Robert W. Payne

STABILITY OF PROTEIN PHARMACEUTICALS: AN UPDATE

In 1989, Manning, Patel, and Borchardt wrote a review of protein stability (Manning et al., Pharm. Res. 6:903–918, 1989), which has been widely referenced ever since. At the time, recombinant protein therapy was still in its infancy. This review summarizes the advances that have been made since then regarding protein stabilization and formulation. In addition to a discussion of the current understanding of chemical and physical instability, sections are included on stabilization in aqueous solution and the dried state, the use of chemical modification and mutagenesis to improve stability, and the interrelationship between chemical and physical instability.

Colloidal behavior of proteins: effects of the second virial coefficient on solubility, crystallization and aggregation of proteins in aqueous solution.

There has been an increasing awareness that proteins, like other biopolymers, are large enough to exhibit colloidal behavior in aqueous solution. Net attractive or repulsive forces have been found to govern important physical properties, such as solubility and aggregation. The extent of intermolecular interactions, usually expressed in terms of the osmotic second virial coefficient, B, is most often measured using static light scattering. More recently, self-interaction chromatography (SIC) has emerged as a method for rapid determination of B in actual formulations, as it uses much less protein and has higher throughput. This review will summarize the relationship of B to crystallization, solubility, and aggregation of proteins in aqueous solution. Moreover, the capability of SIC to obtain B values in a rapid and reproducible fashion will be described in detail. Finally, the use of miniaturized devices to measure B is presented.

Product development issues for PEGylated proteins

Covalent attachment of poly(ethylene) glycol (PEG) groups to proteins, a process commonly called PEGylation, is often used to improve the performance of a protein in vivo. To date, at least eight such PEGylated peptide and protein conjugates have been approved as therapeutic agents and many more have undergone clinical trials. This review examines PEGylation from the perspective of developing a commercially viable drug product. The first section focuses on obtaining a pure and well-characterized drug substance. The latter section discusses formulation and manufacturing issues, with an emphasis on analytical methodology that provides the most detailed description of the purity and stability of PEGylated proteins.

Second virial coefficient determination of a therapeutic peptide by self‐interaction chromatography

Self‐interaction of macromolecules has been shown to play an important role in a number of physical processes, including crystallization, solubility, viscosity, and aggregation. Peptide self‐interaction is not as well studied as for larger proteins, but should play an equally important role. The osmotic second virial coefficient, B, can be used to quantify peptide and protein self‐interaction. B values are typically measured using static light scattering (SLS). Peptides, however, do not scatter enough light to allow such measurements. This study describes the first use of self‐interaction chromatography (SIC) for the measurement of peptide B values because SIC does not have the molecular size limitations of SLS. In the present work, SIC was used to measure B for enfuvirtide, a 36‐amino acid therapeutic peptide, as a function of salt concentration, salt type, and pH. B was found to correlate strongly with solubility and apparent molecular weight. In general, the solubility of enfuvirtide increases with pH from 6 to 10 and decreases as the salt concentration increases from 0 to 0.5M for three different salts. The effect of peptide concentration on B was also investigated and shown to have a significant effect, but only at high concentrations (>80 mg/mL).

Role of Buffers in Protein Formulations

Buffers comprise an integral component of protein formulations. Not only do they function to regulate shifts in pH, they also can stabilize proteins by a variety of mechanisms. The ability of buffers to stabilize therapeutic proteins whether in liquid formulations, frozen solutions, or the solid state is highlighted in this review. Addition of buffers can result in increased conformational stability of proteins, whether by ligand binding or by an excluded solute mechanism. In addition, they can alter the colloidal stability of proteins and modulate interfacial damage. Buffers can also lead to destabilization of proteins, and the stability of buffers themselves is presented. Furthermore, the potential safety and toxicity issues of buffers are discussed, with a special emphasis on the influence of buffers on the perceived pain upon injection. Finally, the interaction of buffers with other excipients is examined.

Structure, stability, and mobility of a lyophilized IgG1 monoclonal antibody as determined using second-derivative infrared spectroscopy.

There are many aspects of stabilization of lyophilized proteins. Of these various factors, retention of native structure, having sufficient amount of stabilizer to embed the protein within an amorphous matrix, and dampening β-relaxations have been shown to be critical in optimizing protein stability during storage. In this study, an IgG1 was lyophilized with varying amounts of sucrose. In some formulations, a small amount of sorbitol was added as a plasticizer. The structure of the protein in dried state was monitored using infrared (IR) spectroscopy. The IR spectra indicated increasing retention of the native structure, which correlated with stability as indicated by size-exclusion chromatography as well as micro-flow imaging. Maximal stability was achieved with a 2:1 mass ratio of sucrose to protein, which is more than that would be expected based on earlier studies. Analysis of both high and low frequency bands associated with intramolecular β-sheet structure provides additional information on the structure of antibodies in the solid state. Finally, there is a correlation between the bandwidth of the β-sheet bands and the enthalpy of relaxation, suggesting that amide I bands can provide some indication of the degree of coupling to the sugar matrix, as well as structural heterogeneity of the protein.

Stability of lyophilized sucrose formulations of an IgG1: subvisible particle formation.

Eight lyophilized formulations of a IgG1 monoclonal antibody (MAb) were prepared containing increasing levels of sucrose. In addition, three of the formulations had sorbitol added at a level of 5% w/w relative to sucrose. The samples were stored for up to 4 weeks at 40°C, which is well below the Tg. Upon reconstitution, the levels of subvisible particles were measured using microflow imaging (MFI). The formulation containing no sucrose contained exceedingly high levels of subvisible particles, accounting for as much as 25% of the weight of the protein. Addition of sucrose markedly decreased the number of subvisible particles, with the maximal sucrose:protein weight ratio being 2:1 (the highest level tested). Addition of sorbitol further decreased subvisible particle levels, even for formulations where the sucrose:protein ratio was relatively high. This suggests that even small amounts of a plasticizer like sorbitol can improve the storage stability of a lyophilized antibody formulation, probably by dampening β-relaxations within the amorphous glass.

Structural Characteristics of Short Peptides in Solution

Short peptides are important biopharmaceuticals as agonistic or antagonistic ligands, aggregation inhibitors, and vaccines, as well as in many other applications. They behave differently from globular proteins in solution. Many short peptides are unstructured and tend to aggregate and undergo structural transition in response to changes in solvent environment, including pH, temperature, ionic strength, presence of organic solvents or surfactants, and exposure to lipid membranes. Such structural transitions are often associated with fibril or β-amyloid formation. These structural characteristics of short peptides have drastic impact on their function, immunogenicity, and storage stability.

Stability of lyophilized teriparatide, PTH(1-34), after reconstitution

The peptide teriparatide, also known as parathyroid hormone (1-34), PTH(1-34), was developed for intranasal delivery, requiring extended stability of the reconstituted product for up to four weeks at room temperature. Lyophilized formulations of PTH(1-34), containing glycine and trehalose and using lactate as the buffer, are stable for months upon storage. However, the physical stability of the peptide after reconstitution unexpectedly varied considerably, depending on peptide concentration and storage temperature, with precipitation seen within two to four weeks in some samples. By comparison, equivalent samples that did not undergo lyophilization did not display any precipitation upon storage in the liquid state for as long as twelve weeks. PTH(1-34) appears to adopt a higher order structure that is perturbed by the combined stresses of freezing and drying, leading to greater propensity to aggregate, which is accentuated at higher peptide concentrations and at higher temperatures. The precipitation seems to be correlated with increased amounts of subvisible particles. This study shows the importance of peptide conformation in long-term stability and illustrates the ability of lyophilization to cause increased propensity to aggregate, even in a peptide.

Denaturation and Aggregation of Interferon-τ in Aqueous Solution

PurposeTo evaluate the different degrees of residual structure in the unfolded state of interferon-τ using chemical denaturation as a function of temperature by both urea and guanidinium hydrochloride.MethodsAsymmetrical flow field-flow fractionation (AF4) using both UV and multi-angle laser light scattering (MALLS). Flow Microscopy. All subvisible particle imaging measurements were made using a FlowCAM flow imaging system.ResultsThe two different denaturants provided different estimates of the conformational stability of the protein when extrapolated back to zero denaturant concentration. This suggests that urea and guanidinium hydrochloride (GnHCl) produce different degrees of residual structure in the unfolded state of interferon-τ. The differences were most pronounced at low temperature, suggesting that the residual structure in the denatured state is progressively lost when samples are heated above 25°C. The extent of expansion in the unfolded states was estimated from the m-values and was also measured using AF4. In contrast, the overall size of interferon-τ was determined by AF4 to decrease in the presence of histidine, which is known to bind to the native state, thereby providing conformational stabilization. Addition of histidine as the buffer resulted in formation of fewer subvisible particles over time at 50°C. Finally, the thermal aggregation was monitored using AF4 and the rate constants were found to be comparable to those determined previously by SEC and DLS. The thermal aggregation appears to be consistent with a nucleation-dependent mechanism with a critical nucleus size of 4 ± 1.ConclusionChemical denaturation of interferon-τ by urea or GnHCl produces differing amounts of residual structure in the denatured state, leading to differing estimates of conformational stability. AF4 was used to determine changes in size, both upon ligand binding as well as upon denaturation with GnHCl. Histidine appears to be the preferred buffer for interferon-τ, as shown by slower formation of soluble aggregates and reduced levels of subvisible particles when heated at 50°C.