PHARM 462

2009

Body

European and International regulatory bodies and their guidelines on different aspects of QA
Full name Guidance on
Focus for Analytical Chemistry in Europe Cooperation of International Traceability in Analytical Chemistry European Cooperation for Accreditation European Committee for Normalization International Union of Pure & Applied Chem. International Standardization Organisation Association of Official Analytical Chemists International Laboratory Accreditation Cooperat. US Food and Drug Administration Method validation Proficiency testing Quality Assurance Accreditation Standardization Method validation Standardisation

Eurachem CITAC EA CEN IUPAC ISO AOAC ILAC FDA

Internal qual. Control Proficiency testing Accreditation

Method validation Method validation Method validation 2009 2

USP
ICH

United States Pharmacopoeia

International Conference on Harmonization

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 Detector Injector Data System

Analyst
Sample
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Method

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Method Life Cycle

Validation

Development

Optimization

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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

p r o v i d e d characteristics: f o r t h e f o l l o w i n g

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 <1225>: 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 <1225>
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 The accuracy, sensitivity, specificity, and reproducibility of test methods employed by the firm

shall be established and documented. Such validation

a nd doc ume ntation m ay be ac com plis he d i n 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

ICH/USP

GMPs (legal)

FDA

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ICH/USP Validation Requirements & Parameters

USP

ICH
Specificity
Linearity Range Accuracy

Specificity Linearity and Range Accuracy Precision Limit of Detection Limit of Quantitation

Precision

Repeatability Intermediate Precision Reproducibility

Ruggedness
Robustness

Limit of Detection Limit of Quantitation

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USP Data Elements Required For Assay Validation

Analytical Performance Parameter Accuracy Assay Category 1 Yes Assay Category 2 Quantitative Yes Limit Tests * Assay Category 3 *

Precision
Specificity

Yes
Yes

Yes
Yes

No
Yes

Yes
*

LOD
LOQ Linearity Range Ruggedness
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No
No Yes Yes Yes

No
Yes Yes Yes Yes

Yes
No No * Yes

*
* * * Yes
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* May be required, depending on the nature of the specific test.

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

Type of Analytical Procedure Accuracy Precision Repeatability Interm. Prec. Specificity LOD LOQ Linearity Range
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Impurity testing Identification No No No Yes No No No No Quantitative Yes Yes Yes Yes No Yes Yes Yes Limit Tests No No No Yes Yes No No No Assay Yes Yes Yes Yes No No Yes Yes
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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 > 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)

Amount Added (mg) Amount Found (mg) Percent Recovery

0.0 50.2
79.6 99.9 120.2 150.4

0.0 50.4
80.1 100.7 119.8 149.7

--100.5
100.6 100.8 99.7 99.5

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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

Should be assessed
using minimum of 9 determinations

over a short interval of

time. Also referred to as Intra-assay precision

(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

Day 1
100.6 100.8

Day 2
99.5 99.9

100.1
100.3 100.5 100.4 Mean = 100.5 RSD = 0.24%

98.9
99.2 99.7 99.6 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

Lab 1
Day 1 Man 1 3 Prep Day 2 Man 2 3 Prep

Lab 2
Day 1 Man 1 3 Prep Day 2 Man 2

Lab 3
Day 1 Man 1 3 Prep Day 2 Man 2 3 Prep

Repeatability test at two different labs.

3 Prep

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

LOQ
Lowest amount of analyte in a sample that can be quantified with suitable

quantitated.
Estimated by Signal to Noise Ratio of 3:1.
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accuracy and precision.

Estimated by Signal to Noise Ratio of 10:1.
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LOD and LOQ Estimated by

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

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LOD and LOQ Estimated by

3.3s DL = S

QL =

10s S

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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Y=bX+a
Statistical estimate of LOD & LOQ

LOD = 3.3 Sbl / b LOQ = 10 Sbl / b

Ybl LOD LOQ

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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 <621>

System Suitability Requirements

Parameters
K R T

Recommendations
In general k 2.0 R > 2, between the peak of interest and the closest potential interferent (degradant, internal STD, impurity, excipient, etc..) T2

N
Repeatability
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In general N > 2000

RSD 2.0% (n 5)
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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 u n v a l i d a t e d 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 Inspection: Laboratory System

Feb July 2002: 212 Inspections (US)
Method Validation 13%
Stability Program 21%

Training/Qual. 4% Controls. General 35%

Inadequate Records 27%

* Reference: Albinus D Sa, FDA, CDER Office of Compliance, from AAPS, Nov. 2002 presentation. 2009

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ICH Update:

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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

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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

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Working Groups
SAFETY EFFICACY

QUALITY

MULTIDISCIPLINARY

STEERING COMMITTEE
Endorses topics, guidelines and monitors progress
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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
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