An alternative assay to hydrophobic interaction chromatography for high-throughput characterization of monoclonal antibodies
An alternative assay to hydrophobic interaction chromatography for high-throughput characterization of monoclonal antibodies
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DOI：
10.1080/19420862.2015.1016694
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发表时间：
2015-05-04
期刊：
影响因子：
5.3
通讯作者：
Xu, Yingda
中科院分区：
文献类型：
--
作者：
Estep, Patricia;Caffry, Isabelle;Xu, Yingda
治疗性单克隆抗体（mAb）的有效性不仅由其生物活性决定，还由其生物物理性质决定。用于快速评估mAb的生物物理性质的测定对于鉴定那些最可能表现出上级性质如高溶解度、低粘度和缓慢血清清除的mAb是有价值的。在高盐浓度下进行以增强疏水相互作用的分析性疏水相互作用色谱（HIC）是用于鉴定具有低疏水性的mAb的有吸引力的测定。然而，该测定是低通量的，因此不适合于处理在抗体发现期间通常产生的大量mAb。因此，我们研究了是否可以开发一种替代的、更高通量的测定法，该测定法基于使用亲和捕获自相互作用纳米颗粒光谱法（AC-荧光光谱法）在高盐浓度下评价抗体自缔合。我们的方法是用多克隆抗人抗体涂覆金纳米颗粒，使用这些缀合物来修饰人mAb，并通过测量抗体缀合物的等离子体波长作为硫酸铵浓度的函数来评估mAb自身相互作用。我们发现，疏水性单克隆抗体，如HIC所确定的，通常显示出显着的自缔合在低至中等硫酸铵浓度，而亲水性单克隆抗体通常只显示出自缔合在高硫酸铵浓度。AC-β和HIC测量值之间的相关性表明，我们的测定法可以以平行方式评估数十至数百种mAb，并且仅需要少量（微克）抗体，将能够早期鉴定具有低疏水性和改善的生物物理特性的mAb候选物。
The effectiveness of therapeutic monoclonal antibodies (mAbs) is governed not only by their bioactivity, but also by their biophysical properties. Assays for rapidly evaluating the biophysical properties of mAbs are valuable for identifying those most likely to exhibit superior properties such as high solubility, low viscosity and slow serum clearance. Analytical hydrophobic interaction chromatography (HIC), which is performed at high salt concentrations to enhance hydrophobic interactions, is an attractive assay for identifying mAbs with low hydrophobicity. However, this assay is low throughput and thus not amenable to processing the large numbers of mAbs that are commonly generated during antibody discovery. Therefore, we investigated whether an alternative, higher throughput, assay could be developed that is based on evaluating antibody self-association at high salt concentrations using affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS). Our approach is to coat gold nanoparticles with polyclonal anti-human antibodies, use these conjugates to immobilize human mAbs, and evaluate mAb self-interactions by measuring the plasmon wavelengths of the antibody conjugates as a function of ammonium sulfate concentration. We find that hydrophobic mAbs, as identified by HIC, generally show significant self-association at low to moderate ammonium sulfate concentrations, while hydrophilic mAbs typically show self-association only at high ammonium sulfate concentrations. The correlation between AC-SINS and HIC measurements suggests that our assay, which can evaluate tens to hundreds of mAbs in a parallel manner and requires only small (microgram) amounts of antibody, will enable early identification of mAb candidates with low hydrophobicity and improved biophysical properties.