Method validation

1. PHARM 462
1
2009

2. 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. Method validation
ISO International Standardization Organisation Standardisation
AOAC
ILAC
Association of Official Analytical Chemists
International Laboratory Accreditation Cooperat.
Internal qual. Control
Proficiency testing
Accreditation
FDA US Food and Drug Administration Method validation
USP United States Pharmacopoeia Method validation
ICH International Conference on Harmonization Method validation
2 2009

3. 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
3
2009

4.  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
4
2009

5. 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.
5
2009

6. 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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2009

7. 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.
7
2009

8. 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
8
2009

9. 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
9
2009

10.  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%
10
2009

11. 11
System Suitability
Sample
Validation
Method
Analyst
Calibration
Pump
Detector
Injector
Data System
2009

12. Method Life Cycle
12
Validation
Development Optimization
2009

13. Verification vs. Validation
 Compendial vs. Non-compendial Methods
 Compendial methods-Verification
 Non-compendial methods-Validation requirement
13
2009

14. 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
14
2009

15. 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”
15
2009

16. 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
16
USP 23 General
Information <1225>
2009

17.  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).
17
21 CFR PART 211 - CURRENT GOOD MANUFACTURING
PRACTICE FOR FINISHED PHARMACEUTICALS
Subpart I-Laboratory Controls
211.165 Testing and release for distribution (e)
2009

18.  The objective of validation of an analytical
procedure is to demonstrate that it is suitable
for its intended purpose
18
ICH Guideline for
Industry
Q2A, Text on
Validation of
Analytical
Procedures
March 1995
2009

19.  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.
19
ICH Guideline for Industry
Q2B, Validation of
Analytical Procedures:
Methodology
2009

20. Today’s Validation Requirements
20
ICH/USP
GMPs
(legal) FDA
2009

21. ICH/USP Validation Requirements &
Parameters
 Specificity
 Linearity
 Range
 Accuracy
 Precision
 Repeatability
 Intermediate Precision
 Reproducibility
 Limit of Detection
 Limit of Quantitation
21
ICH
 Specificity
Specificity
 Linearity and Range
Linearity and Range
 Accuracy
Accuracy
 Precision
Precision
 Limit of Detection
Limit of Detection
 Limit of Quantitation
Limit of Quantitation
 Ruggedness
Ruggedness
 Robustness
Robustness
USP
2009

22. USP Data Elements Required
For Assay Validation
22
Analytical
Performance
Parameter
Assay
Category 1
Assay Category 2
Assay
Category 3
Quantitativ
e
Limit Tests
Accuracy Yes Yes * *
Precision Yes Yes No Yes
Specificity Yes Yes Yes *
LOD No No Yes *
LOQ No Yes No *
Linearity Yes Yes No *
Range Yes Yes * *
Ruggedness Yes Yes Yes Yes
* May be required, depending on the nature of the specific test.
2009

23. 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
23
2009

24. ICH Validation Characteristics vs.
Type of Analytical Procedure
24
Type of
Analytical
Procedure
Identificati
on
Impurity testing
Assay
Quantitativ
e
Limit Tests
Accuracy No Yes No Yes
Precision
Repeatability
No Yes No Yes
Interm.
Prec.
No Yes No Yes
Specificity Yes Yes Yes Yes
LOD No No Yes No
LOQ No Yes No No
Linearity No Yes No Yes
2009

25. 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
25
2009

26. 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.
26
2009

27. 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
27
2009

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

29. 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
29
2009

30. 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
30
2009

31. Accuracy
 Closeness of the test
results obtained by the
method to the true value.
31

32. 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
32
2009

33. Accuracy Data Set (1 of 3)
33
Amount
Added (mg)
Amount
Found (mg)
Percent
Recovery
0.0 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

34. 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
.
34
2009

35. Precision… Considered at 3 Levels
 Repeatability
 Intermediate Precision
 Reproducibility
35
2009

36. Repeatability
 Express the precision
under the same
operating conditions
over a short interval of
time.
 Also referred to as
Intra-assay precision
36
Should be assessed
using minimum of 9
determinations
(3 concentrations/ 3
replicates) or
or
Minimum of 6
determinations at
the 100% level.
2009

37. Intermediate Precision
37
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

38. 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
38
Grand
Mean = 100.0
RSD = 0.59%
Mean = 100.5
RSD = 0.24%
Mean = 99.5
RSD = 0.36%
2009

39. 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
1
Day
2
Day
1
Day
2
Man
1
Man
2
Man
1
Man
2
Man
1
Man
2
3
Prep
3
Prep
3
Pre
p
3
Prep
3
Pre
p
3
Prep
39

40. 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 3:1.
40
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

41. 1. Based in Visual Evaluations
- Used for non-instrumental methods
2. Based on Signal-to Noise-Ratio
- 3:1 for Detection Limit
- 10:1 for Quantitation Limit
3. Based on Standard Deviation of the Response and
the Slope
41
LOD and LOQ Estimated by
2009

42.  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
42
DL =
DL =
3.3s
3.3s
QL =
QL =
10s
10s
S
S S
S
LOD and LOQ Estimated by
2009

43. 43
Ybl
LOD LOQ
Statistical estimate of LOD & LOQ
LOD = 3.3 Sbl / b LOQ = 10 Sbl / b
Y = b X + a
2009

44.  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
44
Robustness
2009

45. 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
45
2009

46. 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)
46
2009

47.  USP 23 <621>
 System Suitability Requirements
47
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

48. 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
48
2009

49. How do we Know the expectations of
the FDA
?
 FDA Form 483
 FDA Warning Letters
 Personal Experiences
49
2009

50. 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.
50
2009

51. 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)
51
2009

52. FDA Systems Based Inspection:
Laboratory System
52
Method
Validation
13%
Training/Qual.
4%
Stability Program
21%
Inadequate
Records
27%
Controls. General
35%
Feb – July 2002: 212 Inspections (US)
* Reference: Albinus D’ Sa, FDA, CDER Office of Compliance, from AAPS, Nov. 2002
presentation.2009

53. ICH Update
:
2009 53

54. 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 54

55. 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 55

56. Working Groups
SAFETY EFFICACY
QUALITY MULTIDISCIPLINARY
STEERING COMMITTEE
Endorses topics, guidelines and monitors progress
2009 56

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