Neutron Reflection Study of Surface Adsorption of Fc, Fab, and the Whole mAb

• Published in: ACS applied materials & interfaces 2017-07, Vol.9 (27), p.23202-23211
• Main Authors: Li, Zongyi, Li, Ruiheng, Smith, Charles, Pan, Fang, Campana, Mario, Webster, John R. P, van der Walle, Christopher F, Uddin, Shahid, Bishop, Steve M, Narwal, Rojaramani, Warwicker, Jim, Lu, Jian Ren
• Format: Article
• Language: English
• ISSN: 1944-8244; 1944-8252
• DOI: 10.1021/acsami.7b06131

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.