A Review: Stability Indicating Forced Degradation Studies

 

Shubhangi V. Sutar*, Veerendra. C. Yeligar, Shitalkumar S. Patil

Ashokrao Mane College of Pharmacy, Peth-Vadgaon, Maharashtra, India. 416112

 

ABSTRACT:

Forced degradation study (FD) studies (stress testing) are an intrinsic part of pharmaceutical product development. It is procedure whereby the natural degradation rate of a product or material is increased by the application of additional stress condition. It manifests chemical behaviour of the molecule which helps in the development of formulation and packaging of pharmaceutical development. It is necessary to specify the specificity of the stability indication methods and provide insight into degradation pathways and degradation products of the drug substance and aid in an elucidation of the structure of the degradation products. This review discusses the regulatory aspects of force degradation and the study of stability and also the analytical hyphenated methods used for the development of the forced degradation study.

 

 

INTRODUCTION:

Product quality, efficacy and safety of drugs have always been a major concern for pharmaceutical industries. Stability of drugs is a quality attribute, which is connected with drug substance or product in terms of strength, purity, identity, safety, apparent physical, chemical, microbiological and biological change, and their effect on biological performance of the drug product. Any change with time in any of the quality attributes of drug product is considered as a potential instability, and assessment of this change becomes mandatory as it is directly related to the safety and efficacy of the drug. Stability testing is done primarily to provide the evidence that the drug substance or the drug product maintains its essential features of quality, identity, purity and strength (within acceptable ranges) throughout the time in which, it is expected to remain safe for further processing or human consumption.

 

Forced Degradation studies provide data to support identification of possible degradants; degradation pathways, intrinsic stability of the drug molecule and validation of stability indicating analytic procedure. Knowledge of stability of molecule help in selecting proper formulation and package and providing proper storage condition and shelf life, which is essential for regulatory documentation. 

 

The FDA and International Conference on Harmonization guideline states that stress testing is deliberate to identify the likely degradation products which more  helps in determination of the inherent  stability of the molecule and establishing degradation pathways, and to validate the stability indicating procedure. But these guidelines are very indefinite in conduct of forced degradation and do not provide particular about the practical approach towards stress testing. Force Degradation studies are regulatory requirement and scientific necessity during drug development. It has become mandatory to perform stability studies of new drug moiety before filing in registration. The FDA and ICH guidance’s state the requirement of stability testing data to understand how the quality of a drug substance and drug product changes with time under the influence of various environmental factors in several conditions.^1-2

 

Regulatory Perspective in Stability Studies:

Efforts were enunciated by the drug regulatory authorities and pharmaceutical industries in Europe, Unites States and Japan to establish harmonized regulation for testing and analysis during research and development of new drugs. These harmonized guidelines came to be known as the International Conference on Harmonization (ICH) which has been incorporated as law in the EU, Japan and in the US. Besides these, other countries are also using them as a de facto force of regulation.

 

The ICH guideline Q1A, exemplifies the core stability recommendations and testing of the features which are susceptible to change during storage, that are likely to influence the quality, safety and efficacy must be done by validated SIM. It is also mentioned that forced decomposition studies at temperatures in 10°C increments above the accelerated temperatures, extremes pH and under oxidative and photolytic conditions should be carried out on the drug substance, so as to establish the inherent stability characteristics and degradation pathways to support the suitability of the proposed analytical procedures. 

 

The ICH Q1B, outlines the photostability testing should be an integral part of stress studies. Generally, an ICH dose of 1.2×106 and 2.4x106 lxh of fluorescent light and 200 Wh/m2 UV light is recommended to estimate the photostability considerations of the given drug substance and products. 

 

The ICH guideline Q3B emphasizes on providing documented evidence that analytical procedures are validated and suitable for the detection and quantitation of degradation products. The guideline gives a clear concept on the threshold levels of degradation products in drug substances, in terms of reporting, identification and quantitation thresholds.

 

The ICH guideline Q6A provides note for guidance on specifications and mentions the requirement of stability-indicating assays under Universal Tests/Criteria for drug substances and drug products. The similar requirement has been laid down in the ICH Q5C guideline on Stability Testing of Biotechnological/Biological Products. Such products have distinguishing characteristics and thus the document outlines the necessity of determining the changes in identity, purity and potency of the product. 

 

Aim and Objective of Forced Degradation Study:

 i.     To determine drug substances and drug products degradation pathways.

ii.     To illuminate the structure of degradation products.

iii.     To differentiate degradation product that are related to drug product from product in formulation.

iv.     To reveal the degradation mechanisms such as hydrolysis, oxidation, thermolysis or photolysis of the drug substance and drug product.^3-4

v.     To understand the chemical properties of drug molecule

vi.     To produce stable formulation.

 vii.     To produce a degradation profile similar to that of what would be observed in a formal stability study under ICH conditions.^5-9

 

When carry out forced degradation:

It is very prime to know when to perform forced degradation studies for the development of new drug substance and new drug product. FDA guidance states that stress testing should be performed in phase III of regulatory submission process. Stress studies should be done in different pH solutions, in the presence of oxygen and light, and at elevated temperatures and humidity levels to determine the stability of the drug substance. These stress studies are conducted on a single batch. The results should be summarized and capitulate  in an annual report. However, starting stress testing early in preclinical phase or phase I of clinical trials is highly vitalize and should be conducted on drug substance to obtain sufficient time for identifying degradation products and structure elucidation as well as optimizing the stress conditions. An early stress study also gives timely recommendations for making changes for better in the manufacturing.¹⁰

 

Most common stress conditions used in Forced degradation study:

The following are general conditions that should be considered when conducting forced degradation studies.

 

Table No.1 Common Conditions used mainly for forced degradation studies .

Solid State	Solution and/or Suspension
Heat	Hydrolysis at various pHs
Heat/humidity	Unbuffered HCl, NaOH, water
Light	Buffer solutions (used to determine if pH adjustment needed to attain maximum stability)
	Oxidative stress testing
	H2O2 (to mimic possible presence of peroxides in excipients)
	Metal ions (to mimic possible exposure during manufacture)
	Radical initiators (to mimic autoxidation)
	Light

 

Fig No.1:  common conditions used in conducting forced degradation study

 

Hydrolytic degradation:

Hydrolysis is a chemical process that includes decomposition of a chemical compound by reaction with water. Under acidic and basic conditions hydrolytic study includes catalysis of ionisable functional groups present in the molecule. Acid or base stress testing involves forced degradation of a drug substance by exposure to acidic or basic conditions which generates primary degradants in desirable range. On the stability of the drug substance the selection of the type and concentrations of acid or base depends. Sodium hydroxide or potassium hydroxide (0.1–1M) for base hydrolysis and Hydrochloric acid or sulphuric acids (0.1–1 M) for acid hydrolysis is suggested as suitable reagents for hydrolysis. The hydrolytic degradation of a new drug in acidic and alkaline condition can be studied by refluxing the drug in 0.1 N HCl or 0.1 N NaOH. If reasonable degradation is seen, testing can be stopped at this point. However in case no degradation is seen under these conditions the drug should be refluxed in acid/alkali of higher strength & for longer duration of time. Alternatively if total degradation is seen after subjecting the drugs to initial condition, acid/alkali strength can be decreased along with decrease in reaction temperature.

 

 

Fig.No.2. Flow chart for performing stress studies for hydrolytic Degradation under acid and alkali condition.

 

Photolytic conditions:

The photo stability testing of drug substances must be evaluated to demonstrate that a light exposure does not result in unacceptable change. Photo stability studies are performed to generate primary degradants of drug substance by exposure to UV or fluorescent conditions. Some recommended conditions for photostability testing are described in ICH guidelines. Samples of drug substance and solid/liquid drug product should be exposed to a minimum of 1.2million lx h and 200 W h/m2 light. The most commonly accepted wavelength of light is in the range of 300– 800 nm to cause the photolytic degradation. The maximum illumination recommended is 6 million lx h. Light stress conditions can induce photo oxidation by free radical mechanism. Functional groups like carbonyls, nitroaromatic, Noxide, alkenes,aryl chlorides, weak C–H and O–H bonds, sulphides and polyenes are likely to introduce drug photosensitivity.

 

Fig.No.3. Flow chart for performing stress studies for photolytic degradation

 

Oxidation conditions:

Hydrogen peroxide is broadly used for oxidation of drug substances in forced degradation studies. Other oxidizing agents such as metal ions, oxygen, and radical initiator (e.g., azobisisobutyronitrile, AIBN) can additionally be used. Choice of an oxidizing agent, its concentration, and process depends on the drug substance. It is reported that subjecting the solutions to 0.1–3% hydrogen peroxide at neutral pH and room temperature for seven days or up to a maximum 20% degradation could potentially produce relevant degradation consequences. Oxidative degradation of drug substance and drug products forms reactive anions and cations. Amines, sulphides and phenols are capable to electron transfer oxidation to give N-oxides, hydroxylamine, sulfones and sulfoxide. The functional groups with labile hydrogen like benzylic carbon, allylic carbon, and tertiary carbon or α-positions with respect to hetero atom to oxidation to form hydro peroxides, hydroxide ketone.

 

Thermal conditions:

The thermal degradation study shall be carried out at more intense conditions than proposed ICH Q1A accelerated test conditions. Solid-state drug product and drug substances samples should be exposed to dry and wet heat, while liquid drug products should be exposed to dry heat. Thermal Studies may be managed at higher temperatures for a shorter period. Effect of temperature on thermal degradation of a substance is studied through the Arrhenius equation:

 

k = Ae-Ea/RT

 

Where, k is specific reaction rate, Ea is energy of activation, A is frequency factor, R is gas constant (1.987cal/deg mole) and T is absolute temperature. Thermal degradation study is carried out at 40–80oC.^11-13

 

Table No.2 Conditions used mainly for forced degradation studies.

Degradation Type	Experimental Conditions	Storage Conditions	Sampling Time (Days)
Hydrolysis	Control API9(No Acid or Base) 0.1M HCl 0.1 M NaOH Acid Control Base Control pH:2,4,6,8	400C, 600C 400C, 600C 400C, 600C 400C, 600C 400C, 600C 400C, 600C	1,3,5 1,3,5 1,3,5 1,3,5 1,3,5 1,3,5
Oxidation	3% H2O2 Peroxide Control Azobisisobutyronitile (AIBN) AIBN Control	250C, 600C 250C, 600C 400C, 600C 400C, 600C	1,3,5 1,3,5 1,3,5 1,3,5
Photolytic	Light 1x ICH Light 3x ICH Light Control	NA NA NA	1,3,5 1,3,5 1,3,5
Thermal	Thermal Heat Chamber Heat Chamber Heat Chamber Heat Chamber Heat Control	600C 600C/75%RH 600C 800C/75%RH 800C Room Temp.	1,3,5 1,3,5 1,3,5 1,3,5 1,3,5

 

Selection of samples:

The duration and intensity of the stress conditions must be decided through experimentation to obtain the sample with the required degradation. Simultaneously, it submits the Placebo (mixture of excipients) according to the manufacturing formula to all the previous stress conditions. For the placebo formulation of a multidrug product containing a drug substance, each must undergo forced degradation. Prepare the test solutions using non-stressed samples, placebo and stressed samples, according to the test method and inject into the HPLC system with detector. Record the chromatograms and calculate the percentage degradation and the percentage of net degradation according to the acceptance criteria. In the case of stable molecules, the percentage of degradation can be difficult to achieve according to acceptance criteria. Therefore, based on the experiments, the study can be concluded and the summary of the experiments documented. Manifest the effectual separation of the analyte from the degradation product and the peaks if any due to the components of the placebo mixture. Ensure that the response of the analyte peak in the test solution is equal to or less than 1 AU (absorbance unit). If it is more, dilute the test solution accordingly and repeat the analysis.

Identification and characterization of pharmaceutical products by selected analytical Hyphenated methods:

Reverse phase HPLC is desire for several reasons, such as its compatibility with aqueous and organic solutions, high precision, sensitivity and ability to detect polar compounds. The separation of the peaks can be carried out by selecting the appropriate column type, the temperature of the column and adjusting the pH of the mobile phase. The highly polar and poorly preserved impurities must be resolved from the solvent front. As part of the development of the method, a gradient elution method with a variable mobile phase composition (very low organic composition to a high organic composition) can be carried out to capture highly eluted highly polar compounds and highly retained non-polar compounds. Stressed samples can also be screened with the gradient method to evaluate the potential elution pattern. The sample solvent and mobile phase should be selected to be compatible with the drug substance, potential impurities and the degradation agents. The preparation of the stress sample should mimic the preparation of the sample outlined in the analytical procedure as close as possible. Sample neutralization or dilution may be necessary for hydrolyzed samples of acid and base. The chromatographic profiles of the samples subjected to tension should be compared with those of the relevant blanks (which do not contain active) and the samples not subjected to tension to determine the origin of the peaks. Blank peaks should be excluded from the calculations. The amount of impurities (known and unknown) obtained under each stress condition should be provided together with the chromatograms (full scale and expanded scale showing all peaks) of blank, stress-free samples and with stressed samples. In addition, chiral drugs should be analyzed with chiral methods to establish the purity and stereochemical stability.

The analytical method of choice must be adequate sensitive to detect impurities at low levels (ie 0.05% of the analyte of interest or less), and the peak responses must be within the range of the detector's linearity. The analytical method must be capable of capturing all impurities formed during a formal stability study at or below the threshold limits of ICH. The identification and characterization of the degradation product should be carried out according to the formal stability results according to the ICH requirements. Conventional methods (eg, column chromatography) or hyphenated techniques (eg, UPLC, LC-MS, GC-MS, SFC-MS, LCNMR, CE-MS) can be used in the identification and characterization of the products of degradation. The use of these techniques can provide a better understanding of the structure of the impurities that could increase the knowledge space of possible structural alerts of genotoxicity and the control of such impurities with stricter limits. It should be noted that the structural characterization of the degradation products is necessary for those impurities that are formed during the stability studies of the formal useful life and are above the limit of the qualification threshold. Various types of detection can be used to analyze stressed samples such as UV and mass spectroscopy. The detector must contain 3D data capabilities, such as diode array detectors or mass spectrometers to detect the lack of spectral homogeneity. The diode array detection also offers the possibility to verify the peak profile for multiple wavelengths. The limitation of the set of diodes arises when the UV profiles are similar for the peak of the analyte and the impurity or the peak of degradation and the level of noise of the system is high to mask the impurities or degradants that elute together. Compounds of molecular weights and similar functional groups such as diastereomers may exhibit similar UV profiles. In such cases, attempts must be made to modify the chromatographic parameters to achieve the necessary separation. An optimal wavelength should be selected to detect and quantify all feasible impurities and degradants. The usage of more than one wavelength may be necessary, if there is no overlap in the UV profile of an analyte and impurity or degradation peaks. A valuable tool in the development of methods is the superposition of separation signals at different wavelengths to discover dissimilarities in the profiles of the peaks.14-15

 

Fig. No.4: Forced degradation process flow chart

 

CONCLUSION:

Forced degradation studies provide information on possible degradation products and degradation pathways of the active ingredients and help to elucidate the structure of the degradants and impurities. The products formed from forced degradation studies are potential degradation products that may or may not be formed under relevant storage conditions but which help in the development of the stability indication method. It is better to start degradation studies earlier in the drug development process to have enough time to get more information about the stability of the molecule. This information in turn will help improve the manufacturing process of the formulation and determine the storage conditions. Since no specific set of conditions is applicable to all pharmaceutical products and pharmaceutical substances, and the normative guide does not specify the conditions that will be used, this study requires the experimenter to use common sense. The objective of any strategy used for forced degradation is to produce the desired amount of degradation, that is, 5-20%.

 

REFERENCES:

1.      ICH guidelines, Q1A (R2): Stability Testing of New Drug Substances and Products (revision2), International Conference on Harmonization.

2.      FDA Guidance for Industry, INDs for Phase 2 and 3 Studies of Drugs, Including Specified Therapeutic Biotechnology Derived Products, Draft Guidance, Food and Drug Administration.

3.      Alsante KM, Hatajik TD, Lohr LL, Santafianos D Sharp TR. Solving impurity/degradation problems: case studies. pp: 380, In; handbook of Isolation and Characterization of impurities in Pharmaceutical, Ahuja S, Alsante K, editors, Academics Press, New York. 2003.

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9.      Baertschi SW, Thatcher SR. Sample presentation for photostability studies: problems and solutions, pp: 445, In; Pharmaceutical Photostability and Stabilization Technology. Piechocki J, Editor, Taylor & Francis, New York. 2006.

10.   Baertschi SW, Alsante KM. Stress testing: the chemistry of the drug degradation, pp: 99, In Pharmaceutical Stress Testing, Baertschi SW, editors, Taylor & Francis, New York. 2005.

11.   D.W. Reynolds, K.L.Facchine, J.F. Mullaney, et al., Available guidance and best practices for conducting forced degradation studies, Pharm. Technol.26(2);(2002):48–56.

12.   Singh S, Bakshi M. Guidance on conduct of stress tests to determine inherent stability of drugs. Phrama Tech. 24; 2000: 1-14.

13.   H. Brummer, How to approach a forced degradation study, Life Sci. Technol. Bull.31; (2011):1–4.

14.   Blessy M., Ruchi D.Patel,Prajesh M. Prajapati, Y.K.Agarwal. Development of Forced degradation and stability indicating studies of drugs-A Review.Journal of Pharmaceutical Analysis.4 (3); 2014:159-165.

15.   Smita Shinde.S.A.Nirmal .A Review on Force Degradation Study for Pharmaceutical Product Development. International journal of Pharmaceutical and chemical sciences .5(2); 2016: 163-166.

 

 

 

 

 

 

Accepted on 02.10.2018           © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(2):885-890.