓度依赖的RSA相关的主要蛋白质-蛋白质界面。令人惊讶的是，V-H结构域、恒定域(C(H)2)和铰链区(C(H)1-C(H)2界面)中的某些肽片段在高蛋白质浓度与低蛋白质浓度下同时显示出显著的局部灵活性增加。这些结果表明，单抗-C在RSA上存在较远距离的动态耦合效应。
There is a need for new analytical approaches to better characterize the nature of the concentration-dependent, reversible self-association (RSA) of monoclonal antibodies (mAbs) directly, and with high resolution, when these proteins are formulated as highly concentrated solutions. In the work reported here, hydrogen exchange mass spectrometry (HX-MS) was used to define the concentration-dependent RSA interface, and to characterize the effects of association on the backbone dynamics of an IgG1 mAb (mAb-C). Dynamic light scattering, chemical cross-linking, and solution viscosity measurements were used to determine conditions that caused the RSA of mAb-C. A novel HX-MS experimental approach was then applied to directly monitor differences in local flexibility of mAb-C due to RSA at different protein concentrations in deuterated buffers. First, a stable formulation containing lyoprotectants that permitted freeze-drying of mAb-C at both 5 and 60 mg/mL was identified. Upon reconstitution with RSA-promoting deuterated solutions, the low vs. high protein concentration samples displayed different levels of solution viscosity (i.e., approx. 1 to 75 mPa.s). The reconstituted mAb-C samples were then analyzed by HX-MS. Two specific sequences covering complementarity-determining regions CDR2H and CDR2L (in the variable heavy and light chains, respectively) showed significant protection against deuterium uptake (i.e., decreased hydrogen exchange). These results define the major protein-protein interfaces associated with the concentration-dependent RSA of mAb-C. Surprisingly, certain peptide segments in the V-H domain, the constant domain (C(H)2), and the hinge region (C(H)1-C(H)2 interface) concomitantly showed significant increases in local flexibility at high vs. low protein concentrations. These results indicate the presence of longer-range, distant dynamic coupling effects within mAb-C occurring upon RSA.