INTRODUCTION:

Forced degradation is the process of subjecting drug compounds to extreme chemical and environmental conditions to determine product breakdown levels and preliminary degradation kinetics, and to identify degradant species. The stress testing practices that companies use can vary significantly and can have a serious impact on the analytical methodology used throughout the industry. Stress testing studies can be conducted to challenge specificity of stability indicating and impurity monitoring methods as part of validation protocol. Forced degradation or stress testing studies are part of the development strategy and are an integral component of validating analytical methods that indicate stability and detect impurities. This relates to the specificity section of the validation studies. It is important to recognize that forced degradation studies are not designed to establish qualitative or quantitative limits for change in drug substance or drug product.

Forced degradation studies are used for multiple purposes, including demonstration of the specificity of separation methods, gaining insight into degradation pathways, and discernment of degradation products in formulations that are related to drug substances versus those that are related to other ingredients of a formulation. Reliable chemical stability testing data can show how a drug product changes over time with influence of environmental factors. The stability of a drug product or a drug substance is a critical parameter, which may affect purity, potency and safety. Changes in drug stability can risk patient safety by formation of a toxic degradation product(s) or deliver a lower dose than expected. Therefore, it is essential to know the purity profile and behaviour of a drug substance under various environmental conditions.

The main purpose of forced degradation testing studies is to evaluate the overall photosensitivity of the material for method development purposes and/or degradation pathway elucidation. This testing may involve the drug substance alone and/or in simple solutions/suspensions to validate the analytical procedures. For development and validation purposes, it is appropriate to limit exposure and end the studies if extensive decomposition occurs. For photostable materials, studies may be terminated after an appropriate exposure level has been used. The initial purpose of forced degradation studies is to investigate stability-related properties of an API and to develop an understanding of the degradation products and pathways. These studies should also be used to evaluate the susceptibility of the drug substance to hydrolysis across a wide range of pH values. Forced degradation studies are used to provide degraded samples for the development of stability-indicating analytical methods for the API. The information developed from a forced degradation study can be utilized in several other areas of development, including analytical methods development, formulation development and storage conditions, manufacturing processing, safety toxicological, identification of possible genotoxic degradants, identification of potential metabolites and API design/discovery.

Forced degradation is synonymous with stress testing and purposeful degradation. Purposeful degradation can be a useful tool to predict the stability of a drug substance or a drug product with effects on purity, potency, and safety. It is imperative to know the impurity profile and behaviour of a drug substance under various stress conditions. Forced degradation also plays an important role in the development of analytical methods, setting specifications, and design of formulations under the quality-by-design (QbD) paradigm. The nature of the stress testing depends on the individual drug substance and the type of drug product (e.g., solid oral dosage, lyophilized powders, and liquid formulations) involved. Forced degradation studies are most beneficial if done early in the drug development process. The reasoning for this is that these studies yield predictive information on the nature of the degradants which are valuable when assessing the appropriate synthesis routes, API salt selection and formulation development. These early studies can help to provide information needed for the following:

1. As a predictive tool to understand degradation pathways and stability-related issues.

2. To predict API stability before real-time stability data is available.

3. Development and validation of stability-indicating methodology.

4. Determination of degradation pathways of drug substances and drug products.

5. Discernment of degradation products in formulations that are related to drug substances versus those that are related to non–drug substances (e.g., excipients).

6. Structure elucidation of degradation products.

7. Determination of the intrinsic stability of drug substances.

8. Helps to identify reactions that cause degradation of pharmaceutical products.

9. To generate a degradation profile that mimics what would be observed in a formal stability study under ICH conditions.

10. To generate more stable formulation.

11. To identify impurities related to drug substances and excipients.

12. To understand the drug molecular chemistry.

13. Selection of storage conditions, packaging and better understanding of the potential liabilities of the drug molecule chemistry. ^[1]

Forced degradation studies can be done on both, the solid state and aqueous solution or suspension forms of the API. Furthermore, the use of analysis at multiple time points allows for approximation of rates of degradation and such testing at early time points can provide a distinction between primary and secondary degradation products. This approach allows for better degradation pathway determination.

Forced degradation studies should be repeated as needed throughout the drug development process. For example, when there are changes in API impurity profile, API salt or polymorph form. When carried out in late development, such studies are referred as confirmatory studies. Confirmatory studies are quantitative in nature. Full mass accountability of the API, its impurities and degradation products are generated from these late stage studies. Furthermore, based upon the outcome of these studies, if necessary, new or orthogonal methods may need to be developed to account for all observed degradation. In addition, confirmatory studies for API are done after finalization of the synthetic route and form of the API. Such studies are typically done in Phase III with one of the registration batches of the API. For drug products, confirmatory studies are done when final formulation(s) and packaging are chosen. After the confirmatory studies are completed, a report on degradation products and pathways is generated and included in or used to support NDA filings.

DEGRADATION CONDITIONS ^{[2, 3]}

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

Solid State

· Heat

· Heat/humidity

· Light

Solution and/or Suspension

· Hydrolysis at various pHs

· Unbuffered HCl, NaOH, water

· 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

SOURCES OF IMPURITIES AND TYPES OF IMPURITIES:

In a typical study, relevant stress conditions are light, heat, humidity, hydrolysis (acid / base influence) and oxidation or even a combination of described parameters. If it is necessary to form degradation products, the strength of stress conditions can vary due to the chemical structure of the drug substance, the kind of drug product, and product specific storage requirements. An individual program has to be set up in order to reach a target degradation of 5 to 20%. A higher level of degradation will be out of the scope of product stability requirements and therefore unrealistic. The scope of the test is to generate degradation products in order to facilitate a method development for determination of the relevant products. Therefore, samples will be stressed in a solid form and/or in solution. Typically, stress tests are carried out on one batch of material. For drug products the placebo should be stressed in a similar way in order to exclude those impurities that are not degradation products (e.g. impurities arising from excipients). Table 1 shows typical stress conditions of API and drug product. Drugs that are poorly soluble in water can be conducted either in suspension or in solution using inert organic co-solvents (e.g., DMSO, acetic acid or propionic acid). It is important to avoid co-solvents that may be reactive with the drug or complicate analysis (e.g. by LC-MS). ^{[7, 8]}

For drug substances, photostability testing should consist of two parts: forced degradation testing and confirmatory testing. The purpose of forced degradation testing studies is to evaluate the overall photosensitivity of the material for method development purposes and/or degradation pathway elucidation. This testing may involve the drug substance alone and/or in simple solutions/suspensions to validate the analytical procedures. In these studies, the samples should be in chemically inert and transparent containers. In these forced degradation studies, a variety of exposure conditions may be used, depending on the photosensitivity of the drug substance involved and the intensity of the light sources used. For development and validation purposes it is appropriate to limit exposure and end the studies if extensive decomposition occurs. For photostable materials, studies may be terminated after an appropriate exposure level has been used. The design of these experiments is left to the applicant’s discretion although the exposure levels used should be justified. The potential for these degradation pathways should be assessed in both drug substance and drug product. These mechanisms can be assessed in a systematic way by exposure to stress conditions of heat, humidity, photo stress, oxidative conditions, and aqueous conditions across a broad range of pH. The ICH guidelines Q3A (R) require identification of each impurity with respect to both chemistry and safety perspective. The chemistry perspective includes classification and identification of impurities, report generation, listing of impurities in specific guidance and a brief discussion of analytical procedures while safety perspectives include specific guidance for qualifying those impurities. The impurities usually encountered in pharmaceuticals are synthesis related, formulation related, and degradation related. These ICH guidelines classify the impurities into the fallowing categories.

1. Impurities associated with active pharmaceutical ingredient (APIs).

2. Impurities that are formed during formulation, formed with ageing and that are related to formulation forms.

Impurities associated with active pharmaceutical ingredients:

According to the ICH guidelines, impurities associated with APIs are classified into the fallowing categories

A. Organic impurities

B. Inorganic impurities

Organic impurities:

· Starting materials

· Byproducts

· Intermediates

· Degradation products

· Residual solvents

· Reaction products with excipients

· Leachables from container closure system

Inorganic impurities

· Reagents, ligands, catalysts

· Heavy metals or other residual metals

· Inorganic salts

Other materials

· Filter aids

· Charcoal

Formulation related impurities:

During formulation, excipients added to API to render the product elegant. In such cases, drug-excipients incompatibility may lead to undesirable products, which can affect the therapeutic efficiency of the product. In addition, other solvents, which is usually present in API or used in the synthesis of API or excipients, may also interact with excipients resulting in impurities.

Dosage form related impurities:

1. Precipitation of main ingredient can occur due to various factors like pH, environment, or leaching. In general, liquid dosage forms are very much susceptible to both degradation and microbial contamination.

Example- Precipitation of imipramine HCl with sodium bisulfate and pH alteration of lidocaine HCl in presence of 5% dextrose solution.

2. Pharmaceutical solids: in the presence of excipients and moisture topochemical and nucleation reactions occur.

Example- Presence of sodium CMC during granulation of papaverine, aminopyrine and salicylic acid tablets causes reduced coloration.

3. Microbial growth resulting from the growth of bacteria, fungi, and yeast in a humid and warm environment may result in oral liquid products that are unusable for human consumption.

Method related impurities:

A known impurity 1-(2, 6 Dichloro phenyl) indoline-2one is formed in the production of a parental dosage form diclofenac sodium ampoules. Formation of this impurity depends on initial pH of the preparation and the conditions of sterilization i.e. autoclave method (121⁰c) that enforces the intramolecular cyclic reaction of diclofenac sodium forming indolineone derivative and sodium hydroxide.^[5]

Environmental related impurities:

The environmental factors that can reduce stability are

i. Temperature:

There is many APIs that are liable to heat or topical temperature. Ex: Vitamins as drug substances are very heat sensitive and degradation frequently leads to loss of potency in vitamin products, especially in liquid formulations. Degradation caused by exposure to temperatures include bond breakage i.e. pyrolysis. Any degradation mechanism that is enhanced at elevated temperatures are thermolytic pathways include Hydrolysis, Dehydration, Isomerization, Decorboxylation, Rearrangement, Polymerization reactions, pyrolysis. In general, rate of reaction increases with increase in temperature. Many APIs are sensitive to heat or topical temperature such as vitamins and peptides. Effect of temperature on thermal degradation of a substance is studied through Arrhenius equation.

K = Ae -Ea/RT

Where K = specific reaction rate

A = frequency factor

Ea = energy of activation

Thermal degradation study is carried out at 40⁰C. The mostly accepted temperature is 70⁰C at low and high humidity for 1-2 months. The use of high-temperatures in predictive degradation studies assumes that the drug molecule will follow the same pathway of decomposition at all temperatures.^[9]

ii. Light (UV light): Light is one of the means by which the formulation degrades because of photolytic reaction. Ex: sunlight having about 8000 foot-candles can destruct nearly 34% of vitamin B in 24 hrs.

iii. Humidity: Humidity is one of the important key factors in case of hygroscopic compounds. Ex: Ranitidine, Aspirin^[9]

Functional group related:

1) Hydrolysis:

Drug degradation that involves reaction with water is called hydrolysis. PH, buffer salts, ionic strength, solvents, complexing agents, surfactants and excipients affect hydrolysis. Hydrolysis reactions are typically acid or base catalyzed. Hydrolysis is the most common degradation chemical reaction over wide range of pH. Water either as solvent or as moisture in the air comes in contact with pharmaceutical dosage form is responsible for degradation of most drugs. Hydrolytic study under acidic and basic condition involves catalyzation of ionisable functional groups present in the molecule.

Example- Aspirin combined with water and hydrolyzed to form salicylic acid and acetic acid. The hydrolytic degradation of new drug in acidic and alkaline condition can be studied by refluxing the drug in 0.1N HCl or 0.1N NaOH. If degradation is seen testing can be stopped at that point. In case of no degradation under these conditions, the drug should be refluxed in acid and alkali of higher strength and for a long duration time. Hydrolysis of most of drugs is depending upon the relative concentration of hydronium and hydroxyl ions.

Example- Anatrazole – degraded in basic pH

Doxophylline – shows degradation in acidic pH

A reaction in which water is an reactant causing precipitation. Hydrolysis is a common phenomenon for ester type of drugs. Esters, amides, lactones, lactams, imides, and carbamates, are susceptible for acid base hydrolysis.

Examples- Aspirin, Chloramphenicol, Barbiturates, Chlordiazepoxide, Oxazepam, Examples includes Aspirin, Benzocaine, Cefolaxime, Cocaine, and Cefpodoxime.

2) Oxidation:

Many drug substances undergo autoxidation i.e. oxidation under normal conditions and involving ground state elemental oxidation. Autoxidation is a free radical reaction that requires free radical initiator to begin the chain reaction. Hydrogen peroxide, metal ions, traces level of impurities in a drug substance act as initiator for oxidation. The oxidative stress testing is initially carried out in 3% H₂O at room temperature for 6 h and it can be increased or decreased to achieve sufficient degradation. The time can also be increased up to 24 hr 3% or decreased up to 30 min with 1% of H₂O The mechanism of oxidative degradation of drug substance involves an electron transfer mechanism to form reactive anions and cations. The mechanism of oxidative degradation of drug substance involves an electron transfer mechanism to form reactive anions and cations. Drugs In pharmaceuticals most common form of oxidative decomposition is oxidation through a free radical chain process.

Example- Amines, sulphide, and phenols are susceptible to electron transfer oxidation. Drugs which prone to oxidation are hydrocortisone, methotrexate, adinazolam, catecholamine conjugated dienes, heterocyclic aromatic rings, nitraso and nitrate derivatives.

a) Rapamycin, an immunosuppressant drug reacts with oxygen to form a complex mixture of monomeric and oligomeric products. Formation of the majority of the rapamycin degradation products could be rationalized with free radical-mediated autoxidation reactions involving alkenes and alcohol sites.

b) Auto-oxidation of ascorbic acid studies reveals that cupric ion known to oxidize ascorbic acid rapidly in to dehydro ascorbic acid and potassium cyanide.

3) Photolysis:

Exposure of drug molecule to light may produce photolytic degradation products. The rate of photo degradation depends upon the intensity of incident light and quantity of light absorbed by drug molecule. Photolytic degradation is carried out by exposing the drug substance to or drug product to a combination of visible and UV light. The photolytic degradation can occur through non-oxidative and oxidative photolytic reaction.

a) The non-oxidative photolytic reaction includes isomerization, diamerization, cyclization, rearrangement, decorboxylation and haemolytic cleavage.

^b)Oxidative photolytic reaction occurs through either single oxidation or triple oxidation mechanism. Ex: Barnidipin.^[1,10]

Photolytic cleavage on aging includes examples of pharmaceutical drugs or products that are prone to degradation on exposure to UV-light. During manufacturing process or packing or on storage drugs like ergometrine, nifedipine, nitroprucide, riboflavin, are liable to photo oxidation. Most of the compounds will degrade as solutions when exposed to high-energy UV exposure. This photo oxidation involves generation of free-radical intermediates, which will degrade the product.

Example- The formulation of ciprofloxacin eye drops 0.3% on exposure to UV-light induces photolysis, which results in formation of ethylene di-amine analogue of ciprofloxacin.

Selection of Stress Conditions

Forced degradation is normally carried out under more severe conditions than those used for accelerated studies. The choice of stress conditions should be consistent with the products decomposition under normal manufacturing, storage, and use conditions, which are specific in each case. The ICH guidance recognize that it is impossible to provide strict degradation guidelines and allows certain freedom in selecting stress conditions for biologics.The choice of forced degradation conditions should be based on data from accelerated pharmaceutical studies and sound scientific understanding of the product’s decomposition mechanism under typical use conditions. A minimal list of stress factors suggested for forced degradation studies must include acid and base hydrolysis, thermal degradation, photolysis, oxidation, and may include freeze-thaw cycles and shear.

Regulatory guidance does not specify pH, temperature ranges, specific oxidizing agents, or conditions to use, the number of freeze-thaw cycles, or specific wavelengths and light intensities. The design of photolysis studies is left to the applicant's discretion although Q1B recommends that the light source should produce combined visible and ultraviolet (UV, 320-400 nm) outputs, and that exposure levels should be justified. Consult the appropriate regulatory authorities on a case-by-case basis to determine guidance for light-induced stress.

Stress conditions

Typical stress tests include four main degradation mechanisms: heat, hydrolytic, oxidative, and photolytic degradation. Selecting suitable reagents such as the concentration of acid, base, or oxidizing agent and varying the conditions (e.g., temperature) and length of exposure can achieve the preferred level of degradation. Over-stressing a sample may lead to the formation of secondary degradants that would not be seen in formal shelf-life stability studies and under-stressing may not serve the purpose of stress testing. Therefore, it is necessary to control the degradation to a desired level. A generic approach for stress testing has been proposed to achieve purposeful degradation that is predictive of long-term and accelerated storage conditions. The generally recommended degradation varies between 5-20% degradation. This range covers the generally permissible 10% degradation for small molecule pharmaceutical drug products, for which the stability limit is 90%-110% of the label claim. Although there are references in the literature that mention a wider recommended range (e.g., 10-30%), the more extreme stress conditions often provide data that are confounded with secondary degradation products.

Table 1. The four climate zones (ICH Stability guidelines)

Zone I	Temperate	21°C ± 2°C/ 45% RH ± 5% RH
Zone II	Subtropical and Mediterranean	25°C ± 2°C/ 60% RH ± 5% RH
Zone III	Hot and Dry	30°C ± 2°C/ 35% RH ± 5% RH
Zone IV	Hot and Humid	30°C ± 2°C/ 65% RH ± 5% RH

Table 2. Type of study with Storage conditions (ICH Stability guidelines)

Study	Storage condition	Min. time period
Long term	25°C ± 2°C/ 60% RH ± 5% RH or 30°C ± 2°C/ 65% RH ± 5% RH	12 months
Intermediate	30°C ± 2°C/ 65% RH ± 5% RH	6 months
Accelerated	40°C ± 2°C/ 75% RH ± 5% RH	6 months

Table 3. Drug products intended for storage in refrigerator (ICH Stability guidelines)

Study	Storage condition	Min. time period
Long term	5°C ± 3°C	12 months
Accelerated	25°C ± 2°C/ 60% RH ± 5% RH	6 months

Table 4. Drug products intended for storage in freezer

Study	Storage condition	Min. time period
Long term	-20°C ± 5°C	12 months

Photostability

Photostability testing should be an integral part of stress testing, especially for photo-labile compounds. Some recommended conditions for photostability testing are described in ICH Q1B Photostability Testing of New Drug Substances and Products. Samples of drug substance, and solid/liquid drug product, should be exposed to a minimum of 1.2 million lux hours and 200-watt hours per square meter light. The same samples should be exposed to both white and UV light. To minimize the effect of temperature changes during exposure, temperature control may be necessary. The light-exposed samples should be analyzed for any changes in physical properties such as appearance, clarity, color of solution, and for assay and degradants. The decision tree outlined in the ICH Q1B can be used to determine the photo stability testing conditions for drug products. The product labelling should reflect the appropriate storage conditions. It is also important to note that the labelling for generic drug products should be concordant with that of the Reference Listed Drug (RLD) and with United States Pharmacopeia (USP) monograph recommendations, as applicable.

Heat:

Thermal stress testing (e.g., dry heat and wet heat) should be more strenuous than recommended ICH Q1A accelerated testing conditions. Samples of solid-state drug substances and drug products should be exposed to dry and wet heat, whereas liquid drug products can be exposed to dry heat. It is recommended that the effect of temperature be studied in 10°C increments above that for routine accelerated testing, and humidity at 75% relative humidity or greater. Studies may be conducted at higher temperatures for a shorter period. Testing at multiple time points could provide information on the rate of degradation and primary and secondary degradation products. In the event that the stress conditions produce little or no degradation due to the stability of a drug molecule, one should ensure that the stress applied is in excess of the energy applied by accelerated conditions (40°C for 6 months) before terminating the stress study.

Acid and base hydrolysis:

Acid and base hydrolytic stress testing can be carried out for drug substances and drug products in solution at ambient temperature or at elevated temperatures. The selection of the type and concentrations of an acid or a base depends on the stability of the drug substance. A strategy for generating relevant stressed samples for hydrolysis is stated as subjecting the drug substance solution to various pH (e.g., 2, 7, 10–12) at room temperature for two weeks or up to a maximum of 15% degradation. Hydrochloric acid or sulphuric acid (0.1M to 1M) for acid hydrolysis and sodium hydroxide or potassium hydroxide (0.1M to 1M) for base hydrolysis are suggested as suitable reagents for hydrolysis. For lipophilic drugs, inert co-solvents can be used to solubilize the drug substance. Attention should be given to the functional groups present in the drug molecule when selecting a co-solvent. Prior knowledge of a compound can be useful in selecting the stress conditions. For instance, if a compound contains ester functionality and is very labile to base hydrolysis, low concentrations of a base can be used. Analysis of samples at various intervals can provide information on the progress of degradation and help to distinguish primary degradants from secondary degradants.

Oxidation:

Oxidative degradation can be complex. Although hydrogen peroxide is used predominantly because it mimics possible presence of peroxides in excipients, other oxidizing agents such as metal ions, oxygen, and radical initiators (e.g., azobisisobutyronitrile) can also be used. Selection of an oxidizing agent, its concentration, and conditions depends on the drug substance. Solutions of drug substances and solid/liquid drug products can be subjected to oxidative degradation. 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 generate relevant degradation products. Samples can be analyzed at different time intervals to determine the desired level of degradation.

Analysis method: The preferred method of analysis for a stability indicating assay is reverse-phase high-performance liquid chromatography. Reverse-phase HPLC is preferred for several reasons, such as its compatibility with aqueous and organic solutions, high precision, sensitivity, and ability to detect polar compounds. Separation of peaks can be carried out by selecting appropriate column type, column temperature, and making adjustment to mobile phase pH. Poorly retained, highly polar impurities should be resolved from the solvent front. As part of method development, a gradient elution method with varying mobile phase composition (very low organic composition to high organic composition) may be carried out to capture early eluting highly polar compounds and highly retained nonpolar compounds. Stressed samples can also be screened with the gradient method to assess potential elution pattern. Sample solvent and mobile phase should be selected to afford compatibility with the drug substance, potential impurities, and degradants. Stress sample preparation should mimic the sample preparation outlined in the analytical procedure as closely as possible. Neutralization or dilution of samples may be necessary for acid and base hydrolyzed samples. Chromatographic profiles of stressed samples should be compared to those of relevant blanks (containing no active) and unstressed samples to determine the origin of peaks. The blank peaks should be excluded from calculations. The amount of impurities (known and unknown) obtained under each stress condition should be provided along with the chromatograms (full scale and expanded scale showing all the peaks) of blanks, unstressed, and stressed samples. Additionally, chiral drugs should be analyzed with chiral methods to establish stereo-chemical purity and stability.

The analytical method of choice should be sensitive enough to detect impurities at low levels (i.e., 0.05% of the analyte of interest or lower), and the peak responses should fall within the range of detector's linearity. The analytical method should be capable of capturing all the impurities formed during a formal stability study at or below ICH threshold limits. Degradation product identification and characterization are to be performed based on formal stability results in accordance with ICH requirements. Conventional methods (e.g., column chromatography) or hyphenated techniques (e.g., LC–MS, LC–NMR) can be used in the identification and characterization of the degradation products. Use of these techniques can provide better insight into the structure of the impurities that could add to the knowledge space of potential structural alerts for genotoxicity and the control of such impurities with tighter limits. It should be noted that structural characterization of degradation products is necessary for those impurities that are formed during formal shelf-life stability studies and are above the qualification threshold limit. Various detection types can be used to analyze stressed samples such as UV and mass spectroscopy. The detector should contain 3D data capabilities such as diode array detectors or mass spectrometers to be able to detect spectral non-homogeneity. Diode array detection also offers the possibility of checking peak profile for multiple wavelengths. The limitation of diode array arises when the UV profiles are similar for analyte peak and impurity or degradant peak and the noise level of the system is high to mask the co-eluting impurities or degradants. Compounds of similar molecular weights and functional groups such as diastereoisomers may exhibit similar UV profiles. In such cases, attempts must be made to modify the chromatographic parameters to achieve necessary separation. An optimal wavelength should be selected to detect and quantitate all the potential impurities and degradants. Use of more than one wavelength may be necessary, if there is no overlap in the UV profile of an analyte and impurity or degradant peaks. A valuable tool in method development is the overlay of separation signals at different wavelengths to discover dissimilarities in peak profiles.

Table 5. Stress conditions for drug substance

Type of study	Conditions	Time
Acid hydrolysis	0.1 N- 1.0 N HCl, RT or higher	1-7 days
Base hydrolysis	0.1 N- 1.0 N NaOH, RT or higher	1-7 days
Thermal hydrolysis	Aqueous solution, 70°C	1-7 days
Oxidative/ solution	O₂ + Initiator (AIBN) in CAN/ H₂O; 80/20,40°C	1-7 days
Oxidative/ solution	0.3-3.0 % H2O/ Ambient in the dark	Few hours to 7 days
Thermal	solid, 70°C	Upto 3 weeks
Thermal/ Humidity	solid, 70°C/ 75% RH	Upto 3 weeks
Photo degradation	Solid, fluorescent and UV light	>2× ICH

Peak purity analysis:

Peak purity is used as an aid in stability indicating method development. The spectral uniqueness of a compound is used to establish peak purity when co-eluting compounds are present. Peak purity or peak homogeneity of the peaks of interest of unstressed and stressed samples should be established using spectral information from a diode array detector. When instrument software is used for the determination of spectral purity of a peak, relevant parameters should be set up in accordance with the manufacturer's guidance. Attention should be given to the peak height requirement for establishing spectral purity. UV detection becomes non linear at higher absorbance values. thresholds should be set such that co-eluting peaks can be detected. Optimum location of reference spectra should also be selected. The ability of the software to automatically correct spectra for continuously changing solvent background in gradient separations should be ascertained.

Termination of study:

Stress testing studies are terminated after ensuring adequate exposure to stress conditions. Typical activation energy of drug substance molecules varies from 12–24 kcal/mol. A compound may not necessarily degrade under every single stress condition. In circumstances, where some stable drugs do not show any degradation under any of the stress conditions, specificity of an analytical method can be established by spiking the drug substance or placebo with known impurities and establishing adequate separation.

Forced degradation in QbD paradigm

A well-designed, forced degradation study is indispensable for analytical method development in a QbD paradigm. A systematic process of manufacturing quality drug products that meet the predefined targets for the critical quality attributes (CQA) necessitates the use of knowledge obtained in forced degradation studies. It helps to establish the specificity of a stability indicating method and to predict potential degradation products that could form during formal stability studies. Incorporating all potential impurities in the analytical method and establishing the peak purity of the peaks of interest helps to avoid unnecessary method re-development and revalidation. Knowledge of chemical behaviour of drug substances under various stress conditions can also provide useful information regarding the selection of excipients for formulation development. Excipients compatibility is an integral part of understanding potential formulation interactions during product development and is a key part of product understanding. Degradation products due to drug-excipient interaction or drug-drug interaction in combination products can be examined by stressing samples of drug substance, drug product, and placebo separately and comparing the impurity profiles. Information obtained regarding drug-related peaks and non-drug-related peaks can be used in the selection and development of more stable formulations. For instance, if a drug substance is labile to oxidation, addition of an antioxidant may be considered for the formulation. For drug substances that are labile to acid or undergo stereochemical conversion in acidic medium, delayed-release formulations may be necessary. Acid/base hydrolysis testing can also provide useful insight in the formulation of drug products that are liquids or suspensions. Knowledge gained in forced degradation studies can facilitate improvements in the manufacturing process.

If a photostability study shows a drug substance to be photolabile, caution should be taken during the manufacturing process of the drug product. Useful information regarding process development (e.g., wet versus dry processing, temperature selection) can be obtained from thermal stress testing of drug substance and drug product. Additionally, increased scientific understanding of degradation products and mechanisms may help to determine the factors that could contribute to stability failures such as ambient temperature, humidity, and light. Appropriate selection of packaging materials can be made to protect against such factors.

GUIDELINES FOR FORCED DEGRADATON STUDY

Impurities in new drug substances (CPMP/ICH/2737/99)

This document are intended to provide guidance for registration applications on the content and qualification of impurities in new drug substances produced by chemical syntheses and not previously registered in a region or member state. It is not intended to apply to new drug substances used during the clinical research stage of development. The following types of drug substances are not covered in this guideline:

· Biological/biotechnological products

· Peptides

· Oligonucleotide

· Radiopharmaceuticals

· Fermentation products

· Semi-synthetic products

· Herbal products, crude products of animal or plant origin.

Impurities in new drug products (CPMP/ICH/2738/99)

This guideline addresses only those impurities in new drug products classified as degradation products of the drug substance or reaction products of the drug substance with an excipients and/or immediate container closure system (referred to as "degradation products"). Generally, impurities present in the new drug substance need not be monitored or specified in the new drug product, unless they are also degradation products. Impurities arising from excipients present in the new drug product, extracted, or leached from the container closure system are not covered by this guideline. ^[5]

Stability Testing of New Drug Substances and Products (ICH Q1A (R2))

Drug substance:

Stress testing a drug substance can help identify the likely degradation products, which can in turn help establish the degradation pathways and the intrinsic stability of the molecule and validate the stability-indicating power of the analytical procedures used. The nature of the stress testing will depend on the individual drug substance and the type of drug product. Stress testing is likely to be carried out on a single batch of the drug substance. It should include the effect of temperatures (in 10⁰C increments [e.g., 50⁰C, 60⁰C] above that for accelerated testing), humidity (e.g., 75% RH or greater) where appropriate, oxidation, and photolysis on the drug substance. Testing should evaluate the susceptibility of the drug substance to hydrolysis across a wide range of pH values when in solution or suspension. Photostability testing should be an integral part of stress testing. Examining degradation products under stress conditions is useful for establishing degradation pathways, developing and validating suitable analytical procedures. It may not be necessary, however, to examine for specific degradation products if previous studies have demonstrated that these products are not formed under accelerated or long-term storage conditions.

Drug product:

The design of formal stability studies for a drug product should be based on the behavior and properties of the drug substance, the results from stability studies on the drug substance, and the experience gained from clinical formulation studies. The likely changes to storage conditions and the rationale for the selection of attributes to be tested in the formal stability studies should be stated. Photostability testing should be conducted on at least one primary batch of the drug product if appropriate.

Stability Testing: Photostability Testing of New Drug Substances and Products (ICH Q1B)

Drug substance:

Photostability testing should consist of two parts: forced-degradation testing and confirmatory testing. The purpose of forced-degradation testing is to evaluate the overall photosensitivity of the material for method-development purposes and/or degradation pathway elucidation. This testing may involve the drug substance alone and/or the substance in simple solutions and suspensions to validate the analytical procedures. In these studies, the samples should be in chemically inert and transparent containers. For forced-degradation studies, various exposure conditions may be used, depending on the photosensitivity of the drug substance and the intensity of the light sources. For development and validation purposes, it is appropriate to limit exposure and end the studies if extensive decomposition occurs. For photostable materials, studies may be terminated after an appropriate exposure level has been used. The design of these experiments is left to the applicant’s discretion although the exposure levels used should be justified. Under forcing conditions, decomposition products may be observed that are unlikely to be formed under the conditions used for confirmatory studies. This information may be useful in developing and validating suitable analytical methods. If in practice it has been demonstrated that they are not formed in the confirmatory studies, these degradation products need not be further examined.

Validation of Analytical Procedures: Methodology (ICH Q2B)

Drug substance/drug product:

If impurity or degradation product standards are unavailable, specificity can be demonstrated by comparing the test results of samples containing impurities or degradation products with a second well-characterized procedure; e.g., pharmacopeial method or other validated analytical procedure (independent procedure). As appropriate, this should include samples stored under relevant stress conditions (light, heat, humidity, acid–base hydrolysis, and oxidation).

Impurities in New Drug Substances (ICH Q3A(R))

Drug substance:

The applicant should summarize the actual and potential impurities most likely to arise during the synthesis, purification, and storage of the new drug substance. This summary should be based on sound scientific appraisal of the chemical reactions involved in the synthesis, impurities associated with raw materials that could contribute to the impurity profile of the new drug substance, and possible degradation products. This discussion can be limited to those impurities that might reasonably be expected based on knowledge of the chemical reactions and conditions involved. In addition, the applicant should summarize the laboratory studies conducted to detect impurities in the new drug substance. This summary should include test results of batches manufactured during the development process and batches from the proposed commercial processes well as the results of stress testing (see ICH Guideline Q1A on Stability) used to identify potential impurities arising during storage. The impurity profile of the drug substance batches intended for marketing should be compared with those used in development and any differences discussed.

Impurities in New Drug Products (ICH Q3B(R))

Drug product:

Analytical procedures should be validated to demonstrate specificity for the specified and unspecified degradation products. As appropriate, this validation should include samples stored under relevant stress conditions: light, heat, humidity, acid/base hydrolysis, and oxidation.

FDA Guidance for Industry: Stability Testing of Drug Substances and Drug Products

Drug substance/drug product:

Stress testing helps determine the intrinsic stability characteristics of a molecule by establishing degradation pathways to identify the likely degradation products, and to validate the stability-indicating power of the analytical procedures used. Stress testing provides data about the forced-decomposition products and decomposition mechanisms of the drug substance. The severe conditions that may be encountered during distribution can be covered by stress testing definitive batches of the drug substance. These studies should establish the inherent stability characteristics of the molecule such as the degradation pathways, lead to identification of degradation products, and hence support the suitability of the proposed analytical procedures. The detailed nature of the studies will depend on the particular drug substance and type of drug product. This testing is likely to be carried out on a single batch of a drug substance. Testing should include the effects of temperatures in 10⁰C increments above the accelerated temperature test condition (e.g., 50⁰C, 60⁰C) and humidity, where appropriate (e.g., 75% or greater). In addition, oxidation and photolysis on the drug substance plus its susceptibility to hydrolysis across a wide range of pH values when in solution or suspension should be evaluated. Results from these studies will form an integral part of the information provided to regulatory authorities. Light testing should be an integral part of stress testing. Some degradation pathways can be complex and under forced conditions, decomposition products may be observed that are unlikely to be formed under accelerated or long-term testing. This information may be useful in developing and validating suitable analytical methods, but it may no always be necessary to examine specifically for all degradation products if, in practice, it has been demonstrated that these are not formed.

FDA Guidance for Industry: Analytical Procedures and Methods Validation

Drug substance/drug product:

Degradation information obtained from stress studies (e.g., products of acid and base hydrolysis, thermal degradation, photolysis, and oxidation) for the drug substance and for the active ingredient in the drug product should be provided to demonstrate the specificity of the assay and analytical procedures for impurities. The stress studies should demonstrate that impurities and degradants from the active ingredient and drug product excipients do not interfere with the quantization of the active ingredient.

FDA Guidance for Industry: Submitting Documentation for the Stability of Human Drugs and Biologics

Drug substance:

A program for the stability assessment might include storage at ambient temperature and under stress conditions. Stress testing conditions ordinarily include temperature (e.g., 5, 50, and 75⁰C), humidity, where appropriate (e.g., 75% or greater) and exposure to various wavelengths of electromagnetic radiation (e.g., 190–780 nm UV and visible ranges), preferably in open containers, where applicable. It is also suggested that the following conditions be evaluated in stability studies on solutions or suspensions of the bulk-drug substance: acidic and alkaline pH, high oxygen atmosphere, and the presence of added substances under consideration for product formulation.

Drug product:

Stress testing the drug product helps identify potential problems during storage and transportation and provides an estimate of the expiration-dating period.

FDA Guidance for Industry: INDs for Phase II and Phase III Studies, Chemistry Manufacturing, and Controls Information

Drug substance:

Performance of stability-stress studies with the drug substance early in drug development is encouraged because these studies provide information crucial to selecting stability indicating analytical procedures for real-time studies. If not performed earlier, stress studies should be conducted during Phase 3 to demonstrate the inherent stability of the drug substance, potential degradation pathways, and the capability and suitability of proposed analytical procedures. Stress studies should assess the stability of the drug substance in different pH solutions, in the presence of oxygen and light, and at elevated levels of temperatures and humidity. These one-time stress studies on a single batch are not considered part of the formal stability program.

Drug product:

Drug products, one-time stress testing can be warranted to assess the potential for changes in the physical (e.g., phase separation, precipitation, aggregation, changes in particular-size distribution) and/or chemical (e.g., degradation and/or interaction of components) characteristics of the drug product. The studies could include testing to assess the effect of high temperature, humidity, oxidation, photolysis and/or thermal cycling. ^[6]

CONCLUSION:

Forced degradation studies, plays important role in formulation development process. A well-designed stress study can provide insight in choosing the appropriate formulation for a proposed product prior to intensive formulation development studies. A thorough knowledge of degradation, including mechanistic understanding of potential degradation pathways, stress conditions etc. are essential for success of these studies. Stress testing can provide useful insight into the selection of physical form, stereo-chemical stability of a drug substance, packaging, and storage conditions. It is important to perform stress testing for generic drugs due to allowable qualitative and quantitative differences in formulation with respect to the RLD, selection of manufacturing process, processing parameters, and packaging materials.

ACKNOWLEDGEMENT:

The authors express their sense of gratitude towards management of Satara College of Pharmacy, Degaon, Satara for providing all obligatory facilities necessary to carry out present work.

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Received on 30.10.2013 Accepted on 25.11.2013

Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 4, Pg 178-188

Test conditions of FPP	Recommended labeling statement
25°C/ 60 % RH (long term)40°C/ 75 % RH (accelerated)	“Do not store above 25°C”
25°C/ 60 % RH (long term)30°C/ 65 % RH (intermediate, failure of accelerated)	“Do not store above 25°C”
30°C/ 65 % RH (long term)40°C/ 75 % RH (accelerated)	“Do not store above 30°C”
30°C/ 75 % RH ( long term)40°C/ 75 % RH (accelerated)	“Do not store above 30°C”
5°C ± 3°C	“Store in refrigerator” (2°C to 8°C)
-20°C ± 5°C	“Store in freezer”