Evaluation of Stability Study of Drug Substance and Drug Product as per Regulatory Guidelines

The term ‘Stability’ can be defined as the period of time in which a product remains stable under the recommended conditions (as different products require different conditions), without compromising its integrity. This means that no physical or chemical changes occur, and the product retains the same quality as when it left the manufacturer. When a company is manufacturing a new product, it needs to perform a stability study.

Stability is a crucial aspect of product development, particularly in industries such as pharmaceuticals, food and beverages, and cosmetics, where product efficacy and safety directly impact consumer health and satisfaction.

The term stability refers to the period during which a product maintains its intended physical, chemical, microbiological, therapeutic, and toxicological specifications when stored under recommended conditions [1]. In other words, a product is considered stable when no significant changes occur in its composition, performance, or appearance throughout its anticipated shelf life.

Stability studies are an integral part of the quality assurance process, designed to evaluate how environmental factors such as temperature, humidity, and light exposure affect a product’s integrity over time [2]. When a new product is manufactured, the initial phase involves testing three primary production batches during the first year of development to establish baseline data. In subsequent years, one batch per strength per year is placed on stability testing to monitor the product’s ongoing quality while on the market [3].

The primary goals of stability studies are to determine the product’s shelf life, identify optimal storage conditions, and confirm that the product remains within specified parameters throughout its lifecycle. This process ensures that consumers receive products that meet established standards of safety, quality, and efficacy up to the point of use [4].

Moreover, stability testing helps manufacturers establish regulatory compliance regarding labeling and quality control documentation. It also aids in decision-making for packaging design, as packaging materials can influence product stability by providing protection against environmental stressors. Stability studies, therefore, serve as a cornerstone of product quality management across diverse sectors.

Regulatory Framework:

• Numerous regulatory agencies provide guidance and enforce requirements for stability testing: International Council for Harmonisation (ICH): For pharmaceuticals, ICH provides several guidelines (e.g., Q1A, Q1B, Q1C) outlining requirements for stability studies, including batch selection, conditions, data analysis, and reporting.
• U.S. Food and Drug Administration (FDA): The FDA mandates rigorous stability testing as part of the New Drug Application (NDA) and Abbreviated New Drug Application (ANDA) processes, and for continued product registration.
• European Medicines Agency (EMA), Japanese Ministry of Health, Labour and Welfare (MHLW), and others: These agencies have harmonized their requirements through ICH participation, ensuring comparability across major markets. 

Objectives of stability study

The main objective of stability testing is to ensure the quality, safety, and efficacy of a drug substance or drug product throughout its shelf life under the influence of various environmental factors such as temperature, humidity, and light. Stability testing is fundamental to understanding how a drug's characteristics evolve over time and to ensure that it maintains its intended performance until its expiry date.

1. Ensure Quality, Safety, and Efficacy

Stability testing guarantees that the drug product retains its purity, potency, safety, and overall quality during storage and use[10]. It evaluates the physical, chemical, microbiological, and biological properties of the drug to ascertain that none of these degrade beyond acceptable limits over time.

2. Determination of Shelf Life and Storage Conditions

One of the primary outcomes of stability testing is to establish a scientifically justified shelf life and to define optimal storage conditions. By studying how environmental factors such as temperature, humidity, and light affect the drug, manufacturers can recommend storage requirements like "Store below 25°C" or "Protect from light" to help maintain drug stability[10].

3. Evaluation of the Stability Profile

Stability testing involves rigorous testing of various parameters such as chemical composition (e.g., potency, degradation products), physical characteristics (e.g., appearance, dissolution rate), microbiological integrity, and biological activity. This comprehensive evaluation helps build a stability profile over time and under different conditions[10].

4. Identification of Degradation Products and Pathways

Testing allows identification of degradation products that may form during storage and helps elucidate degradation pathways. This information is critical to understanding potential risks associated with drug use after prolonged storage and to ensuring safety[10].

5. Regulatory Compliance and Documentation

Stability testing provides the essential data package for regulatory submissions such as New Drug Applications (NDA) or Abbreviated New Drug Applications (ANDA). Regulators require these data to approve marketing authorization and to verify that the product meets quality standards throughout its labeled shelf life[10].

6. Support for Labeling and Marketing Claims

 Data from stability studies back up claims made on product labels, including storage instructions, expiration dates, and specific handling requirements[10]. These claims guide healthcare professionals and consumers in ensuring the drug's effectiveness and safety.

Scope of Stability Study

Stability testing in the pharmaceutical industry encompasses a broad scope, essential for ensuring the consistent quality, safety, and efficacy of drug substances and drug products throughout their shelf life. The scope of stability testing includes multiple dimensions, starting with the types of products tested, environmental conditions, packaging interactions, stability-indicating parameters, special cases, and regulatory compliance.

1. Drug Substances and Drug Products:

Stability testing applies to new drug substances (active pharmaceutical ingredients, APIs) and formulated products such as tablets, capsules, and injections. Both innovator and generic products undergo testing, including biotechnological and biological products like vaccines, peptides, and proteins[5].

2. Environmental Conditions:

 Testing involves various conditions to simulate real-world storage and stress situations:

• Long-term (real-time storage)
• Accelerated (elevated stress)
• Intermediate (moderate stress)
• Stress testing (extreme conditions) to identify degradation pathways and establish analytical methods [6].

3. Packaging:

Studies assess the interaction between drug products and packaging materials (e.g., glass, plastic, blister packs), vital for maintaining product integrity and protection throughout storage and distribution [5].

4. Stability-Indicating Parameters:

Tests cover physical properties such as appearance and dissolution, chemical properties including potency and degradation products, microbiological attributes like sterility, and functional properties such as drug release behavior [2],[8].

5. Special Cases:

Stability testing can include reconstituted products (post-mixing stability), in-use stability for multidose containers, photostability testing to assess light exposure effects, and freeze-thaw studies especially relevant for biological products [5].

6. Regulatory Submissions:

Stability data are a regulatory requirement for Investigational New Drug Applications (INDs), New Drug Applications (NDAs), Abbreviated New Drug Applications (ANDAs), and Common Technical Documents (CTDs). Compliance with ICH guidelines (Q1A-Q1F) and regional authorities such as the US FDA, EMA, and CDSCO is mandatory to support shelf-life determination and labelling [2],[6],[7].

These comprehensive stability studies provide a scientifically based assurance that drug products remain safe and effective during their marketed life, helping manufacturers optimize storage conditions, packaging, and usage recommendations.

Mechanistic Principle of Stability Testing

The mechanistic principle of stability testing of a new drug product centre on understanding the changes in chemical, physical, microbiological, and therapeutic properties over time when subjected to environmental factors such as temperature, humidity, light, and oxygen. The principle involves exposing the drug product to controlled conditions to observe degradation mechanisms, identify degradation pathways such as hydrolysis, oxidation, and photolysis, and determine the kinetics of these degradation reactions (e.g., first-order or zero-order kinetics). This understanding helps elucidate the impact of the drug formulation and packaging on its stability. Ultimately, these studies enable the estimation of shelf life and the assignment of an expiry date, ensuring that the product maintains its intended quality and performance throughout its lifecycle [9],[10].

Stability testing employs stress and accelerated conditions to simulate and predict long-term changes in the drug substance or product. Analytical methods used are stability-indicating, capable of differentiating the drug substance from its degradation products. Kinetic modeling, often based on Arrhenius principles, allows extrapolation from accelerated data to predict behavior under normal storage conditions. This mechanistic approach is essential in establishing robust stability profiles critical for regulatory approval and label claims[9],[10].

Table 1.1.- Types of Mechanisms Considered [2]

Order of reaction ^[12]

Order of a reaction is defined as the number of concentration terms on which the rate of a reaction depends when determined experimentally.

The order of reaction is established with respect to each reactant. This can be verified by plotting log concentration of a reactant on y axis and time on x axis. If a straight line is obtained, then the order will be 'one' with respect to that reactant. The overall order of a reaction is equal to the powers of the concentration terms (m + n) affecting the experimentally determined rate. In contrast to molecularity, it is possible for the order to assume fractional or zero values.

The orders that are common in pharmacy are zero, first-, pseudo first-, and second- orders. These topics will be discussed in detail in the subsequent sections.

Zero Order Reaction :

Zero order reaction is defined as a reaction in which the rate does not depend on the concentration terms of the reactants.

Example:

1. Colour-loss of liquid multisulfonamide preparatioⁿ.  Colour-loss is proportional to decrease in the concentration.
2. Oxidation of vitamin A in an oily solution,
3. Photochemical degradation of chlorpromazine in aqueous solution.

Mechanism : In zero order reaction, the rate must depend on some factor other than the concentration term. The rate limiting factors are solubility as in suspensions or absorption of light as in certain photochemical reactions.

First Order Reaction

First order reaction is defined as a reaction in which rate of reaction depends on the concentration of one reactant.

where c is the concentration of the reactant and kl  is the specific rate constant for first order.

Examples : Decomposition of hydrogen peroxide catalyzed by 0.02 M potassium iodide.

2H2O2→2H2O+O2

The stoichiometric equation involves 2 molecules, i.e., molecularity is two. But the order is found to be first order, based on experimental observation. In the presence of stabilisers, this reaction follows zero order kinetics. In other words, same drug may exhibit different Orders of decomposition under different experimental conditions.

• Acid hydrolysis of ethylacetate and methylacetate
• Inversion of sugar (sucrose)
• Disintegration of radioactive elements

Examples of biological importance are as follows:

1. Diffusion of drug across biological membranes
2. Absorption, distribution, metabolism and excretion of drugs
3. Growth of microorganisms
4. Rate of killing of microorganisms during sterilization

Pseudo First Order Reaction

Pseudo first order reaction is defined as a reaction which is originally a second order, but is made to behave like a first order reaction.

In second order reaction, the rate depends on the concentration terms of two reactants.

Examples:

1. Hydrolysis of ester (methylacetate or ethylacetate) catalyzed by H+ ions. Here, the concentration of H+ ions remains constant Therefore, the rate solely depends on the concentration of ester.
2. Base-catalyzed oxidative degradation of prednisolone in aqueous solution.
3. Hydrolysis (inversion) of sucrose to glucose and fructose in aqueous solution catalysed by acid (water is in large excess) .
4. Acid catalysed hydrolysis of erythromycin oxime .
5. Acid catalysed hydrolysis of digoxin.

Second Order Reaction

Second order reaction is defined as a reaction in which the rate depends on the concentration terms of two reactants each raised to the power one.

In a second-order reaction, the rate of reaction depends on the concentration of two reactant molecules or on the square of one reactant’s concentration. The rate decreases more rapidly compared to first-order reactions, and the half-life is inversely proportional to the initial concentration. This means that as the initial concentration increases, the half-life decreases. Second-order kinetics are typically observed in bimolecular reactions and certain oxidation or substitution processes.

Examples:

1. Alkaline hydrolysis of ester such as methylacetate or ethylacetate.
2. Hydrolysis of chlorbutanol in presence of sodium hydroxide.

Force degradation study

Forced degradation, also known as stress testing, refers to the process of deliberately subjecting a new drug substance or drug product to conditions more severe than those used in accelerated stability studies. This is done to induce degradation rapidly and extensively, which serves several critical purposes in pharmaceutical development. Forced degradation is essential for demonstrating the specificity and robustness of stability-indicating analytical methods by ensuring these methods can distinguish the drug substance from its degradation products. Moreover, it provides valuable insight into the degradation pathways and chemical behavior of the molecule, helping to elucidate the structure of degradation products and understand their formation mechanisms. This information is vital for formulation development and packaging design, helping to optimize product stability and ensure that protective measures in packaging adequately safeguard the drug during its shelf life[13],[14].

Forced degradation (also called stress testing) is an essential part of stability testing that helps:

• Understand degradation pathways
• Identify degradation products
• Establish the molecule’s intrinsic stability
• Develop and validate stability-indicating analyse methods

Unlike formal stability studies conducted under prescribed regulatory conditions (such as ICH guidelines), forced degradation studies are conducted over a shorter time frame and under harsher environmental stresses, including acid/base hydrolysis, oxidation, photolysis, and thermal degradation. These conditions are carefully selected to generate about 5-20% degradation, which is sufficient to identify critical degradation products without overwhelming the analytical systems. Early implementation of forced degradation studies during drug development facilitates timely formulation improvements, appropriate storage condition recommendations, and the development of validated, stability-indicating analytical methods that are essential for regulatory submissions [13],[15].

Objective of forced degradation studies

Forced degradation studies, also known as stress testing, are purposely designed experiments where drug substances and drug products are subjected to conditions harsher than those of accelerated stability studies. The main objective of these studies is to understand the intrinsic stability and degradation behavior of pharmaceutical compounds, thereby supporting drug development, formulation, packaging, regulatory compliance, and quality control.

1. Establishing Degradation Pathways

Forced degradation studies elucidate the specific chemical routes through which drug substances and products break down under stress. Understanding these pathways offers insight into potential degradation products and how environmental or manufacturing factors influence drug stability.

2. Differentiating Drug-Related and Non-Drug-Related Degradants

Because formulations often include excipients and other non-active components, forced degradation helps distinguish impurities that originate from the drug substance versus those arising from the formulation excipients or packaging materials, informing formulation optimizations.

3. Elucidating Degradation Product Structures

Characterizing the chemical structure of degradation products is crucial to evaluate their safety and impact on drug efficacy. Forced degradation facilitates isolation and structural identification of these products by providing stressed samples for analytical methods development.

4. Determining Intrinsic Stability

These studies reveal inherent weaknesses in the chemical stability of drug substances, allowing scientists to predict how molecules will behave in different environments and aiding in mitigating risks early in development

5. Revealing Degradation Mechanisms

Forced degradation exposes mechanisms such as hydrolysis, oxidation, photolysis, thermal decomposition, and others that might be responsible for drug degradation. Understanding these enables better control through formulation or packaging measures

6. Establishing Stability-Indicating Analytical Methods

Validation of analytical methods that precisely measure drug and degradation products is key in regulatory submissions. Forced degradation samples ensure these methods selectively detect the drug substance without interference from degradants

7. Understanding Chemical Properties and Behavior

Information obtained assists in understanding the physicochemical properties, reactivity, and stability profile of the drug molecules under various conditions, supporting drug design and process development

8. Generating More Stable Formulations

By identifying weak points in stability, forced degradation outcomes guide formulation scientists in selecting excipients, adjusting pH, or improving packaging to enhance the drug's shelf life

9. Producing Degradation Profiles Comparable to Long-Term Stability

Forced degradation efforts strive to mimic the degradation kinetics and product profiles observed during formal ICH-guided stability studies, thus providing predictive insights earlier in development

10. Solving Stability-Related Problems

Fig.1.2- Forced dehydration study

 Types of stability types

Stability studies of drug products (formulations) are essential to evaluate how the formulation components, excipients, and packaging affect the product’s safety, efficacy, and quality over time under various storage conditions. These studies are conducted in three main phases:

1. Long-term Stability Study:

This study provides real-time data for establishing the product's shelf life or retest period under actual storage conditions recommended on the product label. Storage conditions are based on climatic zones—for example, 25 °C ± 2 °C/60% RH ± 5% RH for temperate zones (Zone I & II), and up to 30 °C ± 2 °C/75% RH ± 5% RH for hot and very humid zones (Zone IVb, including India and Southeast Asia). Data collection typically begins at 0, 3, 6, 9, and 12 months, continuing annually until the end of the proposed shelf life, which can span 24 to 60 months. A minimum of 12 months of long-term data is required at the time of regulatory submission [2],[5].

2. Accelerated Stability Study:

This phase aims to hasten degradation under more stressful conditions (40 °C ± 2 °C/75% RH ± 5% RH) to quickly identify potential stability issues. The duration is usually 6 months. If no significant change—defined as less than or equal to 5% degradation or no failure in physical or chemical tests—is observed, the accelerated data can be used to support shelf-life extrapolation [2],[6].

3. Intermediate Stability Study:

An intermediate condition study (30 °C ± 2 °C/65% RH ± 5% RH for 12 months) acts as a bridge when the accelerated conditions cause significant instability, helping to ensure stability under conditions between long-term and accelerated testing. This is particularly used to understand whether the product can withstand slightly harsher conditions than those of the long-term storage [2].

These studies collectively support the determination of appropriate storage conditions, shelf-life labelling, and packaging decisions to ensure product quality and patient safety throughout the product lifecycle.

Table 1.2- Stability Study types [12]

Table 1.3- Key difference between Drug Substances vs  Drug product

A stability-indicating assay method (SIAM) is a validated analytical procedure that accurately measures the active pharmaceutical ingredient (API) in a drug substance or drug product without interference from degradation products, impurities, or excipients. The primary purpose of SIAM is to detect chemical degradation over time, monitor the shelf life of drug substances and products, and ensure their safety, efficacy, and quality throughout storage. APIs can degrade due to several environmental factors such as heat, humidity, light, oxidation, or pH changes. Regular assay methods may not distinguish the intact API from its degradants, making SIAM indispensable for stability studies. The International Council for Harmonisation (ICH) guidelines Q1A and Q2(R1) require stability-indicating methods for regulatory approval as they provide critical evidence of a product’s ongoing quality and integrity during its shelf life. SIAM typically involves separation techniques such as high-performance liquid chromatography (HPLC), gas chromatography, or spectroscopic methods coupled with detectors to achieve specificity and sensitivity in detecting degradation products [18],[19],[2].

These methods are vital throughout pharmaceutical development and quality control to ensure that the drug product maintains its intended potency and remains free from interfering impurities caused by degradation. Additionally, SIAM assists in investigating out-of-trend or out-of-specification results, providing a powerful tool for quality assurance processes [18].

• Heat: 50–80 °C (dry heat or oven).
• Humidity: high RH (e.g., 75% RH at 40 °C).
• Photostability: exposure to UV and visible light (per ICH Q1B).
• Oxidation: exposure to hydrogen peroxide or oxygen atmosphere.
• Hydrolysis: acidic, basic, and neutral aqueous solutions.
• Duration -Continued until 10–30% degradation is observed (sufficient to identify products).

Studies include:

• Thermal stress (dry & wet heat)
• Oxidation
• Hydrolysis
• Photolysis
• Freeze–thaw cycles (especially for biologics and injectables).
• Photostability studies (ICH Q1B)
• Performed on both drug product and packaging.
• In-use stability studies
• For multidose products (e.g., eye drops, injections, suspensions).
• Tests stability after opening the container.
• Packaging interaction studies

Checks if container–closure system affects product stability (e.g., leachables, adsorption, permeability).

Characteristics of a Good SIAM

A strong stability-indicating analytical method (SIAM) should be built around a core set of validated performance characteristics that ensure reliability and regulatory acceptability [20].

1. Specificity: Distinguishes API from all degradation products, excipients, and related substances under actual test conditions, including stressed samples. The method should demonstrate that peaks corresponding to degradants do not overlap with the API or with each other across the entire assay run. This is typically shown via forced-degradation studies and peak purity assessments using a diode array detector or mass spectrometry.
2. Accuracy: Reflects the closeness of measured API content to the true amount, verified through recovery studies at multiple levels (e.g., low, medium, high within the analytical range). Acceptable recovery is method- and matrix-dependent but commonly falls within 98–102% for assay methods across many regulatory frameworks.
3. Precision: Demonstrates reproducibility of measurements under varied conditions. This includes repeatability (intra-day, same analyst, same equipment) and intermediate precision (different days, analysts, and equipment). Typically expressed as %RSD and often required to be ≤2% for the assay in the main concentration range.
4. Linearity: Establishes a proportional response of the detector to API concentration across the intended analytical range, often from 50% to 150% of the label claim or method-specified range. Correlation coefficients (r^2) are used to assess fit, with acceptance criteria described in the applicable validation protocol.
5. Robustness: Assesses method reliability under small, deliberate variations in critical parameters such as pH, column temperature, mobile phase composition, flow rate, and detection wavelength. The method should remain capable of delivering acceptable results despite these minor changes.
6. Sensitivity: Defines the method’s ability to detect (LOD) and quantify (LOQ) low levels of degradants. These limits should enable reliable monitoring of potential impurities and degradants throughout stability studies.
7. System suitability: Verifies ongoing performance prior to sample analysis, including resolution between peaks, tailing factors, and plate counts. Acceptance criteria are specified in the validation protocol and are essential to ensure consistent method performance.
8. Range: Defines the interval over which the method produces acceptable accuracy, precision, and linearity. This range should encompass the concentrations encountered during stability testing, including potential degradant levels.
9. Specificity of degradation pathway detection: The SIAM should not only separate API from degradants but also allow identification (or at least characterization) of major degradant classes where possible, supporting interpretation of stability results and potential degradation mechanisms.
10. Documentation and traceability: All method development and validation activities should be thoroughly documented, with justifications for parameter choices, experimental design (including stress conditions), and statistical analyses. Regulatory submissions rely on well-structured dossiers that clearly demonstrate method performance and lifecycle considerations.

Steps in Developing a SIAM

Step 1: Forced Degradation / Stress Testing

Forced degradation, also known as stress testing, is a critical part of stability-indicating method (SIAM) development. Its primary objective is to generate potential degradation products of the active pharmaceutical ingredient (API) to assess the specificity of an analytical method. According to Stability-Indicating Methods for Drug Analysis guideline, stress conditions should be selected based on the chemical nature of the drug substance[20],[2].Typical stress parameters include acid and base hydrolysis (using 0.1–1 N HCl or NaOH to simulate hydrolytic degradation), oxidation (commonly induced by 1–3% hydrogen peroxide), thermal degradation (temperatures ranging from 40°C to 80°C depending on API stability), photolysis (exposure to UV and visible light as per ICH Q1B), and humidity stress (usually 75% RH at 40°C). The level of degradation should ideally range between 5% and 20% to sufficiently challenge the method without completely destroying the analyte.

Step 2: Selection of Analytical Technique

Choosing the appropriate analytical technique is essential for accurate identification and quantification of degradation products[21].Ultra-Performance Liquid Chromatography (UPLC) offers faster analysis with better resolution due to smaller particle sizes in the stationary phase. Coupling chromatographic techniques with mass spectrometry (LC-MS or LC-MS/MS) facilitates identification of unknown degradant structures by providing molecular weight and fragmentation data. UV-Visible spectroscopy can be used when degradation products do not interfere spectrally with the API. For rapid, preliminary assessments, Thin-Layer Chromatography (TLC) or High-Performance TLC (HPTLC) serves as a simple and cost-effective screening method.

Step 3: Method Development

Method development involves the systematic optimization of chromatographic parameters to achieve a stability-indicating profile. Key parameters include the selection of an appropriate column (e.g., C18 reverse-phase), mobile phase composition (such as buffer–organic solvent ratio), pH optimization (to stabilize ionizable drugs), flow rate, and detection wavelength based on the UV-Vis absorption spectrum of the compound[22]. The goal is complete resolution of the API peak from all degradant peaks, verified by peak purity analysis using a Diode Array Detector (DAD) or Mass Spectrometer. The method must ensure no co-elution occurs between analyte and degradation products, establishing true specificity of the method.

Step 4: Method Validation

Validation of the stability-indicating method must follow the International Council for Harmonisation guideline, which defines parameters such as specificity, linearity, accuracy, precision, detection/quantitation limits, robustness, and system suitability[23].Specificity demonstrates that the API is clearly separated from degradation products and excipients. Linearity assesses proportionality of response across a defined range (typically 50–150% of nominal concentration). Accuracy is evaluated by recovery studies, which should fall within 98–102%. Precision is verified through repeatability, ensuring relative standard deviation (%RSD) below 2%. Limits of Detection (LOD) and Quantitation (LOQ) determine the minimum detectable and quantifiable concentrations of degradants. Robustness confirms method reliability against small deliberate variations (e.g., temperature, pH, or mobile phase ratio). System suitability tests, such as evaluating resolution, tailing factor (<2), and number of theoretical plates, ensure performance consistency.

Step 5: Application in Stability Studies

A validated stability-indicating method is then employed in formal stability studies to monitor the drug’s potency and degradation profile over time under prescribed storage conditions. These include long-term (25°C ± 2°C/60% RH ± 5%), intermediate (30°C ± 2°C/65% RH ± 5%), and accelerated (40°C ± 2°C/75% RH ± 5%) conditions[1]. The method quantifies the API content and any degradation products at various time intervals, supporting determination of shelf life (t90 — time at which the API retains 90% of its initial concentration). Data generated from these studies provide a scientific basis for establishing appropriate storage recommendations, expiration dating, and regulatory filing of stability data in accordance with ICH Q1A(R2) guidelines and regulatory requirements across major health authorities[2].

Stability Study Specifications

Stability studies evaluate how a drug substance or product maintains its quality over time under specific environmental conditions (temperature, humidity, light). Specifications focus on critical quality attributes (CQAs) that ensure safety, efficacy, and quality.

Table 1.3- Drug substances specification[25],[26]

Table 1.4- Dosage form specification [27],[28]

A stability study protocol is a documented plan that details the conditions, tests, intervals, and methods used to evaluate the stability of a drug substance or drug product over time.

Purpose of a Stability Study Protocol

1. Determine the shelf life or retest period of the drug substance or drug product by assessing chemical, physical, microbiological, and biological attributes that may change during storage.
2. Identify and characterize degradation products to understand potential pathways and impacts on safety and efficacy.
3. Establish appropriate storage conditions and labeling instructions, ensuring product quality throughout its lifecycle.
4. Ensure regulatory compliance with applicable guidelines such as ICH Q1A(R2), FDA, EMA, and WHO standards for stability testing.

1. Title Page

• Name of the drug substance or dosage form Batch/lot number Study type: long-term, accelerated, intermediate
• Date of study initiation
• Study location and sponsor name
• Study protocol version and date

2. Objective [2]

• To evaluate the stability under long-term, accelerated, and intermediate storage conditions.
• To determine shelf life (t90) and recommend appropriate storage conditions.
• To monitor assay, degradation products, physical attributes, microbiological quality, and other critical quality attributes (CQAs).
• To support labeling claims of expiry date and storage instructions.

3. Scope [29]

• Applies to the Active Pharmaceutical Ingredient (API) or finished dosage form.
• Includes all relevant physical, chemical, microbial, and dissolution tests.
• Covers samples stored in the intended commercial packaging under ICH recommended climatic zones. Includes stability testing of any changes in formulation or manufacturing process.

4. Reference Standards and Guidelines[30]

• ICH Guidelines: Q1A(R2) (Stability Testing), Q1B (Photostability Testing), Q2(R1) (Analytical Validation)
• USP / Ph. Eur. monographs relevant to the drug substance and dosage form
• Analytical reference standards for assay and impurity characterization
• Pharmacopoeial methods and manufacturer’s internal SOPs for stability testing

5. Test Items

• Batch number, quantity, dosage form, packaging, and storage containers
• Storage conditions details: temperature, humidity, light exposure, and packaging type.

6. Storage condition

Table 1.5- Different stability conditions [12]

7. Analytical Methods [2]

• List of analytical techniques used for testing, including validation status
• Assay methods (e.g., HPLC, UV)
• Impurity profiling (e.g., HPLC, LC-MS)
• Physical tests (appearance, dissolution, hardness, friability)
• Moisture content (Karl Fischer)
• Microbial limits (if applicable)pH and solubility tests Polymorphic analysis (DSC, XRD)

8. Acceptance Criteria [2]

• Specifications for each test parameter based on Pharmacopoeial standards or internal criteria
• Assay limits (typically 90-110% of label claim)
• Limits for impurities and degradants as per ICH Q3B guidelines
• Physical parameters within predefined acceptance ranges Microbial limits conforming to Pharmacopoeial limits

9. Data Handling and Statistical Analysis

• Description of data collection and recording procedures Statistical methods for trend analysis and shelf-life estimation (e.g., regression analysis for assay decline)
• Procedures for outlier identification and handling Documentation and reporting format

10. Reporting

• Format and content of stability study reports
• Summary of results at each time point
• Conclusions on shelf life and storage recommendations Any deviations, anomalies, or stability failures documented and explained
• Approval signatures and dates

11. Appendices Copies of analytical method validation reports

• Raw data sheets and chromatograms
• Certificates of analysis for batches tested
• Photostability data (if applicable)
• Glossary of terms and abbreviations

Objective:

Determine the stability of Amlodipine 5 mg tablets under ICH-recommended long-term and accelerated conditions to establish shelf life and storage recommendations.

Test parameters and methods

Table 1.6- Test parameter of Amlodipine Besylate [31]

• Accelerated: 40°C ± 2°C / 75% RH ± 5%, sampled at 0, 1, 3, 6 months
• Long-term: 25°C ± 2°C / 60% RH ± 5%, sampled at 0, 3, 6, 12 months.

Selection of sample

The protocols for the selection of samples are as following

The protocol for stability testing is a pre-requisite for starting stability testing and is necessarily a written document that describes the key components of a regulated and well-controlled stability study. Because the testing condition is based on inherent stability of the compound, the type of dosage form and the proposed container-closure system, the protocol depends on the type of drug substance or the product. In addition, the protocol can depend on whether the drug is new or is already in the market. The protocol should also reflect the regions where the product is proposed to be marketed e.g. if the product is planned to be used in climatic zones I-III, Iva and IVb, the stability program must include all these zones. A well-designed stability protocol should contain the following information[32].

Batches

Stability studies at developmental stages are generally carried out on a single batch while studies intended for registration of new product or unstable established product are done on first three production batches, while for stable and well-established batches, even two are allowed. If the initial data is not on a full-scale production batch, first three batches of drug product manufactured post-approval should be placed on long-term studies using the same protocol as in approved drug application. Data on laboratory scale batches obtained during development of pharmaceuticals are not accepted as primary stability data but constitute supportive information. In general, the selection of batches should constitute a random sample from the population of pilot or production batches[32].

Containers and closures

The testing is done on the product in immediate containers and closures proposed for marketing. The packaging materials include aluminum strip packs, blister packs, Alu-Alu packs, HDPE bottles etc. This may also include secondary packs, but not shippers. Products in all different types of containers/closures, whether meant for distribution or for physician and promotional samples, are to be tested separately. However, for bulk containers, testing in prototype containers is allowed, if it simulates the actual packaging[32].

Orientation of storage of containers

Samples of the solutions, dispersed systems and semi solid drug products for stability studies must be kept upright and positioned either inverted or on the side to allow for full interaction of the product with the container-closure. This orientation helps to determine whether the contact between the drug product or solvent and the closure results in the extraction of chemical substances from the closure components or adsorption of product components in to the container-closure[32].

Sampling time points

Frequency of testing should be such that it is sufficient to establish the stability profile of the new drug substance. For products with a proposed shelf life of at least 12 months, the testing frequency at the long-term storage condition should be every 3 months over the first year, every 6 months over the second year and annually thereafter throughout the proposed shelf-life expiration date. In the case of accelerated storage conditions, a minimum of three time points, including the initial and end points, for example, 0, 3, and 6 months is recommended. When testing at the intermediate storage condition is necessary as a result of significant change at the accelerated storage condition, a minimum of four test points, including the initial and final time points, is recommended, for example, 0, 6, 9 and 12 months[32].

Table 1.7- Test Schedule for stability testing of new products[32].

Sampling Plan

Sampling plan for stability testing involves, planning for the number of samples to be charged to the stability chambers and sampling out of the charged batch so as to cover the entire study. The first step should be the development of the sampling time points followed by the number of samples needed to be drawn at each pull point for complete evaluation of all test parameters and finally adding up to get the total number of samples. For example, there would be a requirement of about 100 tablets per pull out in a long term or accelerated stability studies including 10 each for assay, hardness and moisture determination, 6 each for dissolution and disintegration and 50 for friability. This multiplied by the total number of pull outs will give the total number of tablets required for a study. This is followed by the development of a sampling plan, which includes the selection of the containers representing the batch as a whole but in an unbiased manner. A stratification plan has been suggested whereby from a random starting point every nth container is taken from the filling or packaging line (n is chosen such that the sample is spread over the whole batch)[32].

Test storage conditions

The storage conditions to be selected are based upon the climatic zone in which the product is intended to be marketed or for which the product is proposed to be filed for regulatory approval. General recommendations on the storage conditions have been given by ICH, CPMP and WHO. The abridged/indicative ICH and WHO storage conditions for drug products have been given in Table 1.8 [32].

Table 1.8- Stability test storage conditions for drug Products[32].

The stability test protocol should define the test parameters that would be used for evaluation of the stability samples. The tests that monitor the quality, purity, potency, and identity which could be expected to change upon storage are chosen as stability tests. Therefore appearance, assay, degradation products, microbiological testing, dissolution, and moisture are standard tests performed on stability test samples. Microbiological tests include sterility, preservative efficacy and microbial count as applicable e.g. for liquid injectable preparations. The batches used for stability study must meet all the testing requirements including heavy metals, residue on ignition, residual solvents etc. Some of these are required at the time of product release but not required to be repeated during stability testing[32].

Test methodology

It is always recommended to follow the procedures given in the official compendia, as the results obtained using the official tests, in general find better acceptance. If alternate methods are used, they are required to be duly validated. However, the assay of the drug should be carried out using a stability-indicating method, established by carrying out stress tests on the drug under forced decomposition conditions. This method should be validated for specificity, accuracy, precision and linearity, in the range to which the drug is expected to fall during stability studies. For the assay of degradation products, the validated method should also include the limits of detection/quantification. The methods reported in literature should be used after confirming reproducibility and carrying out minimal validation, say of linearity, range, etc. It is always recommended to prepare a standard test protocol (STP) for each test[32].

Acceptance criteria

All analytical methods are required to be validated before initiating the stability studies. Similarly, the acceptance criteria for the analytical results as well as that for the presence of degradation products should also be fixed beforehand. The acceptance criteria for each test in the stability study are fixed in the form of numerical limits for the results expressed in quantitative terms e.g., moisture pick-up, viscosity, particle size, assay, degradation products, etc. and pass or fail for qualitative tests e.g., odor, color, appearance, cracking, microbial growth, etc. These acceptance criteria should also include individual and total upper limits for degradation products. ICH guideline Q3B(R2) related to impurities in new drug products addresses degradation products in new drug formulations. The degradation products of the active or interaction products from the active ingredients and excipients and/or active and container component should be reported, identified, and/or qualified when the proposed thresholds are exceeded. The reporting threshold of impurities is based upon the intended dose. If the maximum daily dose is less than or equal to 1gm, the limit is 0.1% and if greater than 1, the limit is 0.05%. The identification threshold of impurities is between 1.0-0.1% for the maximum daily dose ranging between 1mg and 2gm [32].

Table 1.9- Selection of samples [2]

The storage conditions to be applied to different stability studies must be strictly controlled within the ranges defined by regulatory guidelines to ensure reliable stability data. The equipment used for stability storage should have the capability to maintain temperature and humidity accurately according to these specifications. During the study, actual temperature and humidity should be continuously monitored, especially when humidity control is required [2],[4].

Short-term deviations or spikes, such as those caused by opening the doors of the storage facility, are generally considered unavoidable and acceptable. However, any excursions due to equipment failure or other reasons should be thoroughly documented and addressed in the study report if they are judged to impact the stability results.

Specifically, excursions that exceed the defined tolerances for more than 24 hours must be described in detail in the stability study report, and an assessment of their effect on product quality should be conducted. This ensures transparency and allows regulators to evaluate the risk of instability under real-world storage conditions.

These requirements are consistent with ICH Q1A(R2) guidelines which state that:

• Stability storage equipment must be capable of controlling the conditions within set tolerance limits.
• Monitoring records of actual temperature and humidity during storage should be maintained.
• Short transient spikes due to typical operations like door openings are acceptable.
• Extended excursions beyond defined limits (over 24 hours) need to be reported with an evaluation of impact on product stability.
• The storage conditions and duration must be sufficient to cover storage, shipment, and subsequent use, ensuring the drug substance and drug product remain within specifications throughout their shelf life[2].

This comprehensive approach helps ensure that the integrity and quality of pharmaceutical products are maintained throughout their marketed shelf-life periods, safeguarding patient safety.

Zones of stability

For the purpose of stability testing, the whole world has been divided into four zones (I - IV) depending upon the environmental conditions the pharmaceutical products are likely to be subjected to during their storage. These conditions have been derived on the basis of the mean annual temperature and relative humidity data in these regions. Based upon this data, long-term or real-time stability testing conditions and accelerated stability testing conditions have been derived. The break-up of the environmental conditions in each zone and also the derived long-term stability test storage conditions, as given by WHO have also been presented. The stability conditions have also been harmonized and adjusted to make them more practical for industry application and rugged for generalized application [32].

The International Council for Harmonisation (ICH) has established five climatic zones recognized globally for stability testing:[2]

• Zone I: Temperate Zone (21°C / 45% Relative Humidity), including regions such as Southern Canada and Europe.
• Zone II: Mediterranean/Subtropical Zone (25°C / 60% RH), covering areas like the Mediterranean, parts of Australia, and the southern USA.
• Zone III: Hot/Dry Zone (30°C / 35% RH), typical of deserts and arid areas including parts of North Africa and the Middle East.
• Zone IVa: Hot Humid/Tropical Zone (30°C / 65% RH), including Southeast Asia and Central Africa.
• Zone IVb: Hot/Higher Humidity Zone (30°C / 75% RH), for regions near the equator and dense rainforests like the Amazon Basin.

These zones are critical to simulate the specific climatic conditions drugs may be exposed to in different regions so that stability testing ensures the drug’s efficacy and safety under those environmental stresses. Testing products under the appropriate climatic zone conditions helps establish reliable shelf life and storage recommendations globally.

Testing frequency determination in stability studies is a critical aspect of drug development and registration, as detailed by the International Council for Harmonization (ICH) Q1A and Q1D guidelines [33]. The frequency of testing depends on the type of study (long-term vs. accelerated) and the use of reduced designs like bracketing and matrixing, which optimize resources based on sound scientific justification. 

Full design testing frequency

The standard testing frequency provides the baseline for stability testing and is applied to all samples in a full study design. 

• Long-Term Storage Conditions
  • • Proposed shelf life of at least 12 months: Testing is typically performed every 3 months during the first year, every 6 months during the second year, and annually thereafter through the proposed shelf life.
    • Proposed shelf life of less than 12 months: More frequent testing may be required to establish a reliable stability profile.
• Accelerated Storage Conditions

• Study duration: 6 months.
• Testing points: A minimum of three time points, including initial, 3 months, and 6 months.

• Intermediate Storage Conditions
  • • Study duration: 12 months.
    • Testing points: A minimum of four time points, including initial, 6, 9, and 12 months. 

Determining testing frequency with bracketing

Bracketing simplifies the stability protocol by reducing the number of different samples that must be tested at each time point, not the time points themselves. The testing schedule remains the standard full design, but it is applied only to the extreme samples. 

Case study: Bracketing testing frequency
A company manufactures an oral solution in three concentrations: 10 mg/mL, 20 mg/mL, and 50 mg/ml. The formulations are identical, differing only in the amount of active pharmaceutical ingredient (API). The proposed shelf life is 24 months. 

1. Determine standard testing frequency
   The standard frequency for a 24-month shelf life is:

• Year 1: 0, 3, 6, 9, 12 months
• Year 2: 18, 24 months
• Total long-term time points: 7 

2. Apply the bracketing principle
   Instead of testing all three strengths at every time point, the bracketing design focuses on the extremes (10 mg/mL and 50 mg/mL). 
3. Define the testing schedule

• 10 mg/mL: Tested at 0, 3, 6, 9, 12, 18, and 24 months.
• 20 mg/mL: Not tested. Its stability is assumed to be bracketed by the 10 mg/mL and 50 mg/mL strengths.
• 50 mg/mL: Tested at 0, 3, 6, 9, 12, 18, and 24 months. 

Result: The testing frequency for the tested batches remains the same as the standard schedule. The resource savings come from eliminating the intermediate sample from all testing time points.

Determining testing frequency with matrixing

Matrixing involves a more complex reduction in testing frequency by testing different subsets of samples at different time points. This is particularly useful when dealing with multiple batches, strengths, or container sizes. 

Case study: Matrixing testing frequency
A company produces tablets in three strengths (S1, S2, S3) from three different batches (B1, B2, B3) for a total of nine factor combinations. The company wants to implement a one-third matrixing design for a proposed 36-month shelf life. 

• 1. Determine the standard testing frequency
     For a 36-month shelf life, the standard long-term time points are: 0, 3, 6, 9, 12, 18, 24, and 36 months. 
  2. Apply the matrixing principle
     The matrixing design requires testing all samples at the initial (0 months) and final (36 months) time points. Intermediate time points are staggered. 
  3. Define the matrixing schedule (one-third reduction) A one-third reduction means that at any given intermediate time point, only one-third of the total batches are tested. 
  4. Evaluate testing frequency
• Individual Batch Frequency: The frequency is no longer uniform for every batch. For example, S1 (B1) is tested at 0, 3, 12, 18, and 36 months, a different set of points than a standard study.
• Overall Time Point Frequency: At intermediate points like 3, 6, 9, 18, and 24 months, only 3 of the 9 batches are tested.
• Justification: This design can be justified if supporting data shows low variability between batches and strengths, demonstrating that a subset of data points is sufficient to model the overall stability trend. 

Drug Product Example

• API: Amlodipine Besylate
• Dosage Form: Tablet
• Strengths: 2.5 mg, 5 mg, 10 mg
• Packaging Configurations:
  • Alu-Alu blister (most protective)
  • PVC/PVDC blister (moderate)
  • HDPE bottle with desiccant (least protective)

Objective: Establish stability profile while reducing unnecessary testing.

Full Factorial Design (Baseline)

If all combinations were tested:

• Strengths: 3
• Packs: 3
• Storage conditions: 3 (25°C/60%RH, 30°C/65%RH, 40°C/75%RH)
• Time points:
  • Long-term: 0, 3, 6, 9, 12, 18, 24 (7 points)
  • Intermediate: 0, 3, 6, 9, 12 (5 points)
  • Accelerated: 0, 3, 6 (3 points)

 Total pulls = 3 × 3 × (7+5+3) = 135 stability pulls.

 Step 1 – Bracketing Application

• Strength Bracketing: Test 2.5 mg (lowest) and 10 mg (highest).
  • Justification (ICH Q1D, Section 2.1): When the same formulation is used and only the API quantity differs, intermediate strengths can be bracketed.
• Packaging Bracketing: Test HDPE bottle (least protective) and Alu-Alu (most protective).
  • Justification (ICH Q1D, Section 2.2): Intermediate packaging configurations can be bracketed if extreme pack types are tested.

Revised pulls = 2 strengths × 2 packs × (7+5+3) = 60 pulls.

Bracketing reduced testing by ~55%.

Step 2 – Matrixing Application

Even after bracketing, 60 pulls remain. To optimize further:

• Matrixing (ICH Q1D, Section 3): At each time point, test only a subset of configurations, with subsets alternating across time points[33].

Matrixing Plan:

• At critical points (0, 6, 12, 24 months long-term; 0, 6, 12 months intermediate; 0, 3, 6 months accelerated) → test all 4 configurations.
• At non-critical points (3, 9, 18 months long-term; 3, 9 months intermediate) → test only 2 configurations, rotating across strengths/packs[33].

 Revised pulls ≈ 45 (instead of 135).

Table 1.11- Testing frequency table [35]

Drug :- Amlodipine besylate

Background

Amlodipine besylate is a dihydropyridine calcium channel blocker used in the management of hypertension and angina pectoris [2]. Like many drug substances, its stability is influenced by temperature and humidity. Being slightly hygroscopic, Amlodipine may undergo physical softening and increased impurity formation under high humidity conditions.

Objective

To determine the appropriate storage condition statement for labeling of Amlodipine Besylate Tablets (5 mg, blister packed) using accelerated and long-term stability data.

Study Design

Dosage Form & Packaging

• Dosage form: Amlodipine Besylate Tablets 5 mg
• Packaging: PVC/Alu blister packs (provides moderate moisture protection)

Storage Condition Decision

Proposed Label Statement

 “Store below 30 °C. Protect from moisture. Keep in the original package.”

• “Store below 30 °C” → Based on Zone IVb long-term stability data (India falls in hot & humid climatic zone).
• “Protect from moisture” → Due to hygroscopicity and physical instability under accelerated humidity.
• “Keep in the original package” → Ensures blister integrity and protection from atmospheric moisture.

Case study 3

How to decide suitable packing material [2]

Step 1 — API characterization

• Chemical structure review — look for hydrolytic, oxidizable, photolabile functional groups.
• Hygroscopicity / moisture sorption — Dynamic Vapor Sorption (DVS) or equilibrium moisture uptake (to quantify % uptake vs RH).
• Thermal behavior — DSC / TGA (detect melting, polymorph transitions, decomposition).
• Crystallinity / polymorphism — XRPD.
• Volatility / headspace GC — for volatiles.
• Partitioning (logP), pKa, solubility — predict interaction with plastics/excipients/liners.
• Stability-indicating analytical method (HPLC/LC-MS) must be available before stability runs — stress testing validates the method. (ICH expectation). 

Step 2 — Forced degradation / stress studies (targeted experiments)

Purpose: Reveal the most damaging stressors quickly so you know which packaging properties matter most.

Standard forced tests (use a small representative batch):

• Thermal (50–80 °C depending on API; short times).
• Humidity (75% RH chamber or DVS).
• Acid/base hydrolysis (if hydrolyzable).
• Oxidation (H?O?, AIBN, or air exposure).
• Photolysis (preliminary) — then follow with ICH photostability confirmatory test (next step).

Output: Identify primary degradants, mechanisms, and analytical markers to follow during packaging tests. ICH Q1A requires stress testing to support stability-indicating methods. 

Step 3 — Photostability (ICH Q1B) — required if photolabile risk exists

Use the confirmatory photostability exposures specified by ICH Q1B: ≥1.2 million lux hours visible light and ≥200 Wh/m² near-UV (or equivalent validated actinometric exposure). If the API shows unacceptable changes, packaging must provide light protection (amber glass, opaque container, alu–alu blister, secondary carton). 

Decision rule: any unacceptable change → include “protect from light” in label and select light-protective packaging.

Step 4 — Shortlist candidate container materials:

• Glass
  • Type I borosilicate — chemically inert, best for volatile/adsorptive APIs and parenteral drug substances.
  • Amber glass — for light protection.
• Plastics
  • HDPE/PP — common for powders; consider moisture permeability and sorption.
  • PVC — common blisters but poor moisture barrier (not ideal for hygroscopic APIs).
• Laminates / Blisters
  • Alu–alu / cold-form aluminum — excellent moisture/oxygen barrier; preferred for very moisture-sensitive solids.
  • PVC/Alu — moderate barrier; cheaper.
• Closures / liners / stoppers — evaluate elastomers for extractables/leachables and swelling.
• Mitigations — use desiccant sachets, nitrogen blanketing, secondary cartons to add protection.

Stability Results

• PVC blister → Not suitable (fails stability criteria).
• HDPE bottle + silica gel → Acceptable, but risk of oxidation remains.
• Amber glass bottle → Suitable for light-sensitive drugs; acceptable.
• Alu-Alu blister → Best stability results, excellent barrier.

Step 5 — Extractables & Leachables (E&L) screening (USP framework)

Two-tiered approach (recommended best practice/ regulatory expectation):

1. Extractables (supplier / worst-case studies) — aggressive solvent extraction and elevated temp to identify substances that could come out of the material (GC-MS, LC-MS, ICP-MS, TOC).
2. Leachables (real-time / stability conditions) — measure what actually migrates into the API under storage conditions and at timepoints.

Use USP chapters and FDA/industry E&L frameworks for study design, AET calculations, and toxicological assessment. If extractables include pro-oxidants or plasticizers at relevant levels, the material may be unsuitable. 

Step 6 — Container-Closure Integrity (CCI)

• Demonstrate the package maintains a physical barrier (no ingress of moisture/oxygen/contaminants) for intended shelf life.
• Use deterministic methods (vacuum decay, pressure decay, helium/tracer gas) where possible; validate against a Maximum Allowable Leak Limit (MALL). CCI is especially critical for sterile products but applies to non-sterile if ingress would affect stability. 

When to run: initial, during stability timepoints (select points), and final shelf-life check.

Step 7 — Formal ICH stability studies in candidate materials

Run parallel stability programs (same batches, same aliquots) in each shortlisted material to generate comparative data.

ICH practical points to follow:

• Batches: at least three primary batches (pilot/ production scale) are recommended for formal stability.
• Conditions & timepoints: Long-term (e.g., 25 °C/60% RH) and Accelerated (40 °C/75% RH); include intermediate when required by climatic zone. Typical timepoints: 0, 3, 6, 9, 12 months (year 1), then 18, 24 months etc., per ICH Q1A. Adjust to intended market climatic zone. 

Tests at each Timepoints:

• Assay (stability-indicating), impurities/ degradants, water content (KF/DVS), appearance/discoloration, residual solvents (if relevant), leachables (selected points), and CCI (where required).

Deliverable: comparative stability reports (assay + impurities vs time) for each packaging candidate.

Step 8 — Evaluate data and apply decision rules

Decision hierarchy (practical):

1. Does the API remain within assay & impurity specs in packaging candidate for intended retest period? If no, reject.
2. Are any leachables above AET / toxicological concern? If yes, reject or re-engineer.
3. Is CCI acceptable (no significant leaks)? If no, reject.
4. Are practical considerations acceptable (cost, supply, scalable filling)? If no, consider alternatives.

If more than one candidate passes, prefer the simplest/commercially robust option (lowest risk for supplier variability).

Regulatory note: justify material choice in regulatory filings with the stress data, E&L reports, CCI data, and stability results. 

Step 9 — Toxicology & safety assessment for leachables

For any identified leachable, quantify and compare to a toxicological threshold (AET/PDE) and perform identification + safety assessment. Follow USP/FDA E&L recommendations. If an unknown leachable is present at concerning levels, consider changing material. 

Step 10 — Final selection, labelling & submission package

Final packaging selection should be supported by: stress maps, photostability report (if relevant), extractables & leachables study, CCI validation report, full stability in final packaging (assay, impurities, water), supplier specs, and a justification narrative. Include required labelling such as “Protect from light” or “Keep tightly closed; store below X °C” where data support it.

Case study 4

Determination of Shelf Life  Drug :- Amlodipine Besylate [36]

1. Objective

The purpose of this study is to determine the shelf life of Amlodipine Besylate bulk drug substance by evaluating:

• Chemical stability (assay of active ingredient)
• Formation of degradation products
• Physical stability (appearance, moisture content, polymorphism)

Definition: Shelf life is the time during which a drug substance remains within its specification limits for potency and purity under specified storage conditions.

2. Drug Substance and Sample Selection

Rationale for selection: Bulk drug is used in formulation; stability data is required for regulatory submissions.

Sampling plan:

• Minimum 3 batches for statistical reliability
• Each batch has enough sample to perform all analytical tests in triplicate
• Time points:
  • Long-term: 0, 3, 6, 9, 12, 18, 24 months
  • Accelerated: 0, 1, 2, 3, 6 months

3. Storage Conditions

Based on ICH guidelines:

• Packaging: Amber glass bottles to prevent light and moisture effects.
• Destructive sampling: Each time point uses a separate container.

4. Analytical Testing

Objective: Detect chemical and physical changes over time.

Acceptance criteria:

• Assay ≥ 90% of initial potency
• Total impurities within regulatory limits
• No significant physical changes

5. Data Collection

• Samples tested at defined time points.
• Example of assay results under accelerated conditions (40°C / 75% RH):

• % Assay vs Time (zero-order)
• ln(% Assay) vs Time (first-order)

Observation: Gradual decrease in potency; degradation is linear in ln(% assay) → first-order kinetics.

6. Kinetic Analysis

Step 1: Determine reaction order

• Most drugs, including Amlodipine Besylate, follow first-order kinetics.
• First-order equation:

Step 2: Calculate degradation rate constant (k)

• Plot ln(% assay) vs time; slope = –k
• Example calculation:

• Slope = (4.574 – 4.605)/6 ≈ –0.0052 month?¹ → k = 0.0052 month?¹

Step 3: Calculate shelf life (t90)

• t90 = time when drug retains 90% potency:

Step 4: Extrapolation (if needed)

• Use Arrhenius equation to predict long-term stability from accelerated data:
• Assumption: Same degradation mechanism at high and normal temperature.

7. Statistical Analysis

• Replicates analyzed to calculate mean, standard deviation, %RSD.
• Linear regression applied to kinetic plot (ln(% assay) vs time).
• Outlier check ensures data reliability.

8. Shelf Life Assignment & Storage

• Based on analysis: Amlodipine Besylate shelf life = 24 months
• Recommended storage:
  • Store below 30°C
  • Protect from moisture and light
  • Use tightly closed amber containers

Case study 5

Evaluation of Stability Study [37]

Objective

The purpose of evaluating a stability study is to:

• Determine how the quality of a drug substance or drug product changes with time under environmental factors like temperature, humidity, and light.
• Establish re-test period (for drug substance) and shelf life (for drug product).
• Recommend appropriate storage conditions and packaging.

Evaluation Process

A. For Drug Substance

Step 1: Study Design

• Storage conditions:
  • Long-term: 25°C ± 2°C / 60% 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)

Step 2: Testing Parameters

B. For Drug Product

Step 1: Study Design

• Same temperature/humidity conditions as the drug substance.
• Include packaged dosage form (e.g., tablets in blisters, bottles, etc.).

Step 2: Testing Parameters

Study Design

1. Drug Substance – Amlodipine Besylate

Packaging: Double polyethylene bags inside HDPE container with screw cap.

Parameters Tested:

• Description (appearance)
• Assay (by HPLC)
• Related substances (impurity profile)
• Moisture content (Karl Fischer)
• pH (for solution)
• Specific optical rotation

2. Drug Product – Amlodipine Tablets 5 mg

Packaging: PVC/Aluminum foil blister packs.

Parameters Tested:

• Appearance (colour, texture, odour)
• Average weight
• Hardness, friability
• Assay (by HPLC)
• Related substances
• Dissolution
• Moisture content
• Microbial limit (if applicable)

5. Analytical Methodology

All analytical methods were validated according to ICH Q2(R1) guidelines for:

• Specificity
• Accuracy
• Precision
• Linearity
• Robustness

Chromatographic Conditions for Assay:

• Column: C18 (250 × 4.6 mm, 5 µm)
• Mobile Phase: Methanol : 0.02M Phosphate buffer (60:40)
• Flow Rate: 1.0 mL/min
• Detection: 238 nm
• Injection Volume: 20 µL

6. Observed Results

6.1 Drug Substance

Interpretation:

• Slight degradation at accelerated conditions.
• Impurities remain <0.5%.
• Stable for at least 12 months under long-term conditions.

6.2 Drug Product (Amlodipine Tablets 5 mg)