Aggregated proteins are a significant concern for biopharmaceutical products because they may be associated with decreased bioactivity and increased immunogenicity. Macromolecular protein complexes can trigger a patient's immune system to recognize the protein as “nonself ” and mount an antigenic response (55). Large macromolecular aggregates also can affect fluid dynamics in organ systems such as eyes (56).
Aggregation is a common problem encountered during manufacture and storage of proteins (16). The potential for aggregated forms is often enhanced by exposure of a protein to liquid–air, liquid–solid, and even liquid–liquid interfaces (57). Mechanical stresses of agitation (shaking, stirring, pipetting or pumping through tubes) can cause protein aggregation. Freezing and thawing can promote it as well. Solution conditions such as temperature, protein concentration, pH, and ionic strength can affect the rate and amount of aggregates observed. Formulation in sucrose can increase aggregation over time because of protein glycation when sucrose is hydrolyzed (58). The presence of certain ligands — including certainions — may enhance aggregation. Interactions with metal surfaces can lead to epitaxic denaturation, which triggers aggregate formation. Foreign particles from the environment, manufacturing process, or container–closure system (e.g., silicone oil) can also induce aggregation (21,59,60). Even handling protein products at compounding pharmacies can induce aggregation 10-fold above initially observed amounts (58).
The impact of aggregation on product potency varies based on the physiochemical attributes of each protein relative to its functional domains and the nature of the activity being measured. Enzymes such as urease and catalase can lose up to 50% of their potency after shaking, fibrinogen clotting activity is decreased after shear stress, and recombinant IL-2 and recombinant interferon activity is substantially affected by aggregation from shaking and shearing (2). Aggregation also affects the mass balance of protein solutions, decreasing the concentration of the target protein. Microaggregated subvisible particles generated anywhere in a manufacturing process can develop into larger particles over time as a product is stored (62). Bevacizumab drug product lost 50% of active IgG after manipulations at the pharmacy triggered significant growth of micron-sized particles in repackaged solutions (58).
Aggregates can be soluble or insoluble, reversible or irreversible, covalent or noncovalent (16). Soluble aggregates are usually reversible: e.g., by altered solution conditions such as changing temperature or osmotic strength or by mild physical disruption such as swirling or filtration. Insoluble aggregates are typically irreversible. Under vigorous physical disruption (e.g., agitation or freezing and thawing) or over time in storage, they can grow into particles that may eventually precipitate. Covalent aggregates form when monomeric proteins become chemically crosslinked, e.g., though disulfide bonds. Although covalent linkages are necessary to stabilize the native tertiary structure of most polypeptide proteins, those that form by degradation can produce undesired crosslinks between protein moieties, which can lead to irreversible aggregation. Noncovalent aggregates are formed when proteins associate and bind based on structural regions of charge or polarity. Because such associates are weak (relative to covalent linkages), they are sensitive to solution conditions and usually reversible.
Mechanism and Factors Involved: Because of the many physical and chemical manipulations required in upstream production and downstream processing, followed by formulation and filling operations, aggregation of protein biopharmaceuticals can be induced during nearly every step of the process including at hold points, shipping, and long-term storage (16,21). Agitation (e.g., shaking, stirring, and shearing) of protein solutions can promote aggregation at the air–liquid interfaces, where protein molecules may align and unfold, exposing their hydrophobic regions for charge-based association (2). Agitation-induced aggregation has been seen in numerous protein products, including recombinant factor XIII, human growth hormone, hemoglobin, and insulin (2). Minimizing foaming caused by agitation during manufacture (as well as during product use) may be critical to preventing significant loss of protein activity or generation of visible particulate matter (62).
Protein concentration also can promote aggregation, with or without agitation events. Results obtained from two PEGylated proteins and one Fc fusion protein demonstrated a direct correlation between protein concentration and aggregation under nonagitated (quiescent) conditions, but researchers found an inverse correlation between protein concentration and aggregation under conditions of shaking, vortexing, and simulated shipping (63).
Antimicrobial preservatives used in multidose formulations also can induce protein aggregation. For example, benzyl alcohol accelerates the aggregation of rhGCSF because it favors partially unfolded conformations of the protein (64). Increasing antimicrobial preservative levels may increase the hydrophobicity of a formulation and could affect a protein's aqueous solubility (62).
Phenol and m-cresol can considerably destabilize a protein: Phenol promotes formation of both soluble and insoluble aggregates, whereas m-cresol can precipitate protein (65).
Freezing and thawing — which can occur multiple times throughout production and use of protein therapeutics — can dramatically affect protein aggregation. Generation of water-ice crystals at a container's periphery (where heat transfer is greatest) can produce a “salting out” effect, whereby the protein and excipients become increasingly concentrated at the slower-freezing center of a container (21). High-salt and/or high-protein concentrations can result in precipitation and aggregation during freezing, which is not completely reversible upon thawing. The effect can be seen with thyroid-stimulating hormone: When stored at −80 °C, 4 °C, or 24 °C for up to 90 days, it remained stable, but when frozen to −20 °C it lost >40% potency in that period, which was attributed to subunit dissociation (66). Multiple freezing and thawing cycles can exacerbate that effect and lead to a cumulative impact on the generation and growth of subvisible and visible particulates. A change in pH can come from crystallization of buffer components during freezing. In one study, potassium phosphate buffers demonstrated a much smaller pH change on freezing than did sodium phosphate buffers (21).
Compendia currently limit the number of particles ≥10 μm and ≥25 μm in size that may be present in injectible pharmaceutical preparations (67,68). However, what levels of subvisible particles (<10 μm) such as protein oligomers are acceptable depends on the physiochemical characteristics and safety/efficacy profile of each product (2). Also, no standards are codified for visible particulates in protein pharmaceutics. Some biotechnology products have visual appearance specifications for drug solutions that include comments such as “essentially free of visible particulates” or “some translucent particles may be present” (69).
Preventive Measures: A protein solution typically can be stabilized against aggregation and precipitation by optimizing solution pH and ionic strength; adding sugars, amino acids, and/or polyols; and using surfactants (70). Comprehensive evaluation of optimal pH and osmotic conditions is a key element of formulation development to prevent protein aggregation or precipitation (71). Irreversible aggregation due to denaturation can be prevented with surfactants, polyols, or sugars (62).
In many cases, nonionic detergents (surfactants) are added to increase stability and to prevent aggregation. The protein–surfactant interaction is hydrophobic, so these compounds stabilize proteins by lowering the surface tension of their solution and binding to hydrophobic sites on their surfaces, thus reducing the possibility of protein–protein interactions that could lead to aggregate formation (72). The nonionic detergents Tween 20 and Tween 80 can prevent formation of soluble protein aggregates with surfactant concentrations below the critical micelle concentrations (CMC) (73). Polysorbate (Tween) 80 added to IgG solutions stabilized small aggregates and prevented them from growing into larger particles (74). Chelating agents also can be used to prevent metal-induced protein aggregation (62,70).