High-throughput thermal stability analysis of a monoclonal antibody by attenuated total reflection FT-IR spectroscopic imaging.
High-throughput thermal stability analysis of a monoclonal antibody by attenuated total reflection FT-IR spectroscopic imaging.
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DOI：
10.1021/ac502529q
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发表时间：
2014-10-07
影响因子：
7.4
通讯作者：
Kazarian, Sergei G.
中科院分区：
文献类型：
--
作者：
Boulet-Audet, Maxime;Byrne, Bernadette;Kazarian, Sergei G.
近年来，诸如单克隆抗体的生物治疗剂的使用显著增加。因此，至关重要的是，生物柴油生产管道尽可能高效。对于生产工艺，主要问题之一是双链抗体聚集的倾向。除了降低生物活性材料的回收率之外，蛋白质聚集还可对药物效力产生重大影响，并引起非常不期望的免疫学效应。因此，必须确定在避免聚集的同时使回收率最大化的加工条件。耐热性是长期聚集倾向的代表。热稳定性测定通常使用各种光谱和散射检测方法进行。在这里，我们评估了宏观衰减全反射傅里叶变换红外（ATR-FT-IR）光谱成像作为一种新的方法，用于单克隆抗体的高通量热稳定性测定的潜力。这种化学特异性可视化方法具有能够区分单体和聚集蛋白的明显优势。衰减全反射特别适用于选择性探测沉淀聚集体聚集的容器底部。通过焦平面阵列检测，我们同时测试了12种不同的缓冲液条件，以评估pH和离子强度对蛋白质热稳定性的影响。Finke模型应用到我们的成像动力学，使我们能够确定成核和自催化生长的速率常数。该分析证明了我们的免疫球蛋白在较高pH和中等离子强度下的更大稳定性，揭示了静电相互作用的关键作用。本文提出的高通量方法具有分析生物治疗药物以及任何其他易于聚集的生物分子的稳定性的巨大潜力。
The use of biotherapeutics, such as monoclonal antibodies, has markedly increased in recent years. It is thus essential that biotherapeutic production pipelines are as efficient as possible. For the production process, one of the major concerns is the propensity of a biotherapeutic antibody to aggregate. In addition to reducing bioactive material recovery, protein aggregation can have major effects on drug potency and cause highly undesirable immunological effects. It is thus essential to identify processing conditions which maximize recovery while avoiding aggregation. Heat resistance is a proxy for long-term aggregation propensity. Thermal stability assays are routinely performed using various spectroscopic and scattering detection methods. Here, we evaluated the potential of macro attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopic imaging as a novel method for the high-throughput thermal stability assay of a monoclonal antibody. This chemically specific visualization method has the distinct advantage of being able to discriminate between monomeric and aggregated protein. Attenuated total reflection is particularly suitable for selectively probing the bottom of vessels, where precipitated aggregates accumulate. With focal plane array detection, we tested 12 different buffer conditions simultaneously to assess the effect of pH and ionic strength on protein thermal stability. Applying the Finke model to our imaging kinetics allowed us to determine the rate constants of nucleation and autocatalytic growth. This analysis demonstrated the greater stability of our immunoglobulin at higher pH and moderate ionic strength, revealing the key role of electrostatic interactions. The high-throughput approach presented here has significant potential for analyzing the stability of biotherapeutics as well as any other biological molecules prone to aggregation.
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影响因子：
2.9
作者：
Cai, SW;Singh, BR
通讯作者：
Singh, BR
影响因子：
3.3
作者：
Boulet-Audet, Maxime;Buffeteau, Thierry;Pezolet, Michel
通讯作者：
Pezolet, Michel
影响因子：
2.6
作者：
Cavallo, Pierpaolo;Pagano, Sergio;Capunzo, Mario
通讯作者：
Capunzo, Mario
影响因子：
2.5
作者：
Chan, K. L. A.;Kazarian, S. G.;Chryssikos, G. D.
通讯作者：
Chryssikos, G. D.
影响因子：
--
作者：
Chan, KLA;Kazarian, SG
通讯作者：
Kazarian, SG