Biological Physics Laboratory, School of Physics and Astronomy, University of Manchester , Oxford Road, Schuster Building, Manchester M13 9PL, United Kingdom.
ISIS Neutron Facility, STFC , Chilton, Didcot OX11 0QZ, United Kingdom.
ACS Appl Mater Interfaces. 2017 Jul 12;9(27):23202-23211. doi: 10.1021/acsami.7b06131. Epub 2017 Jun 27.
Characterizing the influence of fragment crystallization (Fc) and antigen-binding fragment (Fab) on monoclonal antibody (mAb) adsorption at the air/water interface is an important step to understanding liquid mAb drug product stability during manufacture, shipping, and storage. Here, neutron reflection is used to study the air/water adsorption of a mAb and its Fc and Fab fragments. By varying the isotopic contrast, the adsorbed amount, thickness, orientation, and immersion of the adsorbed layers could be determined unambiguously. While Fc adsorption reached saturation within the hour, its surface adsorbed amount showed little variation with bulk concentration. In contrast, Fab adsorption was slower and the adsorbed amount was concentration dependent. The much higher Fc adsorption, as compared to Fab, was linked to its lower surface charge. Time and concentration dependence of mAb adsorption was dominated by Fab behavior, although both Fab and Fc behaviors contributed to the amount of mAb adsorbed. Changing the pH from 5.5 to 8.8 did not much perturb the adsorbed amount of Fc, Fab, or mAb. However, a small decrease in adsorption was observed for the Fc over pH 8-8.8 and vice versa for the Fab and mAb, consistent with a dominant Fab behavior. As bulk concentration increased from 5 to 50 ppm, the thicknesses of the Fc layers were almost constant at 40 Å, while Fab and mAb layers increased from 45 to 50 Å. These results imply that the adsorbed mAb, Fc, and Fab all retained their globular structures and were oriented with their short axial lengths perpendicular to the interface.
表征片段结晶 (Fc) 和抗原结合片段 (Fab) 对单克隆抗体 (mAb) 在空气/水界面吸附的影响，是理解液体 mAb 药物产品在制造、运输和储存过程中稳定性的重要步骤。在此，使用中子反射来研究 mAb 及其 Fc 和 Fab 片段在空气/水界面的吸附。通过改变同位素对比，可以明确确定吸附层的吸附量、厚度、取向和浸渍。虽然 Fc 的吸附在一小时内达到饱和，但它的表面吸附量与体相浓度几乎没有变化。相比之下，Fab 的吸附较慢，且吸附量与浓度有关。与 Fab 相比，Fc 的吸附量高得多，这与其较低的表面电荷有关。mAb 吸附的时间和浓度依赖性主要由 Fab 行为决定，尽管 Fab 和 Fc 行为都对吸附的 mAb 量有贡献。将 pH 值从 5.5 改变到 8.8 并没有显著改变 Fc、Fab 或 mAb 的吸附量。然而，在 pH 8-8.8 之间，Fc 的吸附略有减少，而 Fab 和 mAb 的吸附略有增加，这与 Fab 行为占主导地位一致。随着体相浓度从 5 增加到 50 ppm，Fc 层的厚度几乎保持在 40 Å，而 Fab 和 mAb 层从 45 Å 增加到 50 Å。这些结果表明，吸附的 mAb、Fc 和 Fab 都保留了它们的球状结构，并以短轴向长度垂直于界面取向。
