Forced Degradation to Develop Stability-indicating Methods

Introduction

Forced Degradation (or stress testing) typically involves exposure of drug substances to heat, heat and humidity and light for solid-state studies. For solution-state studies the drug substance is exposed to a range of pH values. The experimental samples produced are then used to demonstrate that a proposed analytical method is “Stability Indicating,” i.e., the method is capable of detecting the loss in content of the active component and subsequent increase in degradation products. Ideally, loss in content of the active component and increase in degradation products should be monitored by a single analytical method. However, in some cases, this is not possible and separate assay and impurity methods have to be developed. This article describes how forced degradation studies are used to develop stability-indicating methods.

Overview of Regulatory Guidance

Forced degradation studies are described in various international guidelines. The International Committee for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) [1] has published a set of guidelines which have been discussed, agreed upon and adopted by the American, European and Japanese regulatory authorities. In the majority of cases, the ICH guidelines only apply to the marketing applications for new products, i.e., they do not apply during clinical development. However, since the conditions used for forced degradation are only defined in general terms, it is possible to apply them for developing stability indicating methods during clinical development. The same forced degradation conditions can then be applied to the drug substance during development and commercialization. The ICH guidelines that are applicable to forced degradation studies are:

• ICH Q1A – Stability Testing of New Drug Substances and Products [2]
• ICH Q1B – Photostability Testing of New Drug Substances and Products [3]
• ICH Q2B – Validation of Analytical Procedures: Methodology [4]

In ICH Q1A, section 2.1.2 (Stress Testing), there are recommended conditions for performing forced degradation studies on drug substances and drug products. The recommendations are to examine the effects of temperature (above that for accelerated testing, i.e., >50°C), humidity (≥75% relative humidity), oxidation and photolysis. Testing in solution should also be performed across a wide pH range either as a solution or suspension. These samples are then used to develop a stability-indicating method.

ICH Q1B gives recommended approaches to assessing the photostability of drug substances and drug products. Forced degradation conditions are specified in Section II (drug substance) and Section III (drug product). Exposure levels for forced degradation studies are not defined, although they can be greater than that specified for confirmatory (stability) testing. The actual design of photostability studies is left to the applicant; however, scientific justification is required where light exposure studies are terminated after a short time, e.g., where excessive degradation is observed. Photostability testing can be performed on the solid or in solution/suspension. These samples are then used to develop a stability indicating method.

Both guidance’s, Q1A and Q1B, note that some of the degradation products formed during forced degradation studies may not actually be observed to form during stability studies, in which case they need not be examined further.

ICH Q2B gives guidance on how to validate analytical methodology and in section B 1.2.2 (impurities not available) there is a recommendation to use samples from forced degradation studies to prove specificity. Specificity is a key factor in determining whether or not the analytical method is stability indicating. Co-elution of peaks or components being retained on the column will underestimate the amount of degradation products formed and could compromise quality and increase risk to the patient.

Choosing Appropriate Experimental Conditions

There are many examples in the literature of experimental conditions for conducting forced degradation studies [5,6] and the structural diversity of drug molecules makes it impossible to specify a generic set of conditions for a forced degradation study.

For an early phase molecule, using a set of standard conditions by first intent makes sense since very little may be known about the intrinsic stability. If early stability data are available which suggest the molecule is labile at a particular condition (e.g., high pH), the conditions can be modified to take into account the instability (e.g., reduced temperature or time of study). Once a set of conditions have been found, they may be repeated whenever a new stability-indicating method is required during development. Therefore, for later-phase molecules, the forced degradation conditions are defined by the earlier work. By reusing the same forced degradation conditions throughout development a consistent approach is maintained.

Figure 1. Potential Degradation Pathways for Compound A

ICH guidelines do not give any guidance as to how much degradation is required in forced degradation studies. If too little stress is applied, some degradation pathways may not be observed which would not challenge the method’s ability to detect and monitor degradation products during stability testing. If too much stress is applied then unrealistic degradation products may be observed and the resulting analytical method may be unsuitable for detecting actual degradation products formed during stability testing. Thus, the actual conditions need to be chosen carefully so that the amount of degradation of the drug substance produced during forced degradation is neither too excessive nor too little. The following example demonstrates the consequences of using excessive forced degradation conditions. Compound A was refluxed with hydrochloric acid and one degradation product, Compound B, was observed (see Figure 1). A stability-indicating method was developed to detect this potential degradation product. However, subsequent work on the drug product demonstrated that Compound B was in fact a secondary degradation product and that Compound C was formed first. The analytical method used was unable to detect Compound C and a new stability-indicating method had to be developed to monitor stability of drug substance and drug product. Compound B was not considered to be a realistic degradation product and there was no longer a requirement to develop a method capable of detecting it.

A survey of forced degradation literature [5,6] suggests that there is wide variation in the levels of degradation achieved by different workers. However, limiting the amount of degradation is scientifically sound as this should maximize chances of forming primary degradation products. By analyzing the forced degradation samples over a number of time-points, it is possible to monitor if secondary degradation products are formed. If the original degradation peaks are observed to decrease and new peaks appear, this suggests that degradation of the original degradation products is occurring. For the majority of drug substances and drug products, the primary degradation products are those that are likely to be observed during stability testing.

Figure 2. HPLC Chromatogram of Compound D After First Time-point

Compound D was being degraded and initially one major degradation product at Relative retention Time (RRT) 0.68 was observed (Figure 2). At a later time-point, the levels of RRT 0.68 decreased and two new degradation products at RRT 0.08 and RRT 0.09 formed (Figure 3). This suggests that the primary degradation product, RRT 0.68, is unstable and under prolonged stressing forms secondary degradation products RRT 0.08 and RRT 0.09.

HPLC with mass spectrometry detection (LC-MS) can provide valuable information on the structural changes occurring as a result of degradation, which can help in the interpretation of the degradation pathways.

Some commonly observed mass changes are shown in Table 1.

Multiple oxygen additions or water losses may indicate the formation of secondary degradation products. If this is suspected to be the case, then the forced degradation study should be repeated, but using milder conditions and/or shorter stressing times.

Figure 3. HPLC Chromatogram of Compound D After Second Time-point

Many drug substances have poor aqueous solubility and require addition of an organic co-solvent to maintain solubility. Solution samples are easier to handle and lend themselves to increased accuracy in analytical testing. ICH Q1A does not specifically state solutions are required for forced degradation studies which then does allow for suspensions to be used if appropriate. However, suspensions will tend to undergo a slower rate of degradation than a solution and typically require whole vial analysis to reduce errors associated with sub-sampling a suspension. The use of co-solvents prevents all of these issues, however, the use of organic co-solvents can lead to the following:

• Increased or decreased rate of degradation
• Reaction products between drug substance and the
• organic co-solvent
• Peaks in the chromatogram arising from the organic co-solvent

Figure 4. Functional Groups that are Likely to React with Methanol

Acetonitrile (MeCN) and dimethyl sulfoxide (DMSO) are two widely used organic co-solvents, which in the majority of cases should be suitable for use in forced degradation studies. However, it has been reported [7] that decomposition of some co-solvent’s can occur under certain conditions so other types of co-solvent may be more appropriate. A general recommendation is to use as little organic co-solvent as possible to minimize any effects. Reaction with the co-solvent can lead to by-products that could be mistaken for degradation products. Hence, alcohols such as methanol are not recommended for use as a co-solvent for forced degradation studies. Methanol has the potential to react with a number of functional groups (Figure 4) forming new compounds which could be mistaken for potential degradation products.

Making the Link Between Forced Degradation Studies and Stability Data

Forced degradation of drug substances has the potential to form many more degradation products than those observed to form during stability testing. However, this minimizes the potential for not detecting the actual degradation products formed during stability testing. Thus, if appropriate forced degradation studies have been performed, the method can be considered to be stability indicating. In such cases, the absence of observed degradation products demonstrates that the drug substance is stable to degradation under the conditions it was stored rather than the method being incapable of detecting degradation products.

The results from the forced degradation studies can also be used to investigate the mechanism of degradation for a drug substance on storage. In turn, this understanding can be used to define the appropriate packaging to minimize or eliminate the degradation of the drug substance.

Compound E was degraded (Figure 5) under a range of typical conditions to form a number of potential degradation products. Of these, only two degradation products, RRT 1.16 and RRT 1.35 were observed to form during stability testing studies.

Figure 5. Forced Degradation Summary of Compound E

For Compound E, only two of the degradation products observed in the forced degradation study were detected in the drug substance during accelerated or real-time stability testing when stored using specified control measures – RRT 1.16 and RRT 1.35. However, it was demonstrated that the HPLC method was capable of detecting all of the potential degradation products from the forced degradation study and was suitable for monitoring the stability of Compound E.

Conclusion

Forced Degradation Studies are an essential component in developing stability-indicating methodology. There are many aspects to cover and the use of good scientific judgement and knowledge is required to ensure that the forced degradation samples produced contain realistic primary degradation products. There is a strong likelihood that many more forced degradation products will be observed than what actually form in accelerated or real-time stability trials. If these criteria are followed, then the analytical methodology has the maximum potential to detect real degradation products formed on accelerated or real-time stability testing. Thus, the absence of observed degradation products can be attributed to the stability of the drug substance rather than deficiencies in the analytical methodology.

References

1. International Conference on Harmonisation; http://www.ich.org/products/guidelines.
2. ICH Harmonised Tripartite Guideline Q1A (R2), Stability Testing of New Drug Substances and Products, February 2003.
3. ICH Harmonised Tripartite Guideline Q1B, Stability Testing: Photostability Testing of New Drug Substances and Products, November 1996.
4. ICH Harmonised Tripartite Guideline Q2 (R1), Validation of Analytical Procedures: Text and Methodology, November 2005.
5. Alsante K.M., Martin L. and Baertschi S., “A Stress Testing Benchmarking Study”, Pharm. Technol., 2003, 27(2), 60-72.
6. Klick S., Muijselaar P.G., Waterval J., Eichinger T., Korn C., Gerding T.K., Debets A.J., Sangervan de Griend C., van den Beld C., Somsen G.W. and De Jong G.J., “Towards a Generic Approach for Stress Testing of Drug Substances and Drug Products”, Pharm. Technol., 2005, 29(2), 48-66.
7. Joshi B.K and Kizzie A.C., American Pharmaceutical Review, 2007, 10(6).