Forced Degradation Studies: Regulatory Considerations and Implementation

Stress testing studies are conducted to challenge specificity of stability-indicating and impurity- monitoring methods as part of validation protocol.

Jul 01, 2005

By Michael Kats, Ph.D.

BioPharm International

Volume 18, Issue 7

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EXPERIMENTS It is one thing to be theoretical and quite another to implement the action plan. This set of stress studies experiments shows the application in practice. The model protein is a covalent homo-dimer (AA) containing 356 amino acids in each chain with a total molecular weight ~92,000 Da of which ~14,000 Da are attributable to carbohydrate attachments.

Table 1. Summary of Analyses of the Stressed Model Protein Drug Substance

We optimized five stress conditions including elevated temperature, UV-VIS (320 to 400 nm) light exposure, H₂O₂-induced oxidation, and high and low pH to generate partial degradation (10 to 30%) of model protein via various degradation pathways. DS buffer solution contained 50 mM sodium chloride and 25 mM phosphate buffer, pH 7.5. In addition, reconstituted DP contained 100 mg/mL of maltose. The purposely degraded samples were used to challenge the ability of stability-indicating methods to separate, detect, quantify, and identify de-gradants. Table 1 is a summary of the results of analytical tests.

Figure 1. SDS-PAGE Analysis of Reduced (R) and Non-reduced (NR) Model protein DP Samples Stressed with UV light and Low pH Markers: Lane 1, Blank: Lanes 2, 7 and 12; Reference material: Lanes 3(R), 6(R), 8(NR) and 11(NR), Low pH stressed: Lanes 4(R) and 9(NR), UV stressed: Lanes 5(R) and 10(NR).

Results from SDS-PAGE are shown in Figure 1. The non-reduced model protein produced a main broad band at ~100 kDa, which represents the intact AA monomer. The reduced subunit (A) produced a main broad band at ~50 kDa. The electrophoretic band migration of the light-stressed protein exhibited additional bands of higher molecular weight species on both reduced and non-reduced SDS gels, indicating formation of higher-molecular-weight covalent aggregates (Figure 1, lanes 5 and 10). Other types of forced degradation did not have a noticeable effect on the protein migration in the gels.

Figure 2. Size Exclusion Chromatography of the Stressed Model Protein

The SEC method separated and quantitated AA monomer, multimeric forms, and lower-molecular-weight species. SEC exhibited almost exclusively an AA form of the intact molecule, usually 99.2 to 99.5% (Figure 2, curve 1). Analyses of the stressed samples demonstrated the presence of elevated amounts of multimeric species for every type of the applied stress condition. The extent of aggregation ranged from 2.6% for the H₂O₂-oxidized sample to 28% for low-pH-stressed protein. Some differences in the relative abundance of higher molecular mass species may indicate different pathways of aggregate formation.

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