Figure 3. Tryptic Peptide Maps of the Model Protein DP Samples (a) Reference Material (b) H2O2–stressed (c) High-pH–stressed

The intact T5 fragment represents a glycopeptide with multiple glycoforms. On the peptide map it eluted as a broad, irregular-shaped peak in the retention time interval 67 to 70 min (Figure 3a). When treated with H₂O₂, this peptide, which contains two Met residues, formed a mixture of degradants with one or two Met-sulfoxides. This mixture of oxidized glycopeptides eluted over broad and overlapping retention intervals, which made chromatographic separation and quantitation of these species not possible.

Table 2. Quantitation of Intact and Sulfoxidized Peptides of the Model Protein Tryptic Map

With the exception of oxidation, other types of stress had little or no effect on specific binding as determined by BIACore and competitive ELISA (Table 1). At the same time, increase of in vitro bioactivity was observed for samples after high- and low-pH stress and UV light exposure. The results indicating sensitivity to light and elevated temperature exposure were used to determine handling, storage, and transportation conditions for the product.

CONCLUSIONS Stress testing studies are conducted to challenge the specificity of stability-indicating and impurity-monitoring methods as part of the validation protocol. Another major goal is to investigate degradation products and pathways. The results of the forced degradation studies are required to be included in a Phase III IND filing. We recommend that you start the study as early as possible to be able to provide valuable information that can be used to improve formulations and the manufacturing process.

The choice of stress conditions should be consistent with product decomposition under normal manufacturing, storage, and use conditions. Recommended stress factors include high and low pH, elevated temperature, photolysis, and oxidation. The extent of the stress applied in forced degradation studies should ensure formation of the desired amount (usually 10 to 20%) of degradation.