Importance of the dynamics of adsorption and of a transient interfacial stress on the formation of aggregates of IgG antibodies

Abstract

It is common knowledge that aggregation of proteins may occur in aqueous solutions under mechanical stress (shaking or high shear), even in solutions that are stable at rest. Addition of surfactants is a practical generic means to prevent this stress-induced aggregation (e.g. in formulations of therapeutic proteins), which suggests that interfaces contribute to destabilization. We studied here the role of interfacial stress by applying brief mechanical impacts on air-water interface, in presence or absence of surfactants, in solutions of immunoglobulin G (IgG), a class of proteins of high importance to the developments of new therapeutics. A variety of surfactants was tested including the neutral ones Tween80, C10-C14 foscholines, alkylaminoxide, surfactin, and two ionic ones TTAB and lauroylsarcosine sodium salt. We determined the presence of aggregates in solution by light scattering. Irrespective of the type of antibody, either human polyclonal or a monoclonal one, we show that the amount of aggregated IgG increases in proportion to the number of impacts on the interface. In absence of stress, we recorded images of oblate aggregates of IgG (ca. 12 nm height and 200-1200 nm in diameter) present at the air-water interface (fluorescence microscopy using anti-Fab or anti-Fc markers, and AFM scans after transfer on freshly cleaved mica). Our results evidence that aggregates are formed at air-water interface, and are brought in solution by transient stresses applied on the water surface. Rupture of interfacial films is an important source of aggregates in solution. Finally, the role of surface dynamics in the protection brought by surfactants is discussed based on comparison of protective efficiencies with dynamic surface tension properties (measured by the maximum bubble pressure method). Our work indicates that better protection is conferred by surfactants showing the faster interfacial dynamics, which corresponds also to conditions of faster lowering of the interfacial energy at short time scale.

Microparticles and Nanoparticles Delivered in Intravenous Saline and in an Intravenous Solution of a Therapeutic Antibody Product

“…The sloughing off and/or mechanical rupture of such films leads to subvisible particles in solution. 21,22 Protein particle formation in IV bags due to protein adsorption to interfaces was also suggested by Kumru, et al , and they observed a reduction in protein particles in the presence of polysorbate 20, an effect attributed to inhibition of protein adsorption to the bag walls and to the air-water interface. 14 …”

Visible and Sub-visible Particle Formation for a Model Bioconjugate

“…While visible bubble formation was minimal at 1 mg/ ml, the contribution of air bubbles to particle formation cannot be entirely ruled out. The disruption of air bubbles could cause additional disruption of the air-water interface that leads to particle formation, as has been demonstrated by disruption of the hydrophobic interface previously (18). An IA of >∼305 mm 2 was found to promote particle formation for this bioconjugate under the studied experimental conditions and time scales.…”

“…Proteins migrate to the air-water interface (11) and unfold (12) causing aggregation and particle formation (13,14); data supporting this concept can be found in the literature (8)(9)(10). Additionally, agitation may be necessary for induction (10,15,16) and can synergistically worsen aggregation (17,18). …”

“…An IA of >∼305 mm 2 was found to promote particle formation for this bioconjugate under the studied experimental conditions and time scales. It is well known that the airwater interface promotes aggregate and particle formation (8,(18)(19)(20), and agitation and/or rupture of the protein gel at the hydrophobic air-water interface leads to increased aggregation (16,17,21). In addition to the presence of an airwater interface, the air-water IA is equally important and a critical IA may promote or inhibit particle formation depending on the therapeutic molecule.…”

“…In addition to the presence of an airwater interface, the air-water IA is equally important and a critical IA may promote or inhibit particle formation depending on the therapeutic molecule. The addition of surfactants at levels near or above the CMC may be used to prevent this air-water interface and agitation-induced particle formation (8,18,(22)(23)(24). Morphological differences in the VPs are in line with previously reported literature (25)(26)(27).…”