Phase Appropriate Method Validation Aryo Boston-Nitto 2

Phase Appropriate Method Validation
Aryo Nikopour
The Southern California Pharmaceutical Discussion Group (SCPDG) of AAPS
Irvine, CA
January 12, 2017

VALIDATION OF ANALYTICAL METHOD

METHOD LIFE CYCLE
Validation
Development Optimization

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.orgwww.ICH.org
(Dates indicate ICH finalization)
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

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.
Noncompendial:
• 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
CategoryCategory
NumberNumber Category NameCategory Name Description of AssayDescription of Assay
I Quantitative
Quantitation of major
components/active ingredients
present at high concentrations.
II
Impurities-
Quantitative Determination of impurities or
degradation products.
II Impurities-Limit
III
Performance
Characteristics
Parameters to be tested depend on
the nature of the test; includes
dissolution testing.
IV Identity

METHOD VALIDATION REQUIREMENTS
USP Assay Category
I
II
III IV
Parameter: Quantitative Limit Tests
Accuracy Y Y • Y N
Precision Y Y N Y N
Intermediate
Precision
Y Y N Y N
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-
clinical PM

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 Characteristics Experimental Details Acceptance Criteria
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
measurement/injections
%RSD ≤2%
Linearity At least 5 Concentration over the range
Assay: 50% to 125% of Specification limit
QL-150% of specification limit
Calibration Model is valid
R ≥0.998
Report Intercept, Slope and %Bias
Detection Limit (DL) DL= 3.3 (DL= 3.3 (σσ/S)/S) S/N≥ 3S/N≥ 3
Quantitation Limit (QL) DL= 10 (DL= 10 (σσ/S)/S) %RSD≤ 15%%RSD≤ 15%

METHOD VALIDATION CHARACTERISTICS
Validation Characteristics Experimental Details Acceptance Criteria
Precision :
Repeatability
Intermediate Precision (Ruggedness)
Reproducibility
6 replicates
6 replicates
%RSD≤ 2%
Overall %RSD (two Analyst)
Accuracy At least 9 determination over 3
concentration level
e.g. 70 to 120% for
For Assay Mean Recovery 97 to 103%
for Impurities : 85% to 115%
Range The range is defined by the results
obtained for linearity, accuracy and
precision
Linearity, accuracy and precision
demonstrated over the range
Solution Stability Determine solution stability of Reference
Standard Solution and Sample over 72
hours
98 to 102 % of control
Robustness Deliberately change critical parameters of
the method
Must meet system suitability and
selectivity requirements

VALIDATION: PHASE I
Drug Product Assay I.D.
Quantitative
Impurities
Limit Test
Selectivity X X X X
Repeatability X X
Accuracy/Precision Recovery at
100%
At 100% of
Reporting
Threshold
Linearity X QL to 200% of
Limit
Range Defined by ALP Defined by 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
Impurities
Limit Test
Selectivity X X X X
Specificity X
Repeatability X X X
Accuracy Recovery at 3
levels
At 100% of
Reporting
Threshold
Linearity X X X
DL/QL DL X QL
Range Define by ALP Defined by ALP

VALIDATION: PHASE III
Assay I.D Quantitative
Impurities
Limit Test
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
Range Defined by ALP Defined by ALP
Robustness X X X

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 forWhat parameters do you measure for
system suitabilitysystem suitability

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

Date NB/Page
Standard B1 (n=6) Injections SST Solution B1 and B2
Area %RSD
Average Tailing
Factor %RSD Retention Time Average Theoretical Plates
Resolution LVF and
DesMethyl-LVF)
Response Factor %
Difference
≤ 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 0.128051 2770 0.168485904
3 Sigma 0.384153 8311 0.505457713
min -0.28 21418 2.88
max 0.48 38040 3.90

SELECTIVITY AND SPECIFICITY
SelectivitySelectivity vs.vs. SpecificitySpecificity

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.

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).

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.

• 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 drug substance molecule.

Defining characteristics of degradation studies:
• Carry out in solution and/or in the solid state.
• Involve conditions more severe than accelerated testing.
• 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.
• Design study based on the formulation (feed, tablet, ointment, etc.).

Chemical Physical Environmental
Acid Agitation Heat
Base
Denaturation,
aggregation, adsorption
and precipitation
Light
(ICH Option I or II)
Oxidation RH
Deamidation Freeze/Thaw
Disulfide Bond Exchange

STRESS STUDY PATHWAYS
Pharmaceutical Biologics
Hydrolytic Hydrolytic
Oxidative Oxidative
Photolytic Aggregation
Thermolytic Deamidation
Disulfide Bond Exchange

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
Light
600 to 800 foot candles
(sources include metal
halides, Hg, Xe lamp, or
UVB fluorescence)
Option II: 74Hours
Option I: 2-4 Hours
Heat/RH 40 °C/ 75% RH and
60 °C
24 to 72 hours
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%).

 Optimize detector setting
 Stress blank, placebo,
standard and sample
 Inject controls
 Extend run time
 Orthogonal Method
Overstress!!Overstress!!

EXAMPLE: PHOTOLYTIC STRESS
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
-2.20
0.00
1.25
2.50
3.80
1-SequenceName:ForcedDegradationLight SampleName:Fresh30mg/mLControl, Sample#12
2-SequenceName:ForcedDegradationLight SampleName:LightStressed30mg/mLSet1, Sample#17
mAU
min
4
3
2
1
Imp1-3.397
Imp3-6.097
Imp5-7.873
Imp6-8.320
DesMethyl-LVF-8.980
Levofloxacin-9.777
Imp7-10.733
Imp9-12.817
Imp10-13.320
Imp11-14.517
Imp14-20.487
WVL:280nm

EXAMPLE: ACID STRESSEXAMPLE: ACID STRESS
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0
-3.40
-2.00
0.00
2.60
1-SequenceName:ForcedDegradationAcid_Base SampleName:Fresh30mg/mLControl, Sample#17
2-SequenceName:ForcedDegradationAcid_Base SampleName:2hrsAcid30mg/mL, Sample#24
mAU
min
5
4
3
2
1
Imp1-2.670
Imp3-3.387
Imp7-8.430
DesMethyl-LVF-9.097
Levofloxacin-9.890
WVL:280nm

EXAMPLE: HEAT STRESSEXAMPLE: HEAT STRESS
0.3 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0
-1.50
0.00
1.50
1-SequenceName:ForcedDegradationHeat SampleName:Fresh50mg/mLControl, Sample#17
2-SequenceName:ForcedDegradationHeat SampleName:4hrHeat,50mg/mLAssay, Sample#24
mAU
min
4
3
2
1
Imp1-2.660
Imp3-4.327
Imp4-6.070
Imp6-8.287
DesMethyl-LVF-8.960
Levofloxacin-9.753
WVL:280nm

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: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: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:Per ICH-Q2A:
DL & QL can be calculated based on the standard deviation of the response (σ)
and the slope of the calibration curve (S) at levels approximating the limits
according to the following formulas:
DL= 3.3 (DL= 3.3 (σσ/S)/S)
QL= 10 (QL= 10 (σσ/S)/S)
The σ can be determined based on the σ of the blank, the residual σ of the
regression line, or the σ of y-intercepts of regression lines.

DETECTION LIMIT (DL)
4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0
-0.100
-0.000
0.100
0.200
0.300
1-LOD_LOQ#1 DiluentBlank UV_VIS_1
2-LOD_LOQ#11 LVFLOD(0.004ug/mL) UV_VIS_1
mAU
min
4
2-Levofloxacin-9.773
3
2
1
WVL:280nm

Quantitation Limit (QL)
4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0
-0.10
0.50
1.00
1.50
1-LOD_LOQ#1 DiluentBlank UV_VIS_1
2-LOD_LOQ#14 LVFLOQ(0.009ug/mL) UV_VIS_1
mAU
min
7
6
5
4
3
2
1
WVL:280nm

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 = my = m••x + bx + b
Where: y = response, x = concentration, m = slope, and
b = y intercept
Percent Bias =
b
(x m) + b
100%
•
×

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.

DETERMINING ACCURACY FOR DRUG SUBSTANCES
• Use reference material.
• Compare procedure results with those of a second, well-characterized procedure.
• Infer from precision, linearity and specificity; 80,100 and 120% levels of label
claim.

DETERMINING ACCURACY FOR DRUG PRODUCTS
• Spike suitable Reference Materials into a Placebo.
• Add known quantities of analyte to the drug product.
• Compare procedure results with those of a second, well-characterized procedure.
• Infer from precision, linearity and specificity; 80, 100 and 120% levels of label claim.

DETERMINING ACCURACY FOR DRUG
PRODUCT RELATED SUBSTANCES
 Add known quantities of impurities to the sample.
Compare procedure results with those of a second, well-characterized procedure.
If impurities are not available, how do
you perform accuracy?

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:Includes:
Repeatability
Intermediate Precision
Reproducibility
Report: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

MINIMUM SPECIFIED RANGES: DRUG SUBSTANCES
Impurity Reporting Thresholds
Maximum
Daily Dose
Qualification
and ID
Threshold
Reporting
Threshold
< 2g/day
0.1% or
1mg/day
(choose the
lower)
0.05%
> 2g/day 0.05% 0.03%

MINIMUM SPECIFIED RANGES: DRUG PRODUCTS
Degradation Product Reporting Thresholds In
New Drug Products:
Maximum Daily
Dose
Reporting
Threshold
≤ 1 g/day 0.1%
> 1 g/day 0.05%

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:Examples of typical RP-HPLC variations:
pH of mobile phase
mobile phase composition
Ionic Strength
Different columns
Column temperature
flow rate

ROBUSTNESS
Parameter
Nominal
Procedure
Condition
Conditions Tested for
Robustness
Determination
MPA*-Buffer constituent pH 4.0 3.9, 4.1
MPA*-Buffer salt
concentration
10 mM Ammonium
Formate
9 mM, 11 mM
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
Change to Method:
Evaluate the effect
Development of
the Method
Validation of the
Method
Method in
Routine use
Redevelopment of
the method required
due to change
Revalidation required
due to change
Change is not covered
by existing validation
Change is covered by
existing validation

REFERENCES
1. Bob Snider, CMC Group
2. ICH Q2 (R1)
3. Current USP <1224>
6. FDA Guidance for Industry
7. Miller, JM., Crowther, JB. 2000. Analytical Chemistry in a GMP
Environment. John Wiley & Sons, Inc.

Questions? Comments?
THANK YOU

Phase Appropriate Method Validation Aryo Boston-Nitto 2

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