Article

Challenges in Analytical Method Development for Drug Products

Dr. Parag Gadkari
Assistant Vice President, Analytical Research and Development, Inventia Healthcare Private Limited

Introduction
Analytical method development has gained a lot of importance these days. This forms an integral part of regulatory filing in Europe and U.S. where tremendous technological advances in Analytical Instrumentation with focus towards reducing time of analysis without compromise in Precision and accuracy of analytical results are bring made. Globally, there are various strategic alliances of pioneers of Analytical Instrument Manufacturers e.g Agilent and Varian. All pharmaceutical organizations are investing a lot in such advanced analytical equipment. Dedicated specialized Functional Department is formed in many of Pharma companies named Analytical R&D due to which Stability Indicating Analytical Method Development is getting more and more challenging.

Method Development Process

The steps involved in method development and method validation depend upon the type of method being developed. However, the following steps are similar to most types of projects: 1. Method Development Plan 2. Gathering background information 3. Laboratory method development 4. Generation of test procedure 5. Validation protocol 6. Laboratory method validation 7. Validated test method generation 8. Validation report

IND (Investigational New Drug Application) and NDA (New Drug Applications) and to set expiration dates for the API or drug product. Before performing stability studies, a stability indicating method is necessary so that any possible degradants generated during storage conditions (such as 5C, 25C/60%RH and 40C/75%RH) can be separated, detected, and quantitated. SIM is useful for checking the quality of the drug substance or product that changes over time in response to environmental factors such as temperature, humidity and light. It establishes the storage and packaging conditions for the drug product. The method should be sensitive to the reportable impurity level. LOQ (Limit of quantiation), which is typically 0.05% of Label Claim, should be established in the method, and the method should be linear from LOQ to typically up to 150% of the nominal standard (std) concentration. for: Stability indicating methods are needed

Regulatory Guidance
To ensure compliance with quality and safety standards, the United States, Europe, Japan, and other countries have published compendia, or pharmacopeias, that describe official test methods for many marketed drug products. For example, compendia analytical methods found in United States Pharmacopeia 25 (USP 25) are legally recognized analytical procedures under section 501 (b) of the Federal Food, Drug, and Cosmetic Act. For these compendia methods, USP provides regulatory guidance for method validation (1). In addition, validation of analytical methods is covered by the United States Code of Federal Regulations (CFR). Specific references are 21 CFR 211.165 (e) and 21 CFR 211.194 (a). As part of this initiative, the International Conference on Harmonization (ICH) has issued guidelines for analytical method validation. Analytical guidance documents recently published by the ICH are: 1. Stability Testing (Q1) 2. Validation of Analytical Procedures (Q2) 3. Impurities in Drug Substances and Drug Products (Q3) 4. Specifications for New Drug Substances and Products (Q6)

Main Categories of Pharmaceutical Analytical Methods

Specific Tests
Tests designed for special quality parameters such as micron size, polymorphism, spatial structure, chirality of drug substance (API) are: 1. X-Ray Diffraction 2. Particle size determination 3. Residual solvent determination by GC/ GC-HS 4. Specific optical rotation 5. TLC for any specific chemical entity

1. Stability studies 2. API release 3. Drug product release 4. Toxicology dosing solutions 5. Excipient compatibility and preformulation 6. Packaging studies 7. Line extension Cases where stability indicating methods are not needed: 1. In process controls 2. Titration 3. Inorganics 4. Limit tests 5. Cleaning validation

Stability Indicating Method

A stability indicating method (SIM) is a quantitative test method that can detect possible degradants and impurities of drug substance (API) and drug products, normally using High Performance Liquid Chromatography. Stability information is needed for regulatory submissions such as

Main process flow of Analytical method development includes :

1. Generating the sample 2. Developing the method 3. Validate the method

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The essential validation parameters included are as follows: 1. Specificity - Ability to measure desired analyte in complex mixture. 2. A c c u r a c y - A g r e e m e n t b e t w e e n measured and real value. 3. Linearity - Proportionality of measured value to concentration. 4. Precision - Agreement between series of measurements. 5. Range - Concentration interval in which method is precise, linear, accurate. 7. Detection Limit - Lowest amount of analyte that can be detected. 8. Quantitaiton Limit - Lowest amount of analyte that can be measured. 9. Robustness - Reproducibility under normal but variable laboratory conditions.

e. Isomerization / Racemization f. Photo degradation

addition of an internal standard or other volumetric manipulation. 4. Solids that must first be dissolved or extracted. Samples that require sample pretreatment to remove interferences and / or protect the column or equipment from damage.

Degradation Phenomenon In Drug Product

a. Drug - Excipient/s Interaction b. Drug - Drug Interaction

Sample Preparation (Critical for Solids, Semi-Solids, Suspension)

Preparations of solution are as follows: 1. Selection of sample size 2. Sample pretreatment for reduction of particle size Factors Enhancing Degradation of Dosage Forms: 1. Changes in mechanical strength due to coating polymers in coated dosage forms 2. Changes in capsule shells with time and storage conditions. 3. Changes indicating release rate from polymeric matrix dosage forms including microspheres. 4. Drug leakage from Liposomes. 5. Changes in Melting Patterns of Suppositories. 6. Aggregation in Emulsions. 7. Moisture adsorption. 8. Discoloration. 9. Transfer of container components onto/into Drug by Adsorption and/or Absorption. 3. Selection of extracting solvent for bringing active ingredient into solution 4. Solubility of any molecule 5. Nature of dosage form 6. Chemical structure

Challenges in Method Development

Analytical method development, validation and transfer are key elements of any pharmaceutical developmental program. Effective method development ensures that laboratory resources are optimized, while methods meet the objectives required at each stage of development. Method validation required by regulatory agencies at certain stages of drug approval process is defined as the process of demonstrating that analytical procedures are suitable for their intended use. Method transfer is the formal process of assessing the suitability of methods in another laboratory. Each of these processes contributes to continual improvement of the methods and results in more efficient drug development. According to International Conference on Harmonization (ICH), the most common types of analytical procedures are: 1. Identification test 2. Quantitative test of the active moiety in samples of API or drug product or other selected components in the drug product 3. Quantitative tests for content of impurities 4. Limit tests for the control of impurities 5. Specificity / Selectivity / Sensitivity a. Chemical Degradation of Drug Substance / Drug Product b. Hydrolysis c. Dehydration d. Oxidation

Instruments used are as follows:

1. Mechanical Shaker 2. Ultra-sonication 3. Homogenization

Filtration technique:
1. Adsorption : Excipient and drug adsorption can hinder filtration of solution 2. A b s o r p t i o n : R e f e r e n c e U S P monograph of Nifidepine formulations (similar molecule, Isradipine also exhibits same effect)

Sample Preparation
Important Information concerning sample composition and properties: Most sample preparations involve the use of organic-aqueous and acid-base extraction techniques. Therefore it is very helpful to understand the chemical, physical properties of the analytes, structurally related compounds, solubility and pKa of the analytes. Solubility in different organic or aqueous solvents determines the best composition of the sample solvent. pKa determines the pH in which the analyte will exist as a neutral or ionic species. This information will facilitate an efficient sample extraction scheme and determine the optimum pH in mobile phase to achieve good separations.

For the sample preparation of liquids:

1. Liquid-liquid extraction 2. Solid phase extraction 3. Precipitation with organic solvents

Selection of Analytical Technique:

As far as separation technique is concerned fast LC/HPLC will be first choice. Other techniques include: 1. For most qualitative and quantitative PDA detector 2. For Non-absorbing impurities ELSD/ CAD 3. F o r s o m e s p e c i f i c m o l e c u l e s Fluorescence/RI 4. Hyphenated techniques LCMS/GC MS/LC-NMR 5. After specificity is established UV Detector is choice

Sample pretreatment
1. Samples come in various forms: 2. Solutions ready for injection. 3. Solutions that require dilution, buffering,

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 Forced degradation (Chemical Stress) Studies

Forced degradation studies typically involve the exposure of representative samples of the drug substance or drug product to the relevant stress conditions of light, heat, humidity, acid/base hydrolysis, and oxidation. These experiments play an important role in the drug development process to facilitate stability indicating method development, drug formulation design, selection of storage conditions and packaging, better understanding of the potential liabilities of the drug molecule chemistry and the resolution of stability related problems. An appropriate sample solvent and mobile phase must be found that affords suitable stability and compatibility with the components of interest as well as the potential impurities and degradants. The method should be carefully examined for its ability to distinguish primary degradants from secondary degradants (degradation products of the primary degradants). An optimal wavelength must be selected that suitably detects all of the critical impurities and degradants. Finally, the method should provide adequate mass balance recovery of the major degradants. Example- Conditions for forced degradation: The goal is to degrade the active moiety to 5-10% in sample for which the conditions can be used as follows: Study Acidic pH Neutral pH Basic pH Oxidation Conditions 0.1N HCl pH 7.0 phosphate buffer 0.1N NaOH O2 Atmosphere, H2O2

Reporting Threshold and Dosage Relation

Maximum Daily Dose Thresholds for Identification TDI-Total Daily Intake < 1 mg 1 10 mg > 10 mg >2g 2g 1.0% or 5 g TDI, whichever is lower 0.5 % or 20 g TDI, whichever is lower 0.2 % or 2 mg TDI, whichever is lower 0.1%

a special procedure. 7. Quantitative calibration. 8. Validate method to release for routine laboratory. Case study: Sample preparation- USP monograph for extended release tablets mentions use of solvent as Acetonitrile: water (2:98) with use of homogenizer. Final estimation on HPLC: Findings: All the brands cannot be analysed by using above solvents and apparatus. Solutions found: Use of organic solvent like ethanol or methanol is essential for dispersion of polymeric matrix in SR formulation. 1. USP monograph of CPH and PPH combination extended release capsules mentioned in old monograph as use of solvent as water and dilute phosphoric acid and shaking by mechanical means. Final estimation on HPLC Findings: All the brands cannot be analysed by using above solvent and apparatus Solution found: Recent monograph has been updated as use of water and dilute phosphoric acid as solvent; however solution is heated till the complete dispersion of matrix. 2. Extraction of drug from fixed dose combinations 2 or 3 activities in Tablet Labeled amount of active ranging from 5mg to 500mg with variable polarities. Findings: All standard solvents like ethanol, methanol, methanolic HCl/NaOH, Not effective to bring into solution. Solution found: Use of Dimethyl formamide in combination with organic solvents and or mobile phase. 3. Challenge Run time For Related Substance (RS) run time exceeds more than 50mins. Findings: Longer run time are costlier. Solutions found: Analytical equipment change like Fast LC (Rapid Resolution Liquid Chromatography) shorten run time resulting in reduction in man power, analysis cost due to lower solvent composition. Reference: BP/EP method for Azithromycin RS.

Reporting Threshold and Dosage Relation

Maximum Daily Dose Threshold for Qualification < 10 mg 10 100 mg 1.0% or 50 g TDI, whichever is lower 0.5 % or 200 g TDI, whichever is lower 0.2 % or 2 mg TDI, whichever is lower 0.1%

> 100 mg 2g > 2g

Stability Indicating Method development Exercise

A stability indicating method (SIM) is a quantitative procedure used to detect a decrease in the amount of the active pharmaceutical ingredient (API) present due to degradation. According to FDA guidelines, a SIM is defined as a validated analytical procedure that accurately and precisely measures active ingredients (drug substance or drug product) free from potential interferences like degradation products, process impurities, excipents, or other stability impurities, and the FDA recommends that all assay procedures for stability studies be stability indicating. The steps involved in stability indicating method developmental exercise are as follows : 1. Information on sample. 2. Need for special HPLC procedure, sample pretreatment, etc. 3. C h o o s e d e t e c t o r a n d d e t e c t o r settings. 4. Chose LC method, preliminary run and estimate best separation conditions. 5. Optimize separation conditions. 6. Check for problems or requirement for

Photolysis (UV) 1000 Watts Hrs/M2 Photolysis 6 x 106 Lux Hrs (Fluorescence)

Reporting Threshold and Dosage Relation

Maximum Daily Dose a Thresholds for Reporting 1g >1g 0.1% 0.05% Threshold b

a-The amount of drug substance administered per day b-Threshold is based on % of Drug Substance

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