Pre-Clinical Development and Protein Chemistry, Regeneron Pharmaceuticals, Inc ., Tarrytown , NY , USA.
Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals Inc ., Ridgefield , CT , USA.
MAbs. 2019 Oct;11(7):1319-1330. doi: 10.1080/19420862.2019.1647744. Epub 2019 Aug 12.
Biotherapeutic proteins are commonly dosed at high concentrations into the blood, which is an inherently complex, crowded solution with substantial protein content. The effects of macromolecular crowding may lead to an appreciable level of non-specific hetero-association in this physiological environment. Therefore, developing a method to characterize the diverse consequences of non-specific interactions between proteins under such non-ideal, crowded conditions, which deviate substantially from those commonly employed for characterization, is vital to achieving a more complete picture of antibody function in a biological context. In this study, we investigated non-specific interactions between human serum albumin (HSA) and two monoclonal antibodies (mAbs) by static light scattering and determined these interactions are both ionic strength-dependent and mAb-dependent. Using biolayer interferometry (BLI), we assessed the effect of HSA on antigen binding by mAbs, demonstrating that these non-specific interactions have a functional impact on mAb:antigen interactions, particularly at low ionic strength. While this effect is mitigated at physiological ionic strength, our data support the notion that HSA in the blood may lead to non-specific interactions with mAbs , with a potential impact on their interactions with antigen. Furthermore, the BLI method offers a high-throughput advantage compared to orthogonal techniques such as analytical ultracentrifugation and is amenable to a greater variety of solution conditions compared to nuclear magnetic resonance spectroscopy. Our study demonstrates that BLI is a viable technology for examining the impact of non-specific interactions on specific biologically relevant interactions, providing a direct method to assess binding events in crowded conditions.
生物治疗性蛋白通常以高浓度给药到血液中，血液是一种固有的复杂、拥挤的溶液，具有大量的蛋白质含量。大分子拥挤的影响可能导致在这种生理环境中产生相当水平的非特异性异源缔合。因此，开发一种方法来描述在这种非理想、拥挤的条件下蛋白质之间非特异性相互作用的多种后果，对于更全面地了解抗体在生物环境中的功能至关重要。在这项研究中，我们通过静态光散射研究了人血清白蛋白（HSA）和两种单克隆抗体（mAbs）之间的非特异性相互作用，并确定这些相互作用既依赖于离子强度，也依赖于 mAb。我们使用生物层干涉（BLI）评估了 HSA 对 mAb 与抗原结合的影响，证明这些非特异性相互作用对 mAb：抗原相互作用具有功能影响，特别是在低离子强度下。虽然这种效应在生理离子强度下得到缓解，但我们的数据支持了这样一种观点，即在血液中的 HSA 可能与 mAbs 发生非特异性相互作用，从而潜在地影响它们与抗原的相互作用。此外，与分析超速离心等正交技术相比，BLI 方法具有高通量优势，并且与核磁共振波谱相比，更适用于更多种类的溶液条件。我们的研究表明，BLI 是一种可行的技术，可以检查非特异性相互作用对特定生物学相关相互作用的影响，提供了一种在拥挤条件下评估结合事件的直接方法。
