Overlooking subvisible particles in therapeutic protein products: gaps that may compromise product quality.

Therapeutic protein products provide unique and effective treatments for numerous human diseases and medical conditions. In many cases, these treatments are used chronically to slow disease progression, reduce morbidity and/or to replace essential proteins that are not produced endogenously in patients. Therefore, any factor that reduces or eliminates the effectiveness of the treatment can lead to patient suffering and even death. One means by which efficacy of therapeutic proteins can be compromised is by an immune response, resulting in antibody-mediated neutralization of the protein’s activity or alterations in bioavailability.1,2 For example, in the case of treatment of hemophilia A, neutralizing antibodies to Factor VIII can cause life-threatening bleeding episodes, resulting in significant morbidity and necessitating treatment with a prolonged course of a tolerance-inducing therapy to reverse immunity.3,4 In other cases, drug-induced antibodies to a therapeutic version of an endogenous protein can cross-react with and neutralize the patient’s endogenous protein. If the endogenous protein serves a non-redundant biological function, such an immune response can have devastating results. For example, pure red cell aplasia can result from neutralizing antibodies to epoetin alpha. 1,2 It is well established that protein aggregates in therapeutic protein products can enhance immunogenicity2, and such an effect is therefore an important risk factor to consider when assessing product quality. The purpose of this commentary is to accomplish the following: provide brief summaries on the factors affecting protein aggregation and the key aspects of protein aggregates that are associated with immunogenicity; emphasize the current scientific gaps in understanding and analytical limitations for quantitation of species of large protein aggregates that are referred to as subvisible particles, with specific consideration of those particles 0.1–10 μm in size; offer a rationale for why these gaps may compromise the safety and/or efficacy of a product; provide scientifically sound, risked based recommendations/conclusions for assessment and control of such aggregate species. Causes of Protein Aggregation Proteins usually aggregate from partially unfolded molecules, which can be part of the native state ensemble of molecules.5 Even though product formulations are developed to maximize and maintain the fraction of the protein molecules present in the native state, significant amounts of aggregates can form, especially over pharmaceutically-relevant time scales and under stress conditions. For example, exposure to interfaces (e.g., air-liquid and solid-liquid), light, temperature fluctuations or minor impurities can induce aggregation. Such exposure can occur during processing steps, as well as in the final product container during storage, shipment and handling. Furthermore, protein particles (visible and subvisible) can be generated from protein alone or from heterogeneous nucleation on foreign micro- and nanoparticles that are shed, for example, from filling pumps or product container/closures.6–8 The levels and sizes of protein particles present in a given product can be changed by many factors relevant to commercial production of therapeutic proteins. Such factors include a change in the type of filling pump during scale-up to commercial manufacturing, changes in formulation or container/closure, and even unintentional changes in the manufacturing process such as alterations in filling pump mechanical parameters or other unforeseen factors.8,9 Thus, unless appropriate quality controls are in place for subvisible particles, a product that was safe and effective in clinical trials may unexpectedly cause adverse events in patients after commercialization. Effects of Aggregate Characteristics on Immunogenicity From work on fundamental aspects of immunology and vaccine development, it is known that large protein assemblies with repetitive arrays of antigens, in which the protein molecules have native conformation, are usually the most potent at inducing immune responses.2,10,11 Furthermore, efforts to develop more effective vaccines have shown that adsorbing antigenic proteins to nano- or microparticles comprised of other materials (e.g., colloidal aluminum salts or polystyrene) can greatly increase immunogenicity.12,13 Applying these lessons to therapeutic protein products, it has been argued that large aggregates containing protein molecules with native-like conformation pose the greatest risk of causing adverse immune responses in patients.2 Thus, for example, particles of therapeutic proteins formed by adsorption of protein molecules onto foreign micro- and nanoparticles might be particularly prone to cause immunogenicity. These particles contain numerous protein molecules, and in the two examples published to date, the adsorbed protein molecules were shown to retain their native conformations.6,8 Unfortunately, lacking are published studies that comprehensively investigate the range of parameters that could influence immunogenicity of aggregates. Because each protein may differ in aggregate formation and consequences, factors that need to be investigated include but are not limited to type, amount and size of aggregates, as well as protein conformation in aggregates, on a case by case basis. Of course, other factors, particularly pertaining to patient status and treatment protocol, are also critical in determining the propensity to generate immune responses. These include immune competence of the patients, route of administration, and dosing frequency and duration. Given the consequences of aggregate-induced immunogenicity in patients, it is important to understand these issues and to reduce the risk to product quality for every therapeutic protein product. Because the exact characteristics and levels of protein aggregates that lead to an enhanced immune response are unclear and may differ among proteins, it is not possible to predict, a priori, the in vivo effects of different sizes, types or quantities of aggregates for therapeutic protein products. In such situations, careful analysis of the relationship between clinical performance and the presence of protein aggregates in relevant clinical trial material may help in the design of suitable control strategies that ensure product quality. However, the validity and utility such correlations are only optimized when the full spectrum of protein aggregate species are thoroughly characterized by multiple and orthogonal techniques.