The dynamics of a protein aggregate mixture are complex and require multiple analytical methods for detection, evaluation, and monitoring. A successful assessment of aggregates in protein-based pharmaceuticals rests on a science-driven, risk-based program that evaluates the protein stability profile and determines the impact that a given aggregate mixture will have on the safety and efficacy of the drug product during its lifecycle. This article provides a regulatory perspective on aggregates in protein-based pharmaceuticals, including their characterization, detection methods, and various control strategies that have been implemented by manufacturers.
Aggregates in biologic products differ in origin, type, and size, and are caused by multiple factors. Of particular interest to regulatory agencies are aggregates that have the potential to enhance immune responses causing adverse clinical effects, or aggregates that may compromise the safety and efficacy of the drug product. Enhanced immune responses to protein aggregates have been reported in animal and clinical studies.1–4 Although classical immune responses to proteinaceous material foreign to humans are expected, the immune system may mount a strong response to aggregated protein preparations that have endogenous counterparts through a tolerance breakdown mechanism. In the mechanism of tolerance breakdown, protein aggregates may serve as facilitators in the formation of protein complexes that trigger B cell production of antibodies against the protein, independent of T-helper cell recruitment. The basis for this type of response comes from the immunon concept, in which antigen presented as a polymeric structure with more than 10 haptens, spaced 5–10 nm apart in a viral-like particle organization, and sized above 100 kDa, can overcome immune tolerance.5,6 Such a scenario may account for the unexpected neutralization of the endogenous protein, and have a profound clinical effect. High molecular weight (HMW) aggregates that conserve most of the native configuration of their monomer counterpart, and that can nucleate haptens in this way, are of most concern for this type of mechanism.7 Alternatively, aggregates displaying non-native protein conformations may be seen by the immune system as neoantigens, which could trigger antibody formation. 7 This article provides a regulatory perspective on aggregates in protein-based pharmaceuticals, their characterization, detection methods, and various controls that have been implemented by drug manufacturers.
CLASSIFICATION OF AGGREGATES
The classification of aggregates is a difficult task and currently, there is no comprehensive classification available. The difficulty resides in the multiple categories within which aggregates can be grouped. Table 1 lists the most common aggregate categories found in biopharmaceuticals. Dimers, reversible aggregates, covalent aggregates, and particulates are all terms used and, for lack of a better classification, help in understanding the type of entity being discussed, but also add to the confusion in the literature.
An alternative classification of aggregates could be based on their sizes, as this may have a direct correlation to potential adverse clinical outcomes. Aggregate size ranges from the soluble dimers and other multimers (approximately 5–10 nm in apparent globular diameter) including high molecular weight (HMW) aggregates through nucleated aggregates that can be either soluble or insoluble, to larger, insoluble species identified as subvisible and visible particulates (approximately 20–50 µm in apparent globular diameter). From the soluble aggregates group, the larger ones such as the HMW species may be more capable of eliciting immunogenic responses that could have an adverse clinical outcome.7 With respect to their molecular weight, aggregates of sizes above 102 kDa may deserve more careful evaluation based on their potential for undesirable immunogenic responses.5,6