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A New Approach to Forced Degradation Studies Using Anhydrous Conditions
Forced-degradation studies, or stress testing, of a drug substance are used to demonstrate the stability-indicating power of analytical procedures (1−5). While regulatory guidance documents emphasize the importance of these studies, the guidance does not specify pH, temperature ranges, specific oxidizing agents, or conditions to use. According to International Conference on Harmonization (ICH) Q1A (R2), the nature of the stress testing will depend on the individual drug substance (1). As such, the design of forced-degradation studies varies from company to company with general experimental designs focused on the four main pharmaceutically relevant degradation mechanisms: thermolytic, hydrolytic, oxidative, and photolytic (6−8). The choice of forced-degradation conditions is based on data from accelerated pharmaceutical studies and sound scientific understanding of the product’s decomposition mechanism (9−10). The experimental designs published to date, however, have only included entirely aqueous systems. The use of organic cosolvents is considered for cases where the drug substances are extremely insoluble, and inert organic cosolvents are selected to avoid reaction with the drug substance under a given set of stress conditions (5). In the case of a Type IIA prodrug (11), whereby water hydrolyzes the drug substance to give the active moiety, forced-degradation experiments using aqueous conditions will only give the degradation products for the active moiety. As a result, additional analytical methods must be used to characterize other potential degradants that could potentially form before hydrolysis of the drug substance to the active moiety. Alternatively, a single normal-phase chromatographic method could be employed where it was shown to be stability-indicating for the drug substance in the case of Type IIA prodrugs. The challenge for this approach is in demonstrating that the method is stability-indicating because traditional forced-degradation experiments, which use aqueous conditions, are incompatible. This article explores the technical challenges associated with conducting forced-degradation experiments under anhydrous conditions.
Anhydrous forced-degradation case study for C1
As the assay and impurity analysis of C1 is dependent on the solvent system, various analytical methods were developed to provide a complete analysis of the drug substance C1 and the active species C2. Forced-degradation studies were conducted on C1 using traditional aqueous conditions and analyzed by reversed-phase high-performance liquid chromatography (RP–HPLC). The degradation products found using this approach were the degradants of C2, the active species, but not the drug substance. Therefore, a second forced degradation study was conducted using anhydrous, reactive organic solutions and analyzed by normal-phase HPLC. The degradation products using this approach were degradants of both C1 and C2. By using anhydrous forced-degradation conditions, a normal-phase HPLC method was demonstrated to be stability-indicating for the drug substance.
Materials. C1 and C2 were synthesized by Millennium Pharmaceuticals (Cambridge, MA). Solvents and reagents, including anhydrous tetrahydrofuran (THF), non-anhydrous THF, hexane, pyridine, and trifluoroacetic acid (TFA) were all HPLC grade. The acetic acid, diisopropylamine and N,N-azobisisobutyronitrile (AIBN) used were analytical reagent grade. The cumene hydroperoxide used was technical grade (80%).
Instruments. The equipment included an Agilent 1100 HPLC equipped with a diode-array detector, chilled autosampler, online degasser, quaternary pump, and high-performance liquid chromatography-mass spectrometry. Photo-UV stability samples were stressed in a Suntest CPS chamber using a xenon lamp.
Method. The C1 drug substance and the active C2 species were separated by gradient using anhydrous (≤ 0.02% water) mobile phase on a cyano column. The sample was prepared by dissolving the C1 drug substance sample in an anhydrous (≤ 0.02% water) organic solvent mixture. The drug substance and active species were separated by the method and quantified by UV detection. The identity of C1 was confirmed by comparison of the C1 peak retention in the sample to that of the external C1 standard.
Degradation conditions. Degradation conditions evaluated included heat, acid, base, oxidation, and light. A stock solution of C1 was used to prepare each stressed solution. For each condition, a control without drug substance was included and treated the same way as the degraded samples. Photo controls included drug substance but were wrapped in aluminum foil to protect from light. The forced-degradation conditions used in this experiment (see Table I) include the following combinations:
Analytical considerations for study execution
Design of anhydrous conditions
Table II: Solubility of C1 in anhydrous, aprotic solvents.
Two different oxidative reagents were used in this study given that radical initiators and oxidative reagents can lead to different degradation products that may or may not be representative of the observed degradants for a product (12). Cumene hydroperoxide was chosen because it is a relatively stable organic peroxide, commercially available with reasonable purity, and readily soluble in organic solvents (13). The radical initiator, AIBN, was chosen because it can be a less reactive oxidant. Additionally, the use of THF in the AIBN stress solution helps to quench alkoxy radical activity that can interfere with the desired peroxy radical (autoxidative) chemistry (14). Interestingly, the same radical initiator reagent, AIBN, was used for anhydrous and aqueous forced-degradation experiments because it can be dissolved in both types of solvents. It also provided an opportunity for overlap in experimentation between the anhydrous and aqueous forced-degradation approaches.
Table III: Assessment and identification of reactive organic solvents/reagents for C1 forced-degradation experiments.
Based on the initial scouting stress conditions, optimized conditions (temperature and length of hold time) were used for the forced-degradation experiment on the C1 drug substance. The conditions were optimized to target 5% to 20% degradation of the main component. Generally, values anywhere between 5% to 20% degradation for the drug substance have been considered reasonable and acceptable for demonstrating specificity of chromatographic assays (15−17). In this case, a low concentration of diisopropylamine was needed to prevent excessive degradation of the main component. The results of the stress testing and overlay of chromatograms at the stress conditions are summarized in Table I and Figure 2.
Results and discussion
Using the anhydrous forced-degradation conditions, sufficient degradation of the C1 drug substance was achieved. Further analysis of the C1 samples stressed under anhydrous conditions was performed using mass spectrometry for major peak identification. The main degradation products from those anhydrous forced-degradation conditions were the hydrolysis degradation product and/or the oxidative degradation products. These degradation products were similar to the ones determined using traditional aqueous forced-degradation conditions; however, other degradation products were also observed in the anhydrous forced degradation conditions that were not observed in the aqueous forced-degradation study. Those additional impurities were identified and found to be additional degradation products of C1 that are detectable by the RP-HPLC method, but not routinely observed in release and stability testing because of the instability of the degradants in aqueous media.
In comparing the aqueous and anhydrous forced-degradation conditions for C1, a higher temperature was used to facilitate degradation under certain conditions to achieve the target degradation within a reasonable time. Different amounts of degradation for C1 were observed for both the aqueous and anhydrous studies, likely due to the type, strength, and mechanism of acid, base, oxidative, or light mediated degradation. Both approaches showed that the hydrolysis degradation product of C1 was the major degradant by heat, acid, and base degradation. Also, the oxidative degradation products of C1 were favored in the peroxide, radical initiated, and UV degradation samples. Both degradation approaches demonstrated that all relevant degradants of C1 result from the degradation of the active molecule, C2. As such, there were no new degradants identified in the anhydrous studies that were not accounted for in the aqueous study. However, exclusive to this approach, the anhydrous forced-degradation study identified moderate amounts of oxidative and hydrolytic degradation products of C1 that were otherwise transient and unstable in the aqueous stress conditions, although not pharmaceutically relevant. The anhydrous forced-degradation studies, therefore, may provide additional information to the aqueous studies traditionally performed for some molecules.
Another advantage includes the ability to better control the extent of degradation, and thus, more easily attain the target 5% to 20%. In the case of the C1 prodrug, the anhydrous forced-degradation study demonstrated that the normal-phase HPLC method was stability-indicating for the drug substance, hence, allowing a single analytical method to be used to characterize the stability of the prodrug.