Phase Appropriate Method Validation

Aryo Nikopour
Irvine, California | January 12, 2017
The Southern California Pharmaceutical Discussion Group (SCPDG) of AAPS
OUTLINE
• What is Validation
• Guidelines
• Method Verification
• Method Transfer
• Phase Approporiate Method Validation
• Method Validation Characteristics
• Mass Balance
VALIDATION OF ANALYTICAL METHOD
Calibration

System
Suitability Validation Analyst

Method
DATA QUALITY TRIANGLE

QC Checks

System
Suitability Tests

Analytical Method
Validation

Analytical Instrument
Qualification
METHOD LIFE CYCLE
Validation

Development Optimization
METHOD VALIDATION
PUBLISHED VALIDATION GUIDELINES
 1978 Current Good Manufacturing Practices (cGMP)
 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”
 2014 CDER/CBER Guidance for Industry: “Analytical Procedure and Method Validation for Drug
and Biologic”
 PDA Technical Report No. 57 : Analytical Method Validation and Transfer for Biotechnology
Products”
GUIDELINES FOR METHOD VALIDATION
www.ICH.org

ICH  Q2(R1): Validation of Analytical Procedures: 
Methodology‐ Nov. 2005
ICH Q3A(R): Impurities in New Drug Substances ‐ Feb. 2002
ICH Q3B(R): Impurities in New Drug Products – Feb. 2003
ICH Q3C: Impurities: Residual Solvents ‐ July 1997
ICH Q5A,D: Biotech/Biological Products ‐ 1997
ICH Q5B,C: Biotech/Biological Products ‐ 1995

(Dates indicate ICH finalization)

VALIDATION IN THE 21 CENTURY

ICH

FDA

USP
METHOD VALIDATION

USP<1225>
• Method Validation

Method • Method Qualification

Qualification

USP<1226>
• Method Verification

USP<1224>
• Method Transfer
CFR
• There are many reason to validate analytical methods:
– Regulatory Requirements
– Good Science
– Quality Control requirements.

• The Code of Federal Regulations (CFR) 311.165c explicitly states that the,
“Accuracy, Sensitivity, Specificity, and Reproducibility of test methods
employed by the firm shall be established and documented.”
ICH GUIDELINE Q2(R1)
• The objective of validation of an analytical procedure is to
demonstrate that it is suitable for its intended purpose,

 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.

 Support the identity, strength, quality, purity, and potency

of the drug substances and drug products.
WHAT IS METHOD VALIDATION?

 Validation is procedure dependent.

 Validation, “Proves” the procedure works as described.
 Validation is product specific.
 Procedures are instrument dependent.
VERIFICATION USP <1226>
• Current USP <1226> Verification of Compendial Procedure

– The Analytical procedures in the current USP 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 the

actual conditions of use.

VERIFICATION

• When using USP analytical procedures, the guidance recommends

that information be provided for the following characteristics:

– Specificity of the procedure

– Stability of the sample solution
– Intermediate precision
METHOD TRANSFER, USP <1224>

• Method Transfer is a documented process that qualifies a

laboratory (Receiving Lab) to use an analytical test
procedure that is originated from the transferring laboratory.

• Types of Method Transfer:

– Comparative Testing
– Co -validation
– Revalidation/Partial Validation
– Transfer Waiver
CLASSIFICATION OF VALIDATED ANALYTICAL METHODS

Compendial (USP 39/ NF 34):

• Legally recognized under section 501 (b) of the Federal Food, Drug, and Cosmetic Act.
• Recommends information be provided for; Specificity, Sample Solution Stability, and
Intermediate Precision.

Non-Compendial:
• Submitted with the NDA/ BLA or ANDA application.
• If the compendial procedure is not stability-indicating, perform an alternative analytical
procedure with complete validation.
USP <1225>ASSAY CATEGORIES
Category
Category Name Description of Assay
Number
Quantitation of major
I Quantitative components/active ingredients
present at high concentrations.
Impurities-
II Determination of impurities or
Quantitative
degradation products.
II Impurities-Limit
Parameters to be tested depend on
Performance
III the nature of the test; includes
Characteristics
dissolution testing.
IV Identity
METHOD VALIDATION REQUIREMENTS
USP Assay Category
II
I III IV
Parameter: Quantitative Limit Tests
Accuracy Y Y  Y N
Precision Y Y N Y N
Intermediate
Y Y N Y N
Precision
Specificity N Y Y N Y
Detection Limit N Y Y  N
Quantitation Limit N Y N  N
Linearity Y Y N  N
Range Y Y   N
Robustness Y Y N N N
Selectivity Y Y N Y Y
System Suitability Y Y N Y N
Solution Stability Y Y N Y N
 May be required, depending on the nature of the specific test.
PHASE APPROPRIATE VALIDATION

Pre- Phase Phase Phase

Clinical I
PM LCM
II III
METHOD VALIDATION READINESS

 Define the application, purpose and scope of the method.

 Define Analytes, Dosage Strength and Sample Matrix.

 Review Method Development Summary Report.

 Evaluate method validation parameters during development.

METHOD VALIDATION CHARACTERISTICS
Validation Experimental Details Acceptance Criteria
Characteristics
Specificity Stress Studies 5-10% Degradation
Selectivity Determine Chromatographic non-interference No inference , minimum resolution
between peaks of interest and
impurities should be >1.5
System Suitability System precision assessed by 6 replicate %RSD ≤2%
measurement/injections
Linearity At least 5 Concentration over the range Calibration Model is valid
Assay: 50% to 125% of Specification limit R ≥0.998
QL-150% of specification limit Report Intercept, Slope and %Bias
Detection Limit (DL) DL= 3.3 (/S) S/N≥ 3

Quantitation Limit (QL) DL= 10 (/S) %RSD≤ 15%

METHOD VALIDATION CHARACTERISTICS
Validation Characteristics Experimental Details Acceptance Criteria
Precision :
Repeatability 6 replicates %RSD≤ 2%
Intermediate Precision (Ruggedness) 6 replicates Overall %RSD (two Analyst)
Reproducibility Comparative Precision/Partial 3 Sigma
Validation 3 Sigma
Accuracy At least 9 determination over 3 For Assay Mean Recovery 97 to
concentration level 103%
e.g. 70 to 120% for for Impurities : 85% to 115%

Range The range is defined by the results Linearity, accuracy and precision
obtained for linearity, accuracy and demonstrated over the range
precision

Solution Stability Determine solution stability of Assay: 98 to 102 % of control

Reference Standard Solution and Impurities: 95 to 105%
Sample over 72 hours
Robustness Deliberately change critical Must meet system suitability and
parameters of the method selectivity requirements
VALIDATION: PHASE I
Quantitative
Drug Product Assay I.D. Limit Test
Impurities
Selectivity X X X X

Repeatability X X

Accuracy/Precision Recovery at At 100% of

100% Reporting
Threshold
Linearity X QL to 200% of
Limit
Range Defined by Defined by ALP
ALP
DL/QL DL QL QL or at Limit

System Suitability X X X X

Solution Stability X X X
VALIDATION: PHASE II
Assay I.D Quantitative Limit Test
Impurities
Selectivity X X X X
Specificity X
Repeatability X X X
Accuracy Recovery at 3 At 100% of
levels Reporting
Threshold

Linearity X X X

DL/QL DL X QL
Range Define by ALP Defined by ALP

System Suitability X X X X

Solution Stability X X X
VALIDATION: PHASE III
Assay I.D Quantitative Limit Test
Impurities
Selectivity X X X X

Specificity X

Repeatability X X X

Intermediate Precision X 2nd Analyst X X

Accuracy X X

Linearity X X

DL/QL DL X QL or at Limit

Range Defined by ALP Defined by ALP

Solution Stability X X X

System Suitability X X X X

Robustness X X X
METHOD VALIDATION
SYSTEM SUITABILITY
Based on the concept that the equipment, electronics, analytical operations and
samples to be analyzed constitute an integral system that can be evaluated as such.

What parameters do you measure for

system suitability?
SYSTEM SUITABILITY
What parameters do you measure for system suitability?

Selectivity Efficiency Capacity

SYSTEM SUITABILITY
Standard B1 (n=6) Injections SST Solution B1 and B2
Average Tailing %RSD Average Resolution LVF and Response Factor
Area %RSD Factor Retention Time Theoretical Plates DesMethyl-LVF) % Difference
Date NB/Page  1% 0.8  Tf  1.4  1% >15000 NLT 2.5  2%
8/18/2006 1494/18 0.1 1.03 0.1 29834 3.27 0.2
8/18/2006 1494/31 0.1 1.03 0 32177 3.28 0.1
8/20/2006 1494/52 0.1 1.03 0.1 27792 3.3 0.7
8/22/2006 1494/72 0.1 1.03 0 26567 3.31 2.7
8/23/2006 1504/1 0 1.03 0.2 27228 3.29 0.8
8/24/2006 1504/8 0.2 1.02 0.1 26535 3.32 1.2
8/25/2006 1504/17 0.1 1.02 0.1 26903 3.31 3.6
9/5/2006 1494/129 0.1 1.03 0.1 27894 3.31 0.5
9/13/2006 1494/171 0.2 1.02 0.1 26916 3.31 0
9/15/2006 1494/181 0.1 1.03 0.1 29553 3.29 0.2
9/15/2006 1494/187 0.1 1.12 0.1 32361 3.47 0.1
9/15/2006 1494/193 0.1 1.11 0.1 27303 3.12 0.2
9/15/2006 1494/199 0.2 1.02 0.1 29424 3.3 0
9/18/2006 1504/37 0.2 1.02 0.1 28020 3.27 0.2
9/18/2006 1504/42 0.6 1.03 0 27627 3.29 1.9
9/18/2006 1461/40 0.1 1.03 0.1 31109 3.66 0.2
10/5/2006 1504/65 0.1 1.04 0.1 36973 3.85 0.2
Average 0.1 1.04 0.1 29729 3.39 0.8
Min 0 1.02 0 26535 3.12 0
Max 0.6 1.12 0.2 37049 3.86 3.6

STDEV 2770 0.1685

3 Sigma 8311 0.5054
min 21418 2.88
max 38040 3.90
GAUSSIAN DISTRIBUTION

C.I. =
CONTROL CHART
SELECTIVITY AND SPECIFICITY

Selectivity vs. Specificity

SELECTIVITY AND SPECIFICITY
Selectivity:
A method’s ability to separate the analyte from other components
that may be present in the sample.
Definition of Selectivity from IUPAC: Selectivity of a method, refers to the
extent to which it can determine particular analytes under given conditions
in mixtures or matrices, simple or complex, without interferences from
other components.
SELECTIVITY AND SPECIFICITY
SELECTIVITY AND SPECIFICITY

Specificity:
A method’s ability to identify and measure absolutely and unequivocally the analyte
in the presence of the other components in the sample, such as; impurities,
degradation products, and excipients.

There must be inarguable supporting data for a method to be considered specific.

Specificity implies identification, purity tests, and assay (content or potency).
SELECTIVITY AND SPECIFICITY

Regulatory Requirements:
Stability indicating methods are not specified, but implied in 21 CFR Part 211.165
and 211.166 (3):

• 211.165 (e) States that the accuracy, sensitivity, specificity, and reproducibility of
test methods employed by the firm shall be established and documented.

• 211.166 (a) (3) Requires that test methods be reliable, meaningful, and specific.
STABILITY INDICATING METHOD (SIM) VS.
STABILITY SPECIFIC METHODS (SSM)

• Stability indicating assays accurately quantitate active ingredients

without interference from:
– Degradation products
– Process impurities
– Excipients
• A stability-specific method is one that meets all of the criteria above
but, in addition, the degradation components are detected and
quantitated.
 Stress
Studies

“Absence of evidence is not evidence of absence”

- Carl Sagan,
The Dragons of Eden: Speculations on the Evolution of Human Intelligence
WHY DO WE PERFORM STRESS STUDIES?
Safety and Efficacy
Forced degradation or stress testing is undertaken to demonstrate specificity when
developing stability-indicating methods, particularly when little information is available
about potential degradation products.
WHY DO WE PERFORM STRESS STUDIES?

• Development and validation of stability-indicating methodology.

• Determination of degradation pathways of drug substances and drug products.
• Discernment of degradation products in formulations that are related to drug substances
versus those that are related to non-drug substances (excipients).
• Structure elucidation of degradation products.
• Determination of intrinsic stability of Active Moiety.
WHY DO WE PERFORM STRESS STUDIES?

Defining characteristics of degradation studies:

• Carry out in solution and/or in the solid state.
• Involve conditions more severe than accelerated stability studies.
• Typically carry out on placebo, drug product, and API.
• Not part of formal stability program.
FORCED DEGRADATION (STRESS STUDIES)

Steps to Approaching Stress Studies in the Lab:

• Investigate the chemical structure and functional group.
• Study chemical and physical properties.
• Study synthetic route.
• Predict stress pathways based on storage conditions and
manufacturing process.
• Identify suitable separation method and detection.
• Orthogonal Approach : develop MS compatible method
• Design study based on the formulation (feed, tablet, ointment, etc.).
FORCED DEGRADATION (STRESS STUDIES)
Chemical Physical Environmental

Acid Agitation Heat

Denaturation,
Light
Base aggregation, adsorption
(ICH Option I or II)
and precipitation

Oxidation RH

Deamidation Freeze/Thaw

Disulfide Bond Exchange

STRESS STUDY PATHWAYS

Pharmaceutical Biologics

Hydrolytic Hydrolytic

Oxidative Oxidative

Photolytic Aggregation

Thermolytic Deamidation

Disulfide Bond Exchange

FORCED DEGRADATION (STRESS STUDIES)
Stress Pathway Condition Time

Acid 0.01N 1 to 24 hours

Base 0.01N 1 o 24 hours

Oxidation 0.3% H2O2 1 to 24 hours

600 to 800 foot candles Option II: 74Hours
Light (sources include metal
halides, Hg, Xe lamp, or Option I: 2-4 Hours
UVB fluorescence)

Heat/RH 40 °C/ 75% RH and 24 to 72 hours

60 °C
Freeze/Thaw -20 °C to 25 °C 3 Cycle of 24 hours
WHAT IS ADEQUATE STRESS?

Overstressing a molecule can lead to degradation profiles that are not

representative of primary degradation and are irrelevant to the stability of
the product.

Stress-testing conditions should be realistic, not excessive (5 – 10%).

FORCED DEGRADATION (STRESS STUDIES)

 Optimize detector setting Overstress!!

 Stress blank, placebo,
standard and sample
 Inject controls
 Extend run time
 Orthogonal Method
EXAMPLE: PHOTOLYTIC STRESS
1 - Sequence Name: Forced De Sample Name: Fresh 30 mg Sample
2 - Sequence Name: Forced De Sample Name: Light Stressed 3 Sample
3 - Sequence Name: Forced De Sample Name: Light Stressed 3 Sample
4 - Sequence Name: Forced De Sample Name: Light Stressed 3 Sample
3.80

Levofloxacin - 9.777
mAU WVL:280 nm

DesMethyl-LVF - 8.980
Imp 6 - 8.320
2.50

Imp 14 - 20.487
Imp 11 - 14.517
Imp 9 - 12.817
Imp 10 - 13.320
Imp 7 - 10.733
Imp 3 - 6.097

Imp 5 - 7.873
Imp 1 - 3.397

1.25

4
0.00
3
2
1
min
2.20
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0
MASS BALANCE1

From ICH Q1 A “Stability Testing of New Drug Substance and Product”

• The process of adding together the assay value and levels of degradation
products to see how closely these add up to 100 percent of the initial value,
with due consideration of the margin of analytical error1.
MASS BALANCE
• Uncertainty in potency

• Loss of volatiles

• Diffusive losses

• Loss of UV chromophore

• Lack of universal detection

• Design of calculation
SOLUTION STABILITY
Purpose:
To determine stability of sample and standard Test solutions to support duration
of run sequence and potential investigation studies.

Procedure:
To evaluate several time intervals; (0, 24, 48, 72 hours), for both stock and
evaluated solution.
ESTABLISHING RANGE
• Range:
– Definition
– Criteria
• Limits of Detection and Quantitation
• Linearity
• Accuracy
• Precision
• Repeatability
DL & QL VERSUS SENSITIVITY
Sensitivity is measured by the slope of the calibration curve:
 More sensitive method, steeper slope: Results in a larger change in the measured
response versus the controlled variable

DL & QL are measured by one of the four methods:

 lowest concentration for which RSD is <5.0%
 plot of standard deviation versus concentration
 95% CI of a best fit
 signal to noise ratio
DETERMINING DL AND QL:

Per ICH-Q2A:

DL & QL can be calculated based on the standard deviation of the response ()
and the slope of the calibration curve (S) at levels approximating the limits
according to the following formulas:
DL= 3.3 (/S)

QL= 10 (/S)

The  can be determined based on the  of the blank, the residual  of the
regression line, or the  of y-intercepts of regression lines.
LINEARITY

The ability of an analytical procedure (within a given range), to obtain test results
which are directly proportional to the concentration (amount) of analyte in the
sample.
LINEARITY CALCULATIONS

y = mx + b
Where: y = response, x = concentration, m = slope, and
b = y intercept

b
Percent Bias =  100%
(x  m) + b
ACCURACY

• The measure of how close the experimental value is to the true value.

− Established across a specified range.

− Also called trueness.

ACCURACY
Determination of Accuracy:

• 9 determinations over 3 concentrations in triplicate preparation.

• The mean is an estimate of accuracy.

• RSD is an estimate of sample analysis precision.

ACCURACY
Should be reported as:

• The percent recovery by the assay of known added amount of analyte in the sample.

• The difference between the mean and the accepted true value together with the
confidence intervals.
PRECISION

The closeness of agreement between a series of measurements, obtained

from a multiple sampling of the same homogeneous sample, under the
prescribed conditions.
PRECISION
Includes:

Repeatability
Intermediate Precision
Reproducibility

Report:

Standard Deviation, Relative Standard

Deviation, Confidence Interval
REPRODUCIBILITY

• Expresses the precision between laboratories.

• Recommended parameters to be evaluated at the second laboratory include:

– Selectivity
– DL/QL
– Repeatability
– System Suitability
RUGGEDNESS
• Degree of reproducibility of test results under a variety of conditions:
−Different Laboratories
−Different Analysts
−Different Instruments
−Different Reagents
−Different Days

Ruggedness ≠ Robustness
ROBUSTNESS

• A measure of a method’s capacity to remain unaffected by small, deliberate variations in

method parameters.
• Provides an indication of a method’s reliability during normal usage.
• Assessed by making small, deliberate changes to the method and evaluating the results.
ROBUSTNESS
Examples of typical RP-HPLC variations:

pH of mobile phase
mobile phase composition
Ionic Strength
Different columns
Column temperature
flow rate
ROBUSTNESS
Conditions Tested for
Nominal Procedure
Parameter Robustness
Condition
Determination
MPA*-Buffer constituent pH 4.0 3.9, 4.1
MPA*-Buffer salt 10 mM Ammonium
9 mM, 11 mM
concentration Formate
Column Temperature 30°C 25°C, 35°C

Detector Wavelength 290 nm 288 nm, 292 nm

Flow Rate 1.0 mL/min 0.9 mL/min, 1.1 mL/min

Injection Volume 20 µL 15 µL, 25 µL

*MPA = Mobile Phase A

METHOD REVALIDATION

Revalidate due to changes in:

Synthesis of the drug substance.

Composition of the drug product.
Analytical procedure.
ANALYTICAL METHOD LIFE CYCLE

Redevelopment of 
Revalidation required  Change is not covered 
the method required 
due to change by existing validation
due to change

Development of Validation of the Method in Change to Method:

the Method Method Routine use Evaluate the effect

Change is covered by 
existing validation
REFERENCES
1. Bob Snider, CMC Group
2. ICH Q2 (R1)
3. Current USP <1224>
4. Current USP <1225>
5. Current USP <1226>
6. FDA Guidance for Industry
7. Miller, JM., Crowther, JB. 2000. Analytical Chemistry in a GMP
Environment. John Wiley & Sons, Inc.
WHAT IS SUCCESS?
THANK YOU
Questions? Comments?