A novel neurotrophic factor, human mesencephalic astrocyte-derived neurotrophic factor (hMANF), is being considered a therapeutic agent for a variety of diseases. However, little, if anything, has been reported about its stability. A preformulation study was conducted to assess the stability of hMANF as a function of pH and temperature. In addition, the effects of buffers and other excipients were evaluated as well. While the chemical and physical stability of hMANF decreases near pH 4, overall, the protein appears to be quite stable, especially near pH 6. Both histidine and phosphate appear to be suitable buffers in this pH range. Some loss of stability was noted above pH 6.5 as well. The stability profile of hMANF was comparable at 1 and 10 mg/mL. The decreased stability at acidic pH is correlated with the loss of the native α-helical conformation, as shown by FTIR spectroscopy. These studies indicate that hMANF is quite stable near pH 6, and formulations capable of exhibiting adequate long-term stability in aqueous solutions should be possible.

While asymmetrical flow field-flow fractionation (AF4) has been widely used for separation of high molecular weight species and even particles, its ability to resolve lower molecular weight species has rarely been explored. Over the course of many projects, we have discovered that AF4 can be an effective analytical method for separating peptides from oligomers and higher molecular weight aggregates. The methodology can be used even for peptides as small as 2 kD in molecular weight. Using multi-angle laser light scattering (MALLS) detection, accurate masses of the parent peptide can be obtained, provided accurate extinction coefficients are provided. It was shown that AF4 can be stability-indicating, suggesting that AF4-MALLS may be a suitable alternative to the use of SEC to monitor the aggregation of peptides.

To 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.

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.

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.

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.

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.

The original article to which this erratum refers was published in 2012; 101:81–91. In the original version of this article, the figure captions were incorrect with the corresponding figures. That error has been corrected below. We regret any confusion this may have caused.[Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9]Figure 2Correlation of peak height of the a− band with the sucrose to protein weight ratio.View Large Image Figure ViewerDownload (PPT)Figure 3Orientation of the exciton bands for idealized β-sheets, resulting in the low frequency a− component near 1640 cm−1 and the high frequency a+ band near 1695 cm−1.View Large Image Figure ViewerDownload (PPT)Figure 4Frequency of the a− band as a function of the sucrose to protein ratio, both in the absence and presence of sorbitol (5% by weight relative to sucrose). The black bar marks the average frequency for the a− band across all of the formulations.View Large Image Figure ViewerDownload (PPT)Figure 5Frequency of the a+ band as a function of the sucrose to protein ratio, both in the absence and presence of sorbitol (5% by weight relative to sucrose).View Large Image Figure ViewerDownload (PPT)Figure 6Width of the a+ and a− bands as a function of the sucrose to protein ratio, both in the absence and presence of sorbitol (5% by weight relative to sucrose).View Large Image Figure ViewerDownload (PPT)Figure 7Correlation of the peak height of the a− band with the degradation rate measured by SEC. This reflects the stability with respect to soluble aggregate formation.View Large Image Figure ViewerDownload (PPT)Figure 8Correlation of the initial peak height of the a− IR band to the total particle count (or subvisible particles) per mL as measured by MFI.View Large Image Figure ViewerDownload (PPT)Figure 9Correlation of the enthalpy of relaxation and the bandwidth of the a− peak.View Large Image Figure ViewerDownload (PPT) Structure, Stability, and Mobility of a Lyophilized IgG1 Monoclonal Antibody as Determined Using Second-Derivative Infrared SpectroscopyJournal of Pharmaceutical SciencesVol. 101Issue 1PreviewThere 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. Full-Text PDF

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.

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.

There have been significant advances in the formulation and stabilization of proteins in the liquid state over the past years since our previous review. Our mechanistic understanding of protein-excipient interactions has increased, allowing one to develop formulations in a more rational fashion. The field has moved towards more complex and challenging formulations, such as high concentration formulations to allow for subcutaneous administration and co-formulation. While much of the published work has focused on mAbs, the principles appear to apply to any therapeutic protein, although mAbs clearly have some distinctive features. In this review, we first discuss chemical degradation reactions. This is followed by a section on physical instability issues. Then, more specific topics are addressed: instability induced by interactions with interfaces, predictive methods for physical stability and interplay between chemical and physical instability. The final parts are devoted to discussions how all the above impacts (co-)formulation strategies, in particular for high protein concentration solutions.’

This chapter contains sections titled: Introduction Composition-Based Approaches Sequence-Based Algorithms Chemometric Methods Lattice Theory and Coarse-Grained Models Mutagenesis Studies Antibody Frameworks Hot Spots and Gatekeepers Conformational Switches Summary References