Method Validation in Regulated Lab

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Waters Korea Ji Yeon Kim

• Method Validation in Regulated Lab

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Laboratory Method Flow
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Why Validate?

• Method validation is completed to insure that an
  analytical methodology is accurate, reproducible
  and rugged over the specific range that an
  analyte will be analyzed.
• Method validation provides assurance of
  reliability.
• FDA Compliance

"The process of providing documented evidence
that something does what it is intended to do."
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The Process of Validation
Method Validation
Instruments
Validation


System Suitability
Software
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Analytical Method Validation

• Initial Method Validation Guidance Issued in 1987
• Guideline for submitting samples and analytical
  data for methods validation. Food and Drug
  Administration, February 1987. US Government
  Printing Office1990-281-79420818.
• Updated in August 2000 (Draft Guidance!)
• Analytical Procedures and Method Validation.
  Fed. Reg. 65(169), 52,776-52,777, 30 August 2000
• LC/GC article also available _at_ www.waters.com
• All Guidances are on FDA Web site
• www.fda.gov/cder/guidance

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Impact of ICH on Method Validation in the USA

• USP 24, Chapter 1225 Updated to Reflect ICH
  Process
• ICH validation guidelines are implemented
• ICH is not Another Regulatory Agency
• USP Guidelines are still appropriate
• Draft Guidance Update
• Other Countries Also Have Incorporated or Adopted
  ICH Guidelines

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FDA Method Validation Compliance1987 Position
Paper

• United States Pharmacopeia or the National
  Formulary (USP/NF) specifications
• Current edition USP 24, NF 19, 2000
• Can still be defined as "The Eight Steps of
  Method Validation" (Chapter 1225)

Update in Process
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USP Method ValidationAnalytical Performance
Characteristics
Precision
Accuracy
Limit of Detection
Limit of Quantitation
Method
Specificity
Validation
Linearity and Range
Ruggedness
Robustness
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ICH Method Validation Analytical Performance
Characteristics
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Analytical Performance CharacteristicsDefinition
of Accuracy

• The closeness of test results to the true value
  obtained by the method.
• Established across the range

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Analytical Performance Characteristics
Determination of Accuracy

• Drug Substance
• Analysis of reference material
• Compare results to a second, well-characterized
  method
• Drug Product
• Analysis of synthetic mixtures spiked with known
  quantities of components
• Compare results to a second, well-characterized
  method
• Determined concurrently with precision, linearity
  and specificity

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Analytical Performance Characteristics
Determination of Accuracy

• Impurities (Quantitation)
• Analysis of samples (Drug substances/Drug
  product) spiked with known amounts of impurities
• If impurities are not available, see specificity
• Recommended Data
• Minimum of 9 determinations over a minimum of 3
  concentration levels covering the specified range
  (e.g. 3 concentrations/3 replicates each)
• Reported as recovery of known, added amount, or
  difference between the mean and true value, with
  confidence intervals

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Analytical Performance CharacteristicsDefinition
of Precision

• Precision
• The measure of the degree of agreement among test
  results when the method is applied repeatedly to
  multiple samplings of a homogeneous sample
• Expressed as RSD for a statistically significant
  number of samples

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Analytical Performance Characteristics
Definition of Precision

• Precision Should Be Performed at Three Levels
• Repeatability
• Intermediate Precision
• Reproducibility

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Analytical Performance Characteristics
Determination of Precision

• Repeatability (Generally the criterion of concern
  in USP analytical procedures)
• Same operating conditions, short time interval
• Inter-assay precision
• Minimum of 9 determinations covering specified
  range of procedure
• (3 levels, 3 reps each), or
• Minimum of 6 determinations at 100 test conc.
• Intermediate Precision (Experimental design
  recommended)
• Within-lab variations (Random events)
• Different days, analysts, equipment
• Reproducibility
• Precision between labs
• Collaborative studies

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Analytical Performance CharacteristicsDefinition
of Specificity

• Specificity (Selectivity)
• The ability to measure accurately and
  specifically the analyte in the presence of
  components that may be expected to be present in
  the matrix
• The degree of interference
• Active Ingredients
• Excipients
• Impurities
• Degradation Products
• Placebo Ingredients

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Specificity (Selectivity)

• Separation
• Resolution
• Determination of separation between peaks
• Plate Count
• Determination of a systems efficiency
• Tailing Factor
• Calculation referencing peak shape
• PDA
• Purity Angle
• Purity Plots

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Analytical Performance CharacteristicsDeterminat
ion of Specificity

• Qualitative Identification Tests
• Demonstrate ability to select between compounds
  of closely related structure
• Confirm positive and negative results
• Assay
• Demonstrate that the results are unaffected by
  spiked impurities or excipients
• Impurities
• Spike the drug product/substance with impurities
  and demonstrate appropriate accuracy and precision

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Analytical Performance CharacteristicsDeterminat
ion of Specificity (Cont.)

• Impurities Are Available
• Demonstrate that the assay is unaffected by the
  presence of spiked materials (impurities and/or
  excipients).
• Impurities Are Not Available
• Compare test results to a second
  well-characterized procedure
• For Assay, compare the two results
• For Impurity Tests, compare impurity profiles
• Peak Purity Test ("diode array, MS")

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More Changes Courtesy of ICHSpecificity
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Peak Purity Calculations

• What is Needed?
• Some Degree of Chromatographic Resolution
• Compounds must have UV Absorbance
• Some Degree of Spectral Differences Between
  Compounds

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Spectral Contrast Theory of Empower

• Comparison of the two vectors A and B -
  calculation of angle ?
• If A and B totally different, ? 90
• If A and B totally identical, ? 0

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PDA and MS Together

PDA and MS together can provide complimentary
useful information.
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Method Validation and PDA/MS
Validation SOP Choice
Detector in Routine Use
Detector to Validate Method Only
Vs.
All Detector Use/Information Considered Robustness
Linearity Precision Etc.
Peak Homogeneity Peak Identity
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Analytical Performance CharacteristicsDefinition
of Detection Limit

• Detection Limit (LOD)
• Lowest Concentration of Analyte in a Sample That
  Can Be Detected (not necessarily quantitated)
• Limit Test Above or Below a Certain Level
• Expressed as Concentration (, ppb)
• Almost never necessary to determine actual
  detection limit
• Rather, limit is shown to be sufficiently low
  (e.g. 0.1)

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Analytical Performance CharacteristicsDeterminat
ion of Detection Limit (DL)

• Visual (Non-Instrumental Methods)
• Signal To Noise Ratio (3 or 21 Generally
  Accepted)
• Detection limit may be based on the standard
  deviation of the response and slope

DL (3.3)STD/S
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Limit of detection/Limit of quantitation
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Analytical Performance CharacteristicsDefinition
of Quantitation Limit

• Limit of Quantitation (LOQ)
• Lowest concentration of analyte in a sample that
  can be determined with acceptable precision and
  accuracy under stated operational conditions
• Expressed as the Concentration of Analyte
• Accuracy
• Precision

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Analytical Performance CharacteristicsDeterminat
ion of Quantitation Limit

• Visual (Non-Instrumental Methods)
• Signal To Noise Ratio (101 is Typical)
• Quantitation limit may be based on the standard
  deviation of the response and slope

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Analytical Performance CharacteristicsDefinition
of Linearity and Range

• Linearity
• The Ability of the Method to Elicit Test Results
  That Are Directly Proportional to Concentration
  Within a Given Range
• Expressed as the Variance of the Slope of the
  Regression Line
• Range
• Interval between upper and lower levels of
  analyte demonstrated by the method
• Precision and Accuracy Expressed in the same
  units as the test results

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Analytical Performance CharacteristicsEvaluation
of Linearity

• Established across the Range of the method
• Dilutions
• Separate Weighings
• Evaluate by Appropriate Statistical Methods (e.g.
  Regression)
• Include Correlation Coefficient, y-Intercept,
  Slope, Residual Sum of Squares, Plot Itself
• Minimum 5 Concentrations

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Analytical Performance CharacteristicsDeterminat
ion of Appropriate Range

• Minimum Specified Ranges
• Assay
• 80-120
• Impurity Test
• From QL to 120 of spec.
• Toxic or more potent impurities commensurate
  with the controlled level
• Content Uniformity
• 70-130 of test concentration
• Dissolution Testing
• /- 20 over specified range

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Analytical Performance CharacteristicsDefinition
of Ruggedness

• 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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Analytical Performance CharacteristicsDefinition
of Robustness

• Robustness
• Measure of The Capacity to Remain Unaffected by
  Small (Deliberate) Variations in Method
  Parameters
• Indication of Reliability During Normal Use

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HPLC Method Validation Process

• Step 1 Software Validation
• Step 2 HPLC System Qualification
• Step 3 Method Validation Using Validated HPLC
  System
• Step 4 Total System Validation Using System
  Suitability

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System Suitability

• System Suitability
• The checking of a system, before or during
  analysis of unknowns, to insure system
  performance.
• No sample analysis is acceptable unless the
  requirements for system suitability have been
  met. (USP Chapter 621)
• Plate Count, Tailing, Resolution
• Determination of reproducibility (RSD)
• For RSD lt 2.0, Five replicates
• For RSD gt 2.0, Six replicates
• System Suitability "Sample"
• A mixture of main components and expected
  by-products utilized to determine system
  suitability
• Whenever There is a Significant change in
  Equipment or Reagents System Suitability Testing
  Should be Performed (USP Chapter 621)

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Recommendations From FDA 1994 Guideline System
Suitability

• Capacity factor
• k' gt 2
• Precision/Injection repeatability
• RSD lt/ 1, n gt/ 5
• Resolution
• Rs gt/ 2 (Major peak and closest eluting)

• Tailing factor
• T lt/ 2
• Theoretical Plates
• In general N gt 2000

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Where Do I Start?!!?
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How is Method Validation Performed?

• Analytical Laboratory Phase
• Following a Test Plan or Protocol
• Usually during 3 to 5 days
• Consist of injecting various concentration levels
  under different operating conditions
• Both on the Bulk (Active form) Formulation
  (Pharmaceutical form)
• Data Reduction Phase
• Statistical Calculations are performed on these
  analysis
• Chromatographic results are always relative
• With statistical approach you can objectively
  evaluate the real variations of the final results

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Example Method Validation Protocol

• Some Basic Assumptions
• Specificity demonstrated by PDA, MS
• Method is developed and optimized to a point..
• Once data is generated, statistics will be used
• Variance
• Confidence levels and limits
• Student, Cochran, Dixon and Fisher tests

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Example Method Validation Protocol
Linearity Day 1-3
LOQ
LOD
Robustness
Level 1 Bulk Drug
Six Blanks Baseline Noise
Parameter 1 Min Max
Level 2 Bulk Drug
Level 3 Bulk Drug
Parameter 2 Min Max
Level 4 Bulk Drug
Parameter 3 Min Max
Level 5 Bulk Drug
Parameter 4 Min Max
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Why is Method ValidationConsidered a Bottleneck?

• Every Method Validation Process Consists of About
  80 to 100 Analyses
• Each of Them Produce About 7 Peak Specific
  Results for a Single Component (Area, RT,
  Resolution)
• These Analyses Result in a total of about 700
  Numbers per Component
• All of These Numbers Need to be Mathematically
  Processed
• Manual (Calculator, Excel Macros etc.)
• Dedicated Method Validation Program

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Different Approaches to theData Reduction Process

• Manual Process
• If all calculations are performed manually
  (typically done using Excel spreadsheets) time
  could lead to 1 week work with 2 persons (One
  person for result entry - and one for
  calculation verification.)
• Using Dedicated Programs (commercialized or home
  build products)
• One day for 2 persons
• One person for result entry verification
• Using Method Validation Software
• Only 15 minutes for only one Single person !!!

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Original USP Method
1
2
0.0
20.0
Time in Minutes
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Original USPMethod on New Column
USP Method Conditions
Column
3.9 by 15cm Symmetry 5µm C18
Mobile Phase
Na
H2PO
pH 5.5/ACN 85/15
4
Flow Rate
1.0 mL/min.
Detection
UV at 280nm
Sample
0.01mg/mL each in water

1 Hydrochlorthiazide
1

2 Triamterene

USP Criteria Resolution gt 3.0,
2

RSD lt 2.0
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Effect of Smaller Particle Size
Method Conditions
Column XTerraMS C18 2.5µm
4.6X30mm Mobile Phase NaH2PO4 pH5.5/ACN
85/15 Flow Rate 1.4 mL/minute Detection
280 nm Sample 0.01 mg/mL each
in water 1.
Hydrochlorthiazide 2.
Triamterene
1
2
Peak Rs RSD Time RSD Area 1 ---
0.1 0.6 2 3.2
0.2 0.4
Time in Minutes
0.0
3.0
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Why Revalidate?

• To Take Advantage of New Technology
• Cost/Benefit Economic Exercise
• Columns, Instrumentation, Methods
• Formulation Changes
• Manufacturing Batch Changes
• Changes in Incoming Raw Material
• Changes in Method

"Validation is a constant, evolving process and
should be considered during method development!"
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Useful Web Sites WithValidation Information

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

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Conclusion

• Defined Validation and its Importance
• Examined The Steps of Method Validation
• Learned How Each Step is Documented and Measured
• System Suitability
• USP/ICH
• Protocol and Pitfalls
• Revalidation
• Software-Automated Method Validation