8.3 Physical degradation

Formulation similarity

unlike the small-molecule drugs that would be highly stable in most lyophilized formulations. Table 8.2 summarizes typical stability problems observed during protein formulation development and potential methods to solve each problem. The list does not represent the complexity of multiple problems that can be experienced with a given protein; as a result, the formulation research should be designed to handle each protein based on its unique stability profile.

Proteins degrade upon physical stresses of many types including hydrophobic surfaces, heating, lyophilization, reconstitution, contact with organic solvents, shaking and many other permutations, and combinations of physical and chemical factors. The final result of physical stress can be denaturation, adsorption on the container walls, precipitation, or aggregation. Aggregation is a common problem encountered during manufacture and storage of proteins. The potential for aggregated forms is often enhanced by exposure to a protein to liquid–air, liquid–solid, and even liquid–liquid interfaces. 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 the amount of aggregates observed. 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. Protein aggregates are formed by mechanisms such as domain swapping, strand association, edge–edge association, or beta strand stacking. The term aggregation usually refers to multimers of proteins, e.g., dimers, trimers, tetramers, all the way to large polymers. The aggregates can be noncovalent or covalent (disulfide-linked), and these can be present as fully soluble in a clear solution, partially insoluble in a turbid solution, or mostly insoluble as a precipitate that collects in the bottom of the container. Nonspecific protein-to-protein association resulting from interactions among solvent-exposed hydrophobic groups can also form aggregates. The covalent aggregation is not reversible. The weakly associated noncovalent aggregate can be reversible, and it usually follows the path of dimers to multimers; the strongly associated noncovalent aggregates are not reversible by dilution and may result in precipitation. Based on the size range, aggregates have been classified into the following categories: (a) submicron (<1 μm in size) particles, which are 293

Biosimilarity: The FDA Perspective

294 conventionally referred to as soluble particles; (b) subvisible particles (1–100 μm in size); and (c) visible particles (>100 μm in size).

There are several models of aggregation. In the native-to-unfolded-toaggregate model, denatured or unfolded molecules aggregate due to hydrophobic interactions. Aggregate formation driven by hydrophobic effect happen when the normally buried hydrophobic regions are exposed. The rate of reaction in this model increases with temperature since unfolding increases with temperature and reactions generally follow first-order kinetics.

In the model native-to-unfolded-to-aggregate wherein the intermediate yields the aggregate, the misfolded intermediates are often thermodynamically stable and can be part of the native-state ensemble. Therefore, aggregation is not an unnatural state of a protein and can occur even under conditions favoring the native state.

The mechanism of protein aggregation involves two stages: a nucleation process which is followed by growth of the nuclei in a critical mass. The level of aggregation can be monitored by the turbidity measurement. However, turbidity is not necessarily an indicator of aggregation. The assembly of initially native and folded proteins can result in irreversible nonnative structures that may contain high levels of nonnative intermolecular β-sheet structures. The onset, rate, and final morphology of the aggregate depend on solution conditions such as pH, salt species, salt concentration, cosolutes, excipients, and surfactants. The exact nature of an aggregate is a function of the relative intrinsic thermodynamic stability of the native state.

Because of the many physical and chemical manipulations required in upstream production and downstream processing, followed by formulation and filling operations, the aggregation of protein biopharmaceuticals can be induced during nearly every step of the process including at hold points, shipping, and long-term storage. 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 the charge-based association. Agitation-induced aggregation has been observed in numerous protein products, including recombinant factor XIII, human growth hormone, hemoglobin, and insulin. 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. The antimicrobial preservatives used in multidose formulations can also induce protein aggregation. For example, benzyl alcohol accelerates the aggregation of rhGCSF because it favors partially unfolded conformations of the protein. Increasing antimicrobial preservative levels may enhance the hydrophobicity of a formulation and could affect a protein’s aqueous solubility. Phenol and m-cresol can considerably destabilize a protein: Phenol promotes the formation of both soluble and insoluble aggregates, whereas m-cresol can precipitate protein.

Formulation similarity

Freezing and thawing that 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 the excipients become increasingly concentrated at the slowly freezing center of a container. The 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. Multiple freezing and thawing cycles can exacerbate that effect and lead to a cumulative impact on the generation and the 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. The causes of aggregation that are related to the process are summarized as follows:

• Fermentation/Expression: Inclusion bodies • Purification: Shear, pH, ionic strength • Filtration: Surface interaction, shear • Fill/Finish: Surface interaction, shear, contamination (e.g., silicone oil) • Freeze/Thaw: Cryoconcentration, pH changes, ice–solution interfaces • Shipping: Agitation, temperature cycling • Lyophilization: Cryoconcentration, pH changes, ice–solution interfaces, dehydration • Administration: Diluents, component materials and surfaces, leachables

Protein aggregation upon oxidation can result in a faster reaction and may show precipitates within a few minutes; it is also highly pH dependent. One of the most difficult parts of formulating proteins comes when they are dispensed in prefilled syringes that have silicone oil lubrication. A 10-fold variability in absorbance at 350 nm in proteins packaged in various brands of syringes is not uncommon. It depends on the quantity and the type of silicone used. A properly folded mAb, for example, can form an intermediate upon hydrophobic interaction with silicone in the syringe resulting in partial unfolding that will first yield to a soluble aggregate followed by a visible precipitation depending on the concentration of silicone to which the mAb is exposed. Subvisible and visible particles together are referred to as insoluble particles. Particles that belong to the size range of 0.1–1 μm are at times referred to as small subvisible particles. 295