New biological entities (NBEs, therapeutic proteins such as interferons or antibodies) are much more complex than new chemical entities (NCEs), the classic “chemical” active ingredients. First, they are much larger. The average molecular weight of antibodies is ~150,000 g/mol. Second, most NBEs contain three-dimensional structural elements — with the protein secondary and tertiary structure being the most prominent, but quaternary structures are also known for some. The 3D structures are essential for correct bioactivity (1), but they are not rigid, “frozen” structures. Most proteins show a certain structural flexibility, which enables their correct molecular interactions (e.g., of an antibody with its antigen or receptor). The extreme conformational states of that flexibility go from the native, correctly folded conformation to a completely denatured state, in which the protein adopts a more or less random conformation. Various interim conformational states can also be adopted.

PRODUCT FOCUS: ALL PROTEINS

PROCESS FOCUS: CHARACTERIZATION

WHO SHOULD READ: FORMULATORS, ANALYTICAL, AND PRODUCT DEVELOPMENT PERSONNEL

KEYWORDS: SPECTROSCOPY, DYNAMIC LIGHT SCATTERING, BIOPHYSICAL CHEMISTRY, ANTIBODIES, PROTEIN ASSOCIATION, PCS

LEVEL: INTERMEDIATE

The large size and complex structure of proteins make them prone to instabilities caused by chemical and biological degradations. In addition, a colloidal (physical) instability is often encountered, which can manifest as protein particle formation, aggregation, association, precipitation, and/or adsorption to materials used in, for example, medical devices, primary packaging, and tubing during filling. Colloidal instability and protein aggregate formation present a major problem for long-term storage stability, shipping, and handling (2).

Aggregates are defined as protein assemblies of higher molecular order formed by unfolded (denatured) and/or partially unfolded monomers. By contrast, protein associates are built up of native monomers (Figure 1) and can be redissolved to yield those native monomers again (1). The term protein particles refers to all protein-containing assemblies, independent of the nature and structure of the protein. Figure 1 represents schematically the formation of protein particles. Protein associates are formed by physical association of native protein monomers, whereas aggregates are made of irreversibly or partially denatured monomers. Protein association — the first step in the formation of protein associates — can be studied using the osmotic second virial coefficient (3,4). It depends strongly on the solution conditions of a formulation.

The size of protein particles varies from dimers to extremely large multimer units. As described below, they can be induced by the presence of nonproteinaceous (extrinsic) particles that act as nucleation sites. The reasons protein particles form in solution are manifold. Induction of physical or thermal stresses, for example, may change the secondary/tertiary structures and lead to denaturation or partial denaturation, followed by formation of particles. Excipients and other ingredients also strongly affect the colloidal stability of a liquid protein formulation — especially so-called “highly concentrated” liquid formulations (HCLFs, with protein concentration ~40–200 mg/mL) requested for subcutaneous application of antibodies, for example. So colloidal stability is a critical issue to consider.

Induction of aggregates may also be due to the presence of extrinsic particles. Chi et al. showed aggregate formation with recombinant human platelet-activating factor acetylhydrolase (rhPAF-AH) in the presence of silica particles (5). Others presented the appearance of protein aggregates induced by silicone oil (6,7). Recently, it was shown that stainless steel particles generated during a filling process using a piston pump can induce IgG particle formation (8). In recent years, the presence of protein particles has been intensively discussed because of their immunogenic potential (9).

A number of techniques are used to characterize protein particles: high-performance size-exclusion chromatography (HP-SEC), light-obscuration microscopy, nephelometry, analytical ultracentrifugation, and field-flow fractionation (FFF). Each has strength and limitations. Because of technical impacts on the samples, correlation of results obtained with different techniques is complicated and in some cases improper (10,11). Additionally, protein particles can cover an extremely broad size range, from a few nanometers up to a few micrometers the latter formed by millions of antibody monomer units. With this particle size range up to six orders of magnitude, no available technique can correctly analyze samples containing such a multitude of sizes. Techniques are especially scarce for subvisual particles. Among the techniques available for particle characterization, photon correlation spectroscopy (PCS) offers the advantage of detecting particles in a broad dynamic range from a few nanometers to a few micrometers (11,12,13).