Analytical method validation for FDA compliance

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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

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• 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

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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.

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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

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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.

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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

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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

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• 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

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System Suitability
Validation
Calibration
Pump
Injector
Detector
Data System
Method
Analyst
Sample
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Method Life Cycle
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Verification vs. Validation

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

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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

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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 lt1225gt Validation
  of Compendial Methods
• 1999 CDER Bioanalytical Method Validation for
  Human Studies
• 2000 CDER Draft Analytical Procedures and
  Method Validation

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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 lt1225gt
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• 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)
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• 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
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• 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
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Todays Validation Requirements
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ICH/USP Validation Requirements Parameters
ICH
USP

• 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

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USP Data Elements Required For Assay Validation
May be required, depending on the nature of the
specific test.
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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

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ICH Validation Characteristics vs. Type of
Analytical Procedure
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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

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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.

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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

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Linearity

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

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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 gt 0.95
• Requires a minimum of 5 concentration levels

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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

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Accuracy

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

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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

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Accuracy Data Set (1 of 3)
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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.

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Precision Considered at 3 Levels

• Repeatability
• Intermediate Precision
• Reproducibility

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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.

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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.

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Repeatability Intermediate Precision
Mean 100.5 RSD 0.24
Mean 99.5 RSD 0.36
Grand Mean 100.0 RSD 0.59
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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

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Detection Limit (LOD)/ Quantitation Limit (LOQ)

• LOD
• Lowest amount of analyte in a sample that can be
  detected but not necessarily quantitated.
• Estimated by Signal to Noise Ratio of 31.

• LOQ
• Lowest amount of analyte in a sample that can be
  quantified with suitable accuracy and precision.
• Estimated by Signal to Noise Ratio of 101.

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LOD and LOQ Estimated by

• Based in Visual Evaluations
• - Used for non-instrumental methods
• Based on Signal-to Noise-Ratio
• - 31 for Detection Limit
• - 101 for Quantitation Limit
• Based on Standard Deviation of the Response and
  the Slope

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LOD and LOQ Estimated by

• 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

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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

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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

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ICH/USP System Suitability

• ICH
• 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)

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• USP 23 lt621gt
• System Suitability Requirements

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??? Re-validation

• When
• 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

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How do we Know the expectations of the FDA?

• FDA Form 483
• FDA Warning Letters
• Personal Experiences

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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.

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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 foruse
  of this statement does not absolvefrom using
  valid, accurate, and
• reproducible methods. (June 2000)

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FDA Systems Based InspectionLaboratory System
Feb July 2002 212 Inspections (US)
Reference Albinus D Sa, FDA, CDER Office of
Compliance, from AAPS, Nov. 2002 presentation.
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Related Site

• www.fda.gov
• www.fda.gov/cder/
• www.waters.com
• www.usp.org
• www.ich.org
• www.aoac.org
• www.pharmweb.net