PHARM 462 2009

European and International regulatory bodies and their guidelines on different aspects of QA Body Full name Guidance on Eurachem Focus for Analytical Chemistry in Europe Method validation CITAC Cooperation of International Traceability in Analytical Chemistry Proficiency testing Quality Assurance EA European Cooperation for Accreditation Accreditation CEN European Committee for Normalization Standardization IUPAC International Union of Pure & Applied Chem. ISO International Standardization Organisation Standardisation AOAC ILAC Association of Official Analytical Chemists International Laboratory Accreditation Cooperat. Internal qual. Control FDA US Food and Drug Administration USP United States Pharmacopoeia ICH International Conference on Harmonization 2009

Method Validation Validation of analytical procedures is the process of determining the suitability of a given methodology for providing useful analytical data. J. Guerra, Pharm. Tech. March 1986 Validation is the formal and systematic proof that a method compiles with the requirements for testing a product when observing a defined procedures. G. Maldener, Chromatographia, July 1989 2009

drug substances and drug products Method validation is the process of demonstrating that analytical procedures are suitable for their intended use and that they support the identity, strength, quality, purity and potency of the drug substances and drug products Method validation is primarily concerned with: identification of the sources of potential errors quantification of the potential errors in the method An method validation describes in mathematical and quantifiable terms the performance characteristics of an assay 2009

Examples of Methods That Require Validation Documentation Chromatographic Methods - HPLC, GC, TLC, GC/MS, etc. Pharmaceutical Analysis - In support of CMC. Bioanalytical Analysis - In support of PK/PD/Clinical Studies. Spectrophotometric Methods – UV/VIS, IR, NIR, AA, NMR, XRD,MS Capillary Electrophoresis Methods - Zone, Isoelectric Focusing Particle Size Analysis Methods - Laser, Microscopic, Sieving, SEC, etc. Automated Analytical Methods - Robots, Automated Analysis. 2009

Considerations Prior to Method Validation Suitability of Instrument Status of Qualification and Calibration Suitability of Materials Status of Reference Standards, Reagents, Placebo Lots Suitability of Analyst Status of Training and Qualification Records Suitability of Documentation Written analytical procedure and proper approved protocol with pre-established acceptance criteria 2009

Validation Step Define the application, purpose and scope of the method. Analytes? Concentration? Sample matrices? Develop a analytical method. Develop a validation protocol. Qualification of instrument. Qualify/train operator Qualification of material. Perform pre-validation experiments. Adjust method parameters and/or acceptance criteria if necessary. Perform full validation experiments. Develop SOP for executing the method in routine analysis. Document validation experiments and results in the validation report. 2009

Purpose of Method Validation Identification of Sources and Quantitation of Potential errors Determination if Method is Acceptable for Intended Use Establish Proof that a Method Can be Used for Decision Making Satisfy FDA Requirements 2009

What is not Analytical Method Validation? Calibration The Process of Performing Tests on Individual System Components to Ensure Proper function For example) HPLC Detector calibration Wavelength Accuracy/ Linear Range/ Noise Level/ Drift 2009

System Suitability Test to verify the proper functioning of the operating system, i.e., the electronics, the equipment, the specimens and the analytical operations. Minimum Resolution of 3.0 between the analyte peak and internal standard peaks Relative Standard Deviation of replicate standard injections of not more than 2.0% 2009

System Suitability Validation Calibration Analyst Method Sample Pump Injector Detector Data System Analyst Method Sample 2009

Method Life Cycle Validation Development Optimization 2009

Verification vs. Validation Compendial vs. Non-compendial Methods Compendial methods-Verification Non-compendial methods-Validation requirement 2009

Compendial Analytical Procedures The Analytical procedures in the USP 25/NF 20 are legally recognized under section 501(b) of the Federal Food, Drug and Cosmetic Act as the regulatory analytical procedures for the compendial items. The suitability of these procedures must be verified under actual conditions of use. When using USP 25/NF 20 analytical procedures, the guidance recommends that information be provided for the following characteristics: Specificity of the procedure Stability of the sample solution Intermediate precision 2009

Published Validation Guidelines 1978 Current Good Manufacturing Practices (cGMPs) 1987 FDA Validation Guideline 1989 Supplement 9 to USP XXI 1994 CDER Reviewer Guidance: Validation of Chromatographic Method 1995 ICH Validation Definitions: Q2A, Text on Validation of Analytical procedures 1997 ICH Validation Methodology: Q2B, Validation of Analytical Procedures: Methodology 1999 Supplement 10 to USP 23 <1225>: Validation of Compendial Methods 1999 CDER “Bioanalytical Method Validation for Human Studies” 2000 CDER Draft “Analytical Procedures and Method Validation” 2009

Regulatory and Compliance Requirements Review Validation of an analytical method is the process by which it is established, by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical applications USP 23 General Information <1225> 2009

The accuracy, sensitivity, specificity, and reproducibility of test methods employed by the firm shall be established and documented. Such validation and documentation may be accomplished in accordance with 211.194(a)(2). 21 CFR PART 211 - CURRENT GOOD MANUFACTURING PRACTICE FOR FINISHED PHARMACEUTICALS Subpart I-Laboratory Controls 211.165 Testing and release for distribution (e) 2009

for its intended purpose The objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose ICH Guideline for Industry Q2A, Text on Validation of Analytical Procedures March 1995 2009

In practice, it is usually possible to design the experimental work such that the appropriate validation characteristics can be considered simultaneously to provide a sound, overall knowledge of the capabilities of the analytical procedure, for instance: Specificity, Linearity, Range, Accuracy, and Precision. ICH Guideline for Industry Q2B, Validation of Analytical Procedures: Methodology 2009

Today’s Validation Requirements ICH/USP GMPs (legal) FDA 2009

ICH/USP Validation Requirements & Parameters Specificity Linearity Range Accuracy Precision Repeatability Intermediate Precision Reproducibility Limit of Detection Limit of Quantitation Specificity Linearity and Range Accuracy Precision Limit of Detection Limit of Quantitation Ruggedness Robustness 2009

USP Data Elements Required For Assay Validation Analytical Performance Parameter Assay Category 1 Assay Category 2 Assay Category 3 Quantitative Limit Tests Accuracy Yes * Precision No Specificity LOD LOQ Linearity Range Ruggedness * May be required, depending on the nature of the specific test. 2009

USP Categories Category 1: Quantitation of major components or active ingredients Category 2: Determination of impurities or degradation products Category 3: Determination of performance characteristics 2009

ICH Validation Characteristics vs. Type of Analytical Procedure Identification Impurity testing Assay Quantitative Limit Tests Accuracy No Yes Precision Repeatability Interm. Prec. Specificity LOD LOQ Linearity Range 2009

Specificity/Selectivity Ability of an analytical method to measure the analyte free from interference due to other components. Selectivity describes the ability of an analytical method to differentiate various substances in a sample Original term used in USP Also Preferred by IUPAC and AOAC Also used to characterize chromatographic columns Degree of Bias (Used in USP) The difference in assay results between the two groups the sample containing added impurities, degradation products, related chemical compounds, placebo ingredients the sample without added substances 2009

Specificity: Impurities Assay Chromatographic Methods Demonstrate Resolution Impurities/Degradants Available Spike with impurities/degradants Show resolution and a lack of interference Impurities/Degradants Not Available Stress Samples For assay, Stressed and Unstressed Samples should be compared. For impurity test, impurity profiles should be compared. 2009

Forced Degradation Studies Temperature (50-60℃) Humidity (70-80%) Acid Hydrolysis (0.1 N HCl) Base Hydrolysis (0.1 N NaOH) Oxidation (3-30%) Light (UV/Vis/Fl) Intent is to create 10 to 30 % Degradation 2009

Linearity Ability of an assay to elicit a direct and proportional response to changes in analyte concentration. 2009

Linearity Should be Evaluated By Visual Inspection of plot of signals vs. analyte concentration By Appropriate statistical methods Linear Regression (y = mx + b) Correlation Coefficient, y-intercept (b), slope (m) Acceptance criteria: Linear regression r2 > 0.95 Requires a minimum of 5 concentration levels 2009

Range Acceptable range having linearity, accuracy, precision. For Drug Substance & Drug product Assay 80 to 120% of test Concentration For Content Uniformity Assay 70 to 130% of test Concentration For Dissolution Test Method +/- 20% over entire Specification Range For Impurity Assays From Reporting Level to 120% of Impurity Specification for Impurity Assays From Reporting Level to 120% of Assay Specification for Impurity/Assay Methods 2009

Accuracy Closeness of the test results obtained by the method to the true value.

Accuracy Should be established across specified range of analytical procedure. Should be assessed using a minimum of 3 concentration levels, each in triplicate (total of 9 determinations) Should be reported as: Percent recovery of known amount added or The difference between the mean assay result and the accepted value 2009

Accuracy Data Set (1 of 3) Amount Added (mg) Found (mg) Percent Recovery 0.0 --- 50.2 50.4 100.5 79.6 80.1 100.6 99.9 100.7 100.8 120.2 119.8 99.7 150.4 149.7 99.5 2009

Precision The closeness of agreement (degree of scatter) between a series of measurements obtained from multiple samplings of the same homogeneous sample. Should be investigated using homogeneous, authentic samples. 2009

Precision… Considered at 3 Levels Repeatability Intermediate Precision Reproducibility 2009

Repeatability Express the precision under the same operating conditions over a short interval of time. Also referred to as Intra-assay precision Should be assessed using minimum of 9 determinations (3 concentrations/ 3 replicates) or Minimum of 6 determinations at the 100% level. 2009

Intermediate Precision Express within-laboratory variations. Expressed in terms of standard deviation, relative standard deviation (coefficient of variation) and confidence interval. Depends on the circumstances under which the procedure is intended to be used. Studies should include varying days, analysts, equipment, etc. 2009

Repeatability & Intermediate Precision Day 1 Day 2 100.6 99.5 100.8 99.9 100.1 98.9 100.3 99.2 100.5 99.7 100.4 99.6 Mean = 100.5 RSD = 0.24% Mean = 99.5 RSD = 0.36% Grand Mean = 100.0 RSD = 0.59% 2009

Reproducibility Definition: Ability reproduce data within the predefined precision Determination: SD, RSD and confidence interval Repeatability test at two different labs. Note: Data not required for BLA/NDA Lab 1 Lab 2 Lab 3 Day 1 Day 2 Day 2 Man 1 Man 2 3 Prep

Detection Limit (LOD)/ Quantitation Limit (LOQ) Lowest amount of analyte in a sample that can be detected but not necessarily quantitated. Estimated by Signal to Noise Ratio of 3:1. LOQ Lowest amount of analyte in a sample that can be quantified with suitable accuracy and precision. Estimated by Signal to Noise Ratio of 10:1. 2009

LOD and LOQ Estimated by Based in Visual Evaluations - Used for non-instrumental methods Based on Signal-to Noise-Ratio - 3:1 for Detection Limit - 10:1 for Quantitation Limit Based on Standard Deviation of the Response and the Slope 2009

LOD and LOQ Estimated by QL = 10s S S = slope of calibration curve s = standard deviation of blank readings or standard deviation of regression line Validated by assaying samples at DL or QL 2009

Ybl LOD LOQ LOD = 3.3 Sbl / b LOQ = 10 Sbl / b Y = b X + a Statistical estimate of LOD & LOQ LOD = 3.3 Sbl / b LOQ = 10 Sbl / b Y = b X + a 2009

Robustness Definition: Capacity to remain unaffected by small but deliberate variations in method parameters Determination: Comparison results under differing conditions with precision under normal conditions Examples of typical variations in LC Influence of variations of pH in a mobile phase Influence of variations in mobile phase composition Different columns (different lots and/or suppliers) Temperature Flow rate 2009

Ruggedness Degree of reproducibility of test results under a variety of conditions Different Laboratories Different Analysts Different Instruments Different Reagents Different Days Etc. Expressed as %RSD 2009

ICH/USP System Suitability Definition: evaluation of equipment, electronic, analytical operations and samples as a whole Determination: repeatability, tailing factor (T), capacity factor (k’), resolution (R), and theoretical Plates (N) 2009

USP 23 <621> System Suitability Requirements Parameters Recommendations K’ In general k’ ≥ 2.0 R R > 2, between the peak of interest and the closest potential interferent (degradant, internal STD, impurity, excipient, etc…..) T T ≤ 2 N In general N > 2000 Repeatability RSD ≤ 2.0% (n ≥ 5) 2009

Re-validation When What Method parameters have been changed The scope of the method has been changed Synthetic methods have been changed Impurity profile has been changed What Preferably everything. Exceptions should be scientifically justified 2009

How do we Know the expectations of the FDA? FDA Form 483 FDA Warning Letters Personal Experiences 2009

483 Observations There was inadequate method validation specificity data to demonstrate that each method was capable of distinguishing the active ingredient from its impurities and degradation products. Specificity studies did not include the minimum stress conditions of acid and base hydrolysis, oxidation, thermal degradation and photolysis, degradation schematic for the active ingredient that identifies the major degradation products was not included for each product. 2009

FDA Waning Letter On addition to the example of modifying both compendial methods and customer supplied methods, we also observed the use of unvalidated in-house methods as well as unvalidated modifications to in-house methods. A statement indicating that the method has not been validated in the particular formulation was included in the certificate of analysis for…use of this statement does not absolve…from using valid, accurate, and reproducible methods. (June 2000) 2009

FDA Systems Based Inspection: Laboratory System Feb – July 2002: 212 Inspections (US) Method Validation 13% Training/Qual. 4% Stability Program 21% Inadequate Records 27% Controls. General 35% * Reference: Albinus D’ Sa, FDA, CDER Office of Compliance, from AAPS, Nov. 2002 presentation. 2009

ICH Update: 2009

A Unique Approach International Conference on Harmonisation (ICH) was created in 1990 Agreement between the EU, Japan and the USA to harmonize different regional requirements for registration of pharmaceutical drug products Unique because joint effort by regulators and associated pharmaceutical industry trade associations 2009

ICH Objectives Identification and elimination of the need to duplicate studies to meet different regulatory requirements More efficient use of resources in the R&D process, as a consequence Quicker access for patients to safe and effective new medicines 2009

Endorses topics, guidelines and monitors progress Working Groups SAFETY EFFICACY QUALITY MULTIDISCIPLINARY STEERING COMMITTEE Endorses topics, guidelines and monitors progress 2009

Related Site www.fda.gov www.fda.gov/cder/ www.waters.com www.usp.org www.ich.org www.aoac.org www.pharmweb.net 2009