title: Weak antibody-cyclodextrin interactions determined by quartz crystal microbalance and dynamic/static light scattering.
abstract: In a quest to elucidate the mechanism by which hydroxypropyl β-cyclodextrin (HPβCD) stabilizes antibodies against shaking stress, two heavily debated hypotheses exist, namely that stabilization is due to HPβCD's surface activity, or due to specific interactions with proteins. In a previous study by Serno et al. (Pharm. Res. 30 (2013) 117), we could refute the first hypothesis by proving that, although HPβCD is slightly surface active, it does not displace the antibody at the air-water interface, and accordingly, its surface activity is not the underlying stabilizing mechanism. In the present study, we investigated the possibility of interactions between HPβCD and monoclonal antibodies as the potential stabilization mechanism using quartz crystal microbalance (QCM) and static as well as dynamic light scattering. In the presence of HPβCD, the adsorption of IgG antibodies in the native state (IgG A) and the unfolded state (IgG A and IgG B) on gold-coated quartz crystals was studied by QCM. Results show that HPβCD causes a reduction in protein adsorption in both the folded and the unfolded states, probably due to an interaction between the protein and the cyclodextrin, leading to a reduced hydrophobicity of the protein and consequently a lower extent of adsorption. These results were supported by investigation of the interaction between the native protein and HPβCD using static and dynamic light scattering experiments, which provide the protein-protein interaction parameters, B22 and kD, respectively. Both B22 and kD showed an increase in magnitude with increasing HPβCD-concentrations, indicating a rise in net repulsive forces between the protein molecules. This is further evidence for the presence of interactions between HPβCD and the studied antibodies, since an association of HPβCD on the protein surface leads to a change in the intermolecular forces between the protein molecules. In conclusion, this study provides evidence that the previously observed stabilizing effect of HPβCD on IgG antibodies is probably due to direct interactions between the cyclodextrin and the protein.
authors.affiliation: Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Härtl
authors.forename: Nitin
authors.initials: N
authors.lastname: Dixit
authors.forename: Ahmed
authors.initials: A
authors.lastname: Besheer
authors.forename: Devendra
authors.initials: D
authors.lastname: Kalonia
authors.forename: Gerhard
authors.initials: G
authors.lastname: Winter
mesh_terms: D000073738:2-Hydroxypropyl-beta-cyclodextrin; D000327:Adsorption; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D005079:Excipients / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000737:chemistry*; D008027:Light; D017510:Protein Folding; D058767:Protein Unfolding; D011506:Proteins / Q000737:chemistry; D058525:Quartz Crystal Microbalance Techniques; D012542:Scattering, Radiation; D047392:beta-Cyclodextrins / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D005079:Excipients; D007074:Immunoglobulin G; D011506:Proteins; D047392:beta-Cyclodextrins; D000073738:2-Hydroxypropyl-beta-cyclodextrin
keywords: Dynamic light scattering (DLS); Hydroxypropyl-beta-cyclodextrin (HPβCD); IgG antibody; Quartz crystal microbalance (QCM); Second virial coefficient (B(22)); Static light scattering (SLS); Unfolded protein