A practical guide to forced degradation and stability studies for drug substances

Forced degradation and stability studies are essential in the pharmaceutical industry, informing integral sections of the chemistry, manufacturing and controls (CMC) section of an investigational medicinal product dossier (IMPD) or investigational new drug (IND) application.

The key aims of forced degradation and stability studies are to:

• Identify degradation products and pathways: Identifying and characterising degradation products of drug substances (DS) under stress conditions, along with mapping their degradation pathways, provides critical insight into potential toxicity. Such toxicity can arise not only from the active pharmaceutical ingredient (API) itself but also from associated impurities, making this assessment essential for ensuring patient safety.

• Verify stability-indicating methods: Forced degradation and stability studies provide confidence that the developed high-performance liquid chromatography (HPLC) method is stability-indicating prior to method validation.

• Assess quality over time: Assessing DS quality over time under the influence of various environmental factors, such as temperature and humidity, provides information on its stability and supports predictions of product performance across the intended shelf life.

• Establishment retest periods and storage conditions: Establishing a retest period for the DS, alongside identifying trends within stability data, supports the selection of appropriate long-term storage conditions.

In this guide, Erica Lamb, Senior Analytical Chemist, outlines Onyx’s approach to forced degradation and stability studies, providing practical insights into study design, execution, data interpretation and how these findings support regulatory submissions and product quality.

The purpose of forced degradation and stability studies

Regulatory guidance from the International Council for Harmonisation (ICH) under section Q1A(R2) suggests stressing the DS under the following conditions:

• Hydrolysis
• Oxidation
• Photostability
• Temperature

These tests should be performed on a single batch of DS that is representative of the final manufacturing process to determine the degradation products and pathways derived from the API and associated impurities. The Food and Drug Administration (FDA) and European Medicines Agency (EMA) provide further guidance for performing these tests, which is underpinned by the ICH.

During these studies, the analytical method used must accurately identify the API without interference from degradation products or impurities, thereby ensuring method specificity in line with ICH Q2(R1). This provides confidence in the suitability of the method prior to validation and enables the determination of related impurity and degradation product levels.

Forced degradation studies provide an in-depth understanding of the degradation pathways of the DS. This knowledge supports improved handling during pre-clinical and pre-formulation studies, facilitates effective formulation development and informs the selection of appropriate packaging and conditions for stability studies.

Designing forced degradation studies

When designing forced degradation studies, it is important to review the emerging chemical and solid form data generated for the molecule that will be stressed, as a ‘one-size-fits-all’ approach is not appropriate. While regulatory guidance outlines a range of stress conditions, simply applying moderate stress, observing no degradation and concluding stability is insufficient.

A quality-by-design approach that involves all development groups is recommended. Where information from material science or chemical development is lacking, additional range-finding work can provide valuable insight. Some of the typical ranges for the specified stress tests to be applied are outlined below:

1. Hydrolytic: This stress condition assesses if the DS will be susceptible to hydrolysis and if hydrolytic degradants will form, particularly from compounds containing functional groups such as esters, amides and lactones.
   • Acidic hydrolysis: Hydrochloric acid (0.1-1.0 M) is typically used at elevated temperatures (40-80°C)
   • Basic hydrolysis: Sodium hydroxide (0.1-1.0 M) is typically used at elevated temperatures (40-80°C)
2. Oxidation: Hydrogen peroxide (3-30%) is used at room or elevated temperatures to encourage oxidative degradation of DS, which contain functional groups such as phenols, thiols and amines.
3. Thermal: The DS is exposed to an elevated temperature in dry or humid conditions to encourage the formation of thermally degraded products. This can aid in identifying thermally degraded products in formal stability studies as well as selecting temperatures for the DS to be stored at during the stability trials.
4. Photolytic: ICH guidelines state that the DS should be exposed to UV and visible light (ICH Q1B(R1)) for no less than 1.2 million lux hours. This assesses the DS susceptibility to photodegradation, which allows insight into what the compound is required to be stored or packaged in.
5. Humidity (where appropriate): If humidity is known to be a potential cause of degradation products, then it should be included as a stress condition in the study, for example, if the DS is known to be hygroscopic. Samples will typically be stored at an elevated temperature and humidity (40°C, 75% relative humidity [RH], respectively) to determine any degradation products from sensitivity to moisture. This information is usually obtained during the solid-state development of the API, with the forced degradation confirming that any degradants formed are detected by the analytical method. This can aid in determining appropriate packaging of the DS for formal stability trials.

These stress conditions are analysed at various time points throughout a seven-day period. Degradation of the DS at 2-20% is targeted to ensure the degradant products are detectable, but the main component has not excessively broken down. If significant degradation is observed (>20%) in a particular condition, the condition will be repeated at a less extreme parameter. A control sample is always analysed alongside the stress conditions to understand what impurities are present in the sample prior to the formation of degradation products.

Forced degradation analysis and data interpretation

Once the solutions for each applicable stress condition are prepared, they are analysed using the developed HPLC parameters (for more information, see our HPLC method development blog). UV and Mass spectrometry (MS) are typically used to detect retention times of the degradants, in addition to the corresponding masses.

The primary objectives of data interpretation include, but are not limited to:

• Peak purity analysis: Analysis of the data should ensure that no degradation products co-elute with the API and that all peaks are well resolved. The peak purity of the API should be >0.995 to confirm the absence of co-eluting impurities and degradants. Retention times of all peaks should be monitored throughout the study to ensure they remain consistent; otherwise, this could suggest method instability. The MS spectra obtained in the analysis should also be carefully scrutinised, particularly for the API to check for coeluting peaks.

• Mass balance assessment: Assay standards should be freshly prepared and injected alongside the sample solutions at various time points throughout the forced degradation study to be able to calculate mass balance. Mass balance (typical acceptance criteria of 90-110%) is an important parameter, as deviations may indicate volatilization, adsorption, or the presence of undetectable or unidentified degradation products. As the UV response of degradants often differs from that of the API, mass balance can sometimes fall outside the acceptance criteria, particularly when a key degradant has a markedly different response. Where undetected degradation products are suspected, the assay value for the main component can also be calculated relative to the assay standard to quantitatively determine the extent of degradation attributable to these impurities.

• Identification of major degradants: If major degradants are observed in the analysis, MS should be used for structural elucidation. Further analysis, such as NMR, may be used to aid the identification of the major degradant products.

• Reporting: A report should be generated summarising the experimental work and the data that have been obtained. Structures will be postulated and included in the report for key degradants noted in the study.

Designing stability studies

The key parameters to test in a stability study are thermal stability and sensitivity to moisture under its intended storage conditions. The stability studies should be conducted on the DS packaged in a container closure system that is the same as or simulates the packaging proposed for storage and distribution, and typically consists of double low-density polyethylene bags packaged inside a high-density polyethylene container. Desiccant is, on occasion, placed in between the polythene bags if a DS is identified as being hygroscopic. It is worth investigating the compounds’ propensity for hygroscopicity prior to a study. Slightly hygroscopic compounds are often handled easily as a bulk solid, but when stored in the small quantities used for a stability study, they may gradually absorb moisture to an unacceptable level. The study design must allow for this.

The studies typically include long-term testing under recommended storage conditions and accelerated testing under stressed conditions to predict potential degradation pathways. The conditions that the samples are subjected to are summarised below:

• Accelerated conditions – 40°C/75% RH: Speeds up degradation to predict long-term stability and identify degradation pathways.

• Long-term storage conditions – 25°C/60% RH (climate zone II): Simulates normal storage and directly determines the retest period of a DS. Actual conditions can vary depending on which climate zone the compound will be stored.

• Refrigerated/freezer conditions – 2-8°C/-20°C: For compounds with known stability issues. Initial stability studies often include at least one of these conditions, where the samples will only be tested if significant degradation has occurred in the long-term storage samples.

Sampling intervals are usually at 0, 1, 3, 6, 9, 12, 18, 24 and 36 months for long-term studies, and 0, 1, 3 and 6 months for the accelerated conditions, which tend to only last 6 months. For early-phase compounds, the initial development batch should be set down in addition to any GMP material produced; ideally, 6 months of accelerated data should be available for the development batch to inform retest dates for the GMP material. For later phase work, typically, at least three process typical batches of the DS are set down in the study, which have been produced at an appropriate scale. In addition, post-compound approval, at least one batch a year will be set down on stability and monitored to confirm stability performance as per ICH Q1.

The table below displays the time points in a study for a DS with little stability data. The conditions in the protocol are driven by the data collected in development and the 2-8°C condition is often not included if sufficient data has been acquired through the development of the DS.

	Timepoint
Condition	1 month	3 months	6 months	9 months	12 months	18 months	24 months	36 months
-20°C	X2	X2	X2	X2	X2	X2	X2	X2
2-8°C	X2	X2	X2	X2	X2	X2	X2	X2
25°C/60%RH	X	X	X	X	X1	X	X1	X1
40°C/75%RH	X	X	X

X1 – Microbial testing to be performed.
X2 – Only to be tested if failure at 25°C/60%RH.

Typical tests included in the protocols are:

• Appearance
• Assay and chemical purity by HPLC
• Impurities by HPLC
• Water content by Karl Fischer titration
• Physical characteristics (such as X-ray powder diffraction)

Additional testing can be added to the protocols in particular circumstances, examples include:

• Chiral purity by HPLC
• Potential mutagenic impurity content
• Water activity

The majority of these are usually tested at each time point, with the physical characteristics being tested intermittently (e.g. at 6, 12, 24 and 36 months). The testing conducted is informed by the forced degradation study, which may have identified particular impurities to test for during the stability study and other information gained during the development of the DS.

At the end of the study, a report is generated summarizing the tests that have been performed throughout the study. The primary/secondary packaging, storage and shipping conditions are informed by the stability study data. The retest period is also dictated by the stability data.

When planning retest periods, it is important to note that they must not exceed the duration of the planned stability study and are only valid when no degradation is observed. For example, a 36-month retest period can be assigned based on 24-month stability data showing no changes, but only if the stability study also runs to 36 months, as shown in the table below.

Acceptable accelerated real-time data (40°C/75%RH)	Acceptable real-time data (25°C/60%RH)	Recommended retest period assignments
6 months	6 months	12 months
	12 months	24 months
	24 months	36 months
	36 months	36 months

 

From degradation pathways to clinical confidence

Forced degradation and stability studies are fundamental to pharmaceutical development, providing the critical data that underpins the CMC section of IMPD and IND submissions while ensuring compliance with ICH guidance. By confirming method specificity and robustness, forced degradation supports the identification of potentially toxic degradants. In parallel, stability studies establish retest periods and appropriate storage conditions, safeguarding product quality, efficacy and patient safety across the lifecycle.

At Onyx, we apply a structured, phase-appropriate, data-driven approach to deliver reliable insights that strengthen regulatory submissions, accelerate clinical trial applications and ensure confidence in investigational products.

Contact our team today to learn how we can support your forced degradation and stability studies with phase-appropriate strategies that strengthen your regulatory submissions.