Recent Advances in Studying Interfacial Adsorption of Bioengineered Monoclonal Antibodies

Abstract

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.

Keywords: antibody; co-adsorption; mAbs; neutron reflection; self-assembly; structural unfolding; surface adsorption; surfactant.

Figure 1

An illustration of a basic syringe system with a stainless steel needle, a glass barrel and lubricated rubber plunger.

Figure 2

(a) Molecular model of COE-3 taking after the native human IgG1κ. Side view, front view and bottom view projected from the space filled IgG1κ model. (b) The schematic depiction of the molecule, its key domains and the cleavage of the hinge to produce Fab and Fc fragments via papain digestion.

Figure 3

Simple schematic of J.A. Woollam 2000 M spectroscopic ellipsometer, including (from left to right) a QTH deuterium light source and a 75W xenon light source, followed by a fixed calcite Glan-Taylor polarizer. Next is a continuously rotating compensator then a fixed calcite Glan-Taylor polarizer analyser. Finally, a back-thinned silicon CCD array detector is used to measure UV and visible light and an InGaAs photodiode array detector is used to measure near infrared.

Figure 4

Simulated data showing increasing adsorbed amounts at the SiO₂/water interface. There is sensitivity to the adsorbed amount in the phase information, Δ, as well as a subtle but distinct shift in the Ψ data set. The simulation used a silicon substrate with a native oxide layer of 13Å and a Cauchy protein model in H₂O.

Figure 5

Schematic depictions of monoclonal antibodies (mAb) adsorption on surface of water: (a) null reflecting water (NRW) (determining the whole layer thickness), (b) H₂O:D₂O = 1:1 (close to the SLD of mAb, determining the thickness of the region above water) and (c) D₂O (determining the extent of immersion in water).

Figure 6

Schematic representation of mAb adsorption at the oil/water interface using (a) silicon as the substrate for the oil (excluding the silicon oxide layer for simplicity). There is very low contrast between the protein and other silicon matched components. (b) Sapphire is used as the substrate. The oil and other components are contrast matched to the substrate, this results in a much larger signal from the protein than from the silicon system.

Figure 7

Schematic diagram of protein adsorbed to the oil/water interface using a sapphire substrate. (a) H₂O water phase for information about the entire interface. (b) D₂O:H₂O 1:1 highlights mixing between the protein and the oil. (c) All the systems components (except the protein) contrast matched to sapphire to extract the adsorbed amount of protein at the interface.

Figure 8

The equilibrated amount of adsorption in mg/m² (a) and nmol/m² (b) plotted against concentration for Fc (▲), Fab (♦) and the whole mAb COE-3 (●). (c) A schematic representations of the surface adsorbed COE-3 layers, Fab represented in red and Fc in purple. Reproduced with permission from Z. Li, ACS Applied Materials & Interfaces, 2017.

Figure 9

Plots of thickness × SLD (τρ/10⁻⁶ Å⁻²) versus the concentration of surfactant (expressed as the fraction of CMC) for both h-Surf and d-Surf, with the concentration of COE-3 fixed at 50 ppm. The product from the binary mixture of d-Surf and COE-3 is marked in blue diamonds ((τρ)_d-Surf) and that from the mixture of h-Surf and COE-3 in red squares ((τρ)_h-Surf). The τρ data from d-Surf alone are shown in green triangles. Reproduced with permission from Z. Li, mAbs; published by Taylor & Francis, 2017.

Figure 10

Schematic depiction of COE-3 adsorption at 0.050 mg/mL (a) on its own with a thickness close to 50 Å and most part of the layer being immersed in water, (b) co-adsorption with nonionic PS 80 surfactant (blue heads) when its concentration is below 1/20 CMC. (c) As the surfactant concentration moves above 1/20 CMC, the mAb is completely expelled by the surfactant.

Figure 11

A schematic representation of the use of isotopic contrasts to highlight the structure of the interfacial layers formed by self-assembly of PS80-7EO on top of a COE-3 layer at the SiO₂/water interface. Schematic (a) depicts the system in D₂O solvent coupled with hydrogenated (H-PS80-7EO) nonionic surfactants, (b) depicts the system in CMAb (contrast matched to COE-3 with SLD = 2.56 × 10⁻⁶ Å⁻²) solvent coupled with hydrogenated (H-PS80-7EO) nonionic surfactants, (c) depicts the system in D₂O solvent coupled with head deuterated (D-Head PS80-7EO) nonionic surfactant and (d) depicts the system in CMAb (contrast matched to COE-3 with SLD = 2.56 × 10⁻⁶ Å⁻²) solvent coupled with head deuterated (D-Head PS80-7EO) nonionic surfactants. The model schematics were based on simultaneous fittings of 4 reflectivity profiles measured under the 4 isotopic contrasts at the SiO₂/water interface. COE-3 adsorption at 0.010 mg/mL mixed with 0.20 mg/mL protonated or deuterated PS80-7EO.