senior scientist in analytical development, small molecules at Millennium Pharmaceuticals,Cambridge, MD

Laila Kott

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


In this case study, drug substance C1 was used because it is classified as a Type IIA prodrug. The drug substance C1 hydrolyzes under aqueous conditions to give the biologically active species C2. When C1 is dissolved in anhydrous, aprotic solvents, the molecule remains in its original form. When dissolved in aqueous systems, however, C1 converts into C2 as described in 

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. 

 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 

-azobisisobutyronitrile (AIBN) used were analytical reagent grade. The cumene hydroperoxide used was technical grade (80%). 

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. 

 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 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 

Control solution: 1 mg/mL C1 in 40/60 (v/v) anhydrous THF/hexane

Heat solution: 1 mg/mL of C1 in 40/60 (v/v) anhydrous THF/hexane

Acid solution: 0.1 N acetic acid in 40/60 (v/v) anhydrous THF/hexane

Base solution: 0.0001 N diisopropylamine in 40/60 (v/v) anhydrous THF/hexane

Oxidation solution: 0.1% cumene hydroperoxide in 40/60 (v/v) anhydrous THF/hexane

Radical initiation solution: 10 mM AIBN in 40/60 (v/v) anhydrous THF/hexane

Photo solution: 1 mg/mL of C1 in 40/60 (v/v) anhydrous THF/hexane.     

Table I: Anhydrous forced-degradation results. THF is tetrahydrofuran, AIBN is 

1 mg/mL API in 40/60 (v/v) anhydrous   THF/hexane

1 mg/mL API and 0.1 N acetic acid in   40/60 (v/v) anhydrous THF/hexane

1 mg/mL API and 0.0001 N diisopropylamine   in 40/60 (v/v) anhydrous THF/hexane

1 mg/mL API and 10 mM AIBN in 40/60   (v/v) anhydrous THF/hexane

1 mg/mL API in 0.1% cumene   hydroperoxide in 40/60 (v/v) anhydrous THF/hexane

Analytical considerations for study execution


In the case of aqueous forced degradation studies, most companies follow a standardized approach with pre-set conditions such as 0.1 N HCl or NaOH and a target drug substance concentration (6). In the case of this non-aqueous forced-degradation study, stress conditions required careful consideration with regard to drug solubility, reactivity with the drug substance, safety, and compatibility with the separation method and detection technique. For the prodrug C1, organic reagents needed to have ≤ 0.02% water present to prevent hydrolysis and maintain the form of the molecule. Mixtures of organic and aqueous solvents were, therefore, not appropriate. 


The identification of reactive organic solvents/reagents with minimal water content included assessment of C1 solubility (see 

), compatibility, chemical reactivity with the drug substance, and safety handling (see 

). The criteria for good, fair, and poor was based on the feasibility of the experimentation. The drug substance solubility in the selected conditions was a crucial consideration because the drug substance was not soluble in many organic solvents. The drug substance was highly soluble in dimethyl sulfoxide (DMSO) and THF, which is why many of the systems considered contained these solvents. The normal-phase HPLC method diluent utilized a mixture of THF and hexane. As such, the HPLC diluent was a preferred solvent option for the anhydrous forced-degradation solution conditions. Another consideration was the miscibility of the solvent with the organic reactive reagent. In general, non-nucleophilic organic acids and bases were considered to avoid the potential hydrolysis of the drug substance. In addition, organic acids and bases that are commercially available, reasonably pure, and safe when heated were criteria used to select reagent candidates. 

Table II: Solubility of C1 in anhydrous, aprotic solvents.

Articles

In this article, the authors demonstrate that a normal-phase chromatographic method was stability-indicating for a water-sensitive prodrug. The stress conditions using aqueous and non-aqueous conditions were also compared to understand the information obtained with each approach.