Recent Advances in Studying Interfacial Adsorption of Bioengineered Monoclonal Antibodies.

Monoclonal antibodies (mAbs) are an important class of biotherapeutics; as of 2020, dozens are commercialized medicines, over a hundred are in clinical trials, and many more are in preclinical developmental stages. Therapeutic mAbs are sequence modified from the wild type IgG isoforms to varying extents and can have different intrinsic structural stability. For chronic treatments in particular, high concentration (≥ 100 mg/mL) aqueous formulations are often preferred for at-home administration with a syringe-based device. MAbs, like any globular protein, are amphiphilic and readily adsorb to interfaces, potentially causing structural deformation and even unfolding. Desorption of structurally perturbed mAbs is often hypothesized to promote aggregation, potentially leading to the formation of subvisible particles and visible precipitates. Since mAbs are exposed to numerous interfaces during biomanufacturing, storage and administration, many studies have examined mAb adsorption to different interfaces under various mitigation strategies. This review examines recent published literature focusing on adsorption of bioengineered mAbs under well-defined solution and surface conditions. The focus of this review is on understanding adsorption features driven by distinct antibody domains and on recent advances in establishing model interfaces suitable for high resolution surface measurements. Our summary highlights the need to further understand the relationship between mAb interfacial adsorption and desorption, solution aggregation, and product instability during fill-finish, transport, storage and administration.

Interfacial Adsorption of Monoclonal Antibody COE-3 at the Solid/Water Interface.

Spectroscopic ellipsometry (SE) and neutron reflection (NR) data for the adsorption of a monoclonal antibody (mAb, termed COE-3, pI 8.44) at the bare SiO2/water interface are compared here to the simulations based on Derjaguin-Landau-Verwey-Overbeek theory. COE-3 adsorption was characterized by an initial rapid increase in the surface-adsorbed amount (Γ) followed by a plateau. Only the initial rate of the increase in Γ was strongly correlated with the bulk concentration (0.002-0.2 mg/mL), with Γ at the plateau being about 2.2 mg/m2 (pH 5.5). Simulations captured COE-3 adsorption at equilibrium most accurately, the point at which the outgoing flux of molecules within the adsorbed plane matched the adsorption flux. Increasing the buffer pH from 5.5 to 9 increased Γ at equilibrium to ∼3 mg/m2 (0.02 mg/mL COE-3), revealing a dominant role for lateral repulsion between adsorbed mAb molecules. In contrast, increasing the buffer ionic strength (pH 6) reduced Γ, which was captured by simulations accounting for electrostatic screening by ions, in addition to mAb/SiO2 attractive forces and lateral repulsion. NR data at the same bulk concentrations corroborated the SE data, albeit with slightly higher Γ due to longer adsorption times for data acquisition; for example, at pH 9, Γ was 3.6 mg/m2 (0.02 mg/mL COE-3), equivalent to a relatively high volume fraction of 0.5. An adsorbed monolayer with a thickness of 50-52 Å was consistently determined by NR, corresponding to the short axial lengths of fragment antigen-binding and fragment crystallization and implying minimal structural perturbation. Thus, the simulations enabled a mechanistic interpretation of the experimental data of mAb adsorption at the SiO2/water interface.

Coadsorption of a Monoclonal Antibody and Nonionic Surfactant at the SiO2/Water Interface.

During the formulation of therapeutic monoclonal antibodies (mAbs), nonionic surfactants are commonly added to attenuate structural rearrangement caused by adsorption/desorption at interfaces during processing, shipping, and storage. We examined the adsorption of a mAb (COE-3) at the SiO2/water interface in the presence of pentaethylene glycol monododecyl ether (C12E5), polysorbate 80 (PS80-20EO), and a polysorbate 80 analogue with seven ethoxylates (PS80-7EO). Spectroscopic ellipsometry was used to follow COE-3 dynamic adsorption, and neutron reflection was used to determine interfacial structure and composition. Neither PS80-20EO nor C12E5 had a notable affinity for COE-3 or the interface under the conditions studied and thus did not prevent COE-3 adsorption. In contrast, PS80-7EO did coadsorb but did not influence the dynamic process or the equilibrated amount of absorbed COE-3. Near equilibration, COE-3 underwent structural rearrangement and PS80-7EO started to bind the COE-3 interfacial layer and subsequently formed a well-defined surfactant bilayer via self-assembly. The resultant interfacial layer comprised an inner mAb layer of about 70 Å thickness and an outer surfactant layer of a further 70 Å, with distinct transitional regions across the mAb-surfactant and surfactant-bulk water boundaries. Once formed, such interfacial layers were very robust and worked to prevent further mAb adsorption, desorption, and structural rearrangement. Such robust interfacial layers could be anticipated to exist for formulated mAbs stored in type II glass vials; further research is required to understand the behavior of these layers for siliconized glass syringes.

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

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.

Antibody adsorption on the surface of water studied by neutron reflection.

Surface and interfacial adsorption of antibody molecules could cause structural unfolding and desorbed molecules could trigger solution aggregation, resulting in the compromise of physical stability. Although antibody adsorption is important and its relevance to many mechanistic processes has been proposed, few techniques can offer direct structural information about antibody adsorption under different conditions. The main aim of this study was to demonstrate the power of neutron reflection to unravel the amount and structural conformation of the adsorbed antibody layers at the air/water interface with and without surfactant, using a monoclonal antibody 'COE-3' as the model. By selecting isotopic contrasts from different ratios of H2O and D2O, the adsorbed amount, thickness and extent of the immersion of the antibody layer could be determined unambiguously. Upon mixing with the commonly-used non-ionic surfactant Polysorbate 80 (Tween 80), the surfactant in the mixed layer could be distinguished from antibody by using both hydrogenated and deuterated surfactants. Neutron reflection measurements from the co-adsorbed layers in null reflecting water revealed that, although the surfactant started to remove antibody from the surface at 1/100 critical micelle concentration (CMC) of the surfactant, complete removal was not achieved until above 1/10 CMC. The neutron study also revealed that antibody molecules retained their globular structure when either adsorbed by themselves or co-adsorbed with the surfactant under the conditions studied.