Container Surfaces Control Initiation of Cavitation and Resulting Particle Formation in Protein Formulations After Application of Mechanical Shock.

Mechanical shock may cause cavitation in vials containing liquid formulations of therapeutic proteins and generate protein aggregates and other particulates. To test whether common formulation components such as protein molecules, air bubbles, or polysorbate 20 (PS20) micelles might nucleate cavitation, a high-speed video camera was used to detect cavitation in vials containing antibody formulations after application of controlled mechanical shock using a shock test. Higher concentrations of subvisible particles were found in formulations where cavitation had occurred. Bubbles trapped on vial surfaces were a primary site for cavitation nucleation; other potential cavitation nuclei were ineffective. The incidence of cavitation events observed after application of mechanical shock was lower in type I glass vials than in cyclic olefin polymer vials or in SiOPlas™ cyclic olefin polymer vials and correlated with the surface roughness of the different vials. To reduce the incidence of cavitation and the adsorption of mAb on glass-water and silicone oil-water interfaces and thus minimize protein damage due to cavitation, PS20, a common nonionic surfactant, was added to formulations. Addition of PS20 to formulations in glass and silicone oil-coated glass vials significantly reduced both incidence of mechanical shock-induced cavitation and the particle formation that resulted from cavitation events.

Subvisible Particles in IVIg Formulations Activate Complement in Human Serum.

When administered intravenously, various particles and nanomedicines activate complement, potentially leading to infusion reactions and other adverse drug reactions. Particles form within formulations of therapeutic proteins due to stresses incurred during shipping, handling, and administration to patients. In this study, IVIg solutions were stored in multiple types of vials and prefilled syringes and exposed to agitation and freeze-thaw stresses to generate particles. The stressed samples were added to human serum to determine whether these particles activated complement. Subvisible IVIg particles ranging in size between 2 and 10 microns activated complement in a fashion that was linearly dependent on the number of particles dosed, whereas little correlation was found between doses of larger particles (>10 microns) and complement activation. Activation of complement by subvisible particles of IVIg followed the alternative pathway, as shown by the release of complement cascade factor Bb and the production of the anaphylatoxins C3a and C5a without generation of C4a. The number and the morphology of subvisible particles formed depended on the applied stress, formulation, and on the container material. But the capacity of the 2- to 10-micron-sized particles to activate complement in human serum appeared to depend only on particle concentration.

Immunogenicity of Structurally Perturbed Hen Egg Lysozyme Adsorbed to Silicone Oil Microdroplets in Wild-Type and Transgenic Mouse Models.

Silicone oil microdroplets may act as adjuvants, promoting unwanted immune responses against both foreign and self-proteins. Proteins often unfold upon adsorption to silicone oil microdroplets, but it is unclear how such unfolding might affect the immune response. In this study, we found that hen egg lysozyme (HEL) readily adsorbed to silicone oil microdroplets and perturbed the conformation of HEL. We compared the immune response to injections of HEL formulated in the presence and absence of silicone oil microdroplets in both wild-type mice and transgenic littermates that express a soluble form of HEL (sHEL), thus rendering them immunologically tolerant to this nominal self-antigen. Following 2 subcutaneous injections of a HEL formulation containing silicone oil microdroplets, wild-type mice exhibited a stronger IgG1 antibody response against HEL compared to the response in wild-type mice that administered an oil-free HEL formulation. However, when HEL was subcutaneously administered to sHEL-transgenic mice, immunological tolerance to sHEL was not broken in the presence of silicone oil microdroplets. Thus, although structural perturbations in proteins adsorbed to silicone oil microdroplets may augment the immune response, in the case of endogenously expressed proteins, such structural perturbations may not be sufficient to result in a breach of immunological tolerance.