Physical Stability

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In solution, the structure is not infinitely stable in the folded state (18,36), and therefore the structure is affected by production conditions, stress during preparation of the formulation, etc.

Denaturation

The term "denaturation" is used to denote "a process (or sequence of processes) in which the spatial arrangement of the polypeptide chains within the molecule is changed from that typical of the native protein to a more disordered arrangement" (33), where "spatial arrangemenf' can be replaced by configuration, conformation, or state of folding. The effect from denaturation can be alterations of specific parameters, such as solubility, loss of activity, or unfolding. In the following sections, denaturation and unfolding are used interchangeably.

One way to describe the unfolding process is the two-state model shown in Figure 4. It is an equilibrium, a single-transition step between the folded native (N) and the disordered, unfolded or denatured (D) species (36-38). An intermediate step, the formation of possible intermediates (I), can be present between the transformation from N to D. The intermediate state (I) has often been described as the molten globule, for example, for growth hormone (39). It is a stable compact, partly denatured species, which retains some ordered secondary structure but not the tertiary structure of the native protein (35,36,40,41). The aggregate (A) formed may occur from irreversible changes to the unfolded species (18,42^14).

Reversible denaturation also occurs, but the resulting structure formed may not always regain the biological activity (40). Many conditions lead to denaturation and not

Native (N)

Intermediate (I)

Denatured (D)

Native (N)

Intermediate (I)

Denatured (D)

\ . /

Fibril

Aggregate (A)

Fibril

Figure 4 The protein aggregation process. It is an equilibrium between the folded native (N) and the disordered, unfolded or denatured (D) species. An intermediate step, the formation of possible intermediates (I), can be present between the transformation from N to D. The aggregate (A) formed may occur from irreversible changes to the unfolded species and can result in formations of fibrils (F).

all proteins respond to these conditions in a similar manner. During production for example, proteins do undergo denaturation-renaturation cycles during extraction and purification, and proteins can be unfolded by various factors such as temperature, pH, and pressure. In addition, the characteristics of the denatured state can vary significantly (25). Some of the frequently applied denaturing conditions include dénaturants such as, urea or guanidine hydrochloride, heat or other types of stress (45-47).

Aggregation and Fibrillation

Aggregation is used to describe either soluble or insoluble protein assemblies caused by either covalent or noncovalent interactions. The noncovalent interactions can occur between folded; associated, or unfolded proteins; aggregation. This self-association can occur because of changes in the environment such as pH, protein concentration, ionic strength, etc. (25). Aggregates on the other hand, are formed from unfolded or partly unfolded proteins (48). The aggregate formation may involve the formation of covalent bonds, for example, disulfide shuffling, or the formation of noncovalent bonds, for example, hydrophobic interactions (49), where the noncovalent aggregate can be disrupted by denaturing agents, for example, sodium dodecyl sulphate or guanidine hydrochloride (18).

These aggregates contain normative structures, for example, intermolecular P-sheet (50), and can cause irreversible changes to the unfolded species, such as precipitation, aggregation, or fibrillation (18,35,43), where precipitation is the macroscopic equivalent of aggregation (40,42). Aggregation is dependent not only on the protein concentration but also on the stress caused by shaking (49,51). The structure of the aggregated proteins can be more or less well defined, and an example of well-defined structures is the formation of insulin fibrils (52,53).

Adsorption

Apart from the unfolding of the protein, physical stability or degradation also includes undesirable adsorption to different surfaces and interfaces, which can induce unwanted

Adsorption Unfolding

Figure 5 Changes of the conformation of proteins upon adsorption to surfaces. An example of the structural changes, where insulin is believed to adapt to the surface by changing the overall structure exposing the more hydrophobic residues buried in the interior of the protein.

structural changes in the solution (40). Upon adsorption at the surface, the protein structure undergoes a change from its globular configuration in solution to an extended chain structure, that is, surface denaturation (34,35,45). Proteins are amphiphilic molecules that adsorb at interfaces. They are also flexible molecules that adapt to their surroundings, accommodating changes in the environment that they are exposed to at the interface. Proteins readily adsorb at the interface in a manner dependent on bulk concentration, the diffusion coefficient through the solvent, affinity toward the interface, time, and available surface area (54,55). Upon adsorption, proteins may unfold, and this causes a rearrangement in the secondary and tertiary structure (31,56,57). Some proteins exhibit larger resilience against the structural rearrangement at the interface than others; these are often referred to as "hard" while those more structurally labile proteins are referred to as "soft" (58). The soft and less rigid proteins, for example, bovine serum albumin (BSA), unfold almost completely upon adsorption to surfaces (56,58).

An example of the structural changes is shown in Figure 5, where insulin is believed to adapt to the surface by changing the overall structure exposing the more hydrophobic residues buried in the interior of the protein (18,56,57,59,60).

Various surfaces occur during the production, purification, and preparation of proteins as well as in the formulation process, for example, exposure to glass surfaces in vials and air-liquid interfaces in the formulation. All these types of surfaces have various adsorption properties. A major distinction is made between hydrophilic and hydrophobic surfaces as well as air-liquid, liquid-liquid, and solid-liquid surfaces. Naturally, the type of interface also influences the observed structural changes (56,60,61).

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