Analytical method validation approaches

from Development to Launch

BM Rao, Ph.D. 21st July, 2011

Topics

Analytical Method Validation ( What, Why, When and How much)

Drug Development Phases Validation Requirement (Innovator Vs Generics & regulatory Perspective) Validation Prerequisites Validation Activity Flow Validation Parameters System Suitability

Out of Acceptance case studies

Recent FDA 483s & Warning Letters References Summary recommendations & Conclusions Q & A session

Glossary
RT : Reporting Threshold DL : Detection Limit QL : Quantitation Limit SL : Specification Limit API : Active Pharmaceutical Ingredient DS : Drug Substance DP : Drug Product IND : Investigational New Drug CTA : Clinical Trial Application NDA : New Drug Application MAA : Marketing Authorization Application Sample Matrix: Other possible ingredient of drug product except the API

What is Method Validation

Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use
The FDA defines the term as : Established documented evidence which provides a high degree of assurance that a

specific process will consistently produce a product meeting its pre-determined

specifications and quality attributes. - General Principles of Validation (1987) ICH guideline : A documented program that provides a high degree of assurance that a specific process, method, or system will consistently produce a result meeting pre-determined acceptance criteria. - Q7A-GMP for active pharmaceutical ingredients (2000) EU-guideline : Action of proving, in accordance with GMP-principles that any procedure, process,

equipment, material, activity or system actually leads to the expected results.

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Why Method Validation

To obtain reliable analytical results & comply with international regulations Essential component of the measure that laboratories should employ to ensure that they produce accurate and reliable results Universally recognized comprehensive system of quality assurance 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 To meet accreditation requirement Ensure that the test method give correct results Customers want to be assured of the correctness of result Objective evidence for defense against challenges

When Method Validation

Method validations are required when

New method is developed

Existing method is significantly modified (optimized) Existing validated method is applied to a different sample matrix

Method Validation how much is adequate

Validation is always a balance between costs, risks and technical possibilities

Depends on Phase of drug for which method is to be used The critically of the measurement The scope of the method

Method Validation how much contd..

Test method description
Standard methods with performance data (e.g. compendia method USP/EP etc.) in-house developed methods Published in the literature without any performance data Published in the literature with performance data

Validation or Verification requirements

Verification of performance, but validation may be required if any changes made Full validation Full validation Verification of performance but more likely full validation required

Changes in implementation of previously validated method i.e. changes to equipment, reagents, lab environment or Verification staff. Existing validated method applied to different matrices, different concentration ranges Existing validated method applied to additional analytes Commercial Test Kits - collaboratively tested, third party evaluation (e.g. AOAC) Commercial Test Kits - no performance data available, incomplete or not applicable Validation - extent will vary - e.g. having similar properties to those of representative matrices Full Validation Verification Full Validation

Different Phases During New Drug Development

Pre-phase-I Phase-I Phase-II
Phase-II A Phase-II B

Phase-III

Filing / Approvals NDA

Pre-phase-I Phase-I Phase-IIA

: Pharmacology and toxicology studies : Testing the simplest formulation of drug in healthy volunteers : Evaluation of drug for the clinical effectiveness in the target patient
population (for fixing the proper and safe dosage range)

Phase-II B and Phase-III : Testing in thousands of patients with proposed marketed

formulation after the establishment of safe and clinical effectiveness (Late Phase Development)

Filing / Approvals : Regulatory submissions Early phase Late phase

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Drug Product Development and GMP

Standard screening methods Validated Method

Full characterization Full GMP

21 CFR 210, 211

Phase II A
Phase I

Phase II B

Phase III

Pre-clinical

Early Phase Late Phase Clinical Monitoring Program

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PHASED APPROACH TO ANALYTICAL VALIDATION

Pre- Phase1 to Phase I Limited validation, focussing on key method attributes eg. specificity, limits of quantitation and linearity Up to Phase IIA Starting to include accuracy and precision data to support specifications From Phase IIB to Phase III Full validation according to ICH guidelines will be completed for all analytical methods prior to submission of marketing applications

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Innovator Vs Generics
Innovator R & D on API Preclinical trials
Clinical trials pre -phase I to IIa
(Early Development)

Generics -

Method validation summary

Full Method validation Validated methods Validated methods

Validated methods: GMP and GLP

Clinical trials phase IIb to III (Late Development) Post marketing phase IV Entering of Generics; Pharmaceutical development, Comparability with Innovator

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Validation Parameters defined in ICH & USP

USP ( USP 33, NF 28)
Specificity
Linearity & Range Accuracy Precision

ICH (Q2 (R1))

Specificity
Linearity Range Accuracy Precision (Repeatability, Intermediate Precision, Reproducibility) Detection Limit Quantitation Limit Robustness
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Limit of Detection

Limit of Quantitation Ruggedness Robustness

Assessment of method validation requirements

What analytes should be detected? What are the expected concentration levels? What are the sample matrices? Are there interfering substances expected, and, if so, should they be detected and quantified? Are there any specific legislative or regulatory requirements? Should information be qualitative or quantitative? What are the required detection and quantitation limits? What is the expected concentration range? What precision and accuracy is expected? How robust should the method be?

Which type of equipment should be used? Is the method for one specific instrument, or should it
be used by all instruments of the same type? Will the method be used in one specific laboratory or should it be applicable in all laboratories at one side or around the globe? What skills do the anticipated users of the method have?

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Prerequisites for analytical method validation

Six Ms Machine Man
qualified

Methods
characterised

calibrated

robust skilled qualified suitable

documented

Reference standards

Vibrations

Time Irradiations

Quality of the analytical method

Analysts support

Temperature Quality
Humidity

Supplies

Material

Milieu

Management
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Validation Activity Flow

Approved SOP for method validation Validation Protocol Validation Specifications

Initiate the event to Identify the root cause

Not meeting Acceptance Criteria

Execution of analytical activity

Define Corrective action

Validation Report
Summary of the observed events

Meeting the acceptance criteria

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Required Validation Parameters

Identification Impurities quantitative limit Assay

Accuracy
Precision Repeatability (System Repeatability & Analysis Repeatability) Intermediate precision Reproducibility Specificity Detection Limit

+
+

+ + + + + +

+ + + -

Quantitation Limit
Linearity Range

+ +
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SPECIFICITY
Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc. Specificity is demonstrated by representative chromatograms of appropriate solutions which may include but not limited to , reference, selectivity batch, stressed sample, placebo and stressed placebo solutions that contains all compounds for which specificity has to be proven.

Most common techniques are used to determine specificity: Photo-diode array detector LC-MS
The chromatographic signal does not indicate any impurity in either peak. Spectral evaluation, however, identifies the peak on the left as impure.

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ACCURACY & PRECISION

Accuracy The closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. Precision The closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.

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ACCURACY
Should be established across specified range of analytical procedure. Should be assessed using a minimum of 3 concentration levels covering the specified range in presence of sample matrix, each in triplicate (total of 9 determinations). Should be evaluated as Percent recovery of known amount added.
Concentration in % w.r.t. nominal sample concentration 70-80-100-120-130 QL-SL- atleast 1.2 times SL 70-100-130 20b-100-120 QL-SL- atleast 1.2 times SL Concentration Concentration % Recovery 1 % Recovery 1 70% 70% 100.6 100.6 100.2 100.2 99.0 99.0 99.9 99.9 100% 100% 99.7 99.7 99.9 99.9 100.2 100.2 99.9 99.9 120% 120% 99.7 99.7 99.4 99.4 99.2 99.2 99.4 99.4

Test Type Assay Related Substances a Content Uniformity Dissolution Residual Solvent

% Recovery 2 % Recovery 2
% Recovery 3 % Recovery 3 Mean Recovery Mean Recovery

a: using impurity for specified impurity and using active for unspecified degradants b: it should be below the value at 1st timepoint of profile. Ex. extended release it may be below 20%.

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PRECISION
The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility. Precision should be investigated using homogeneous, authentic samples. However, if it is not possible to obtain a homogeneous sample it may be investigated using artificially prepared samples or a sample solution. Repeatability (System Repeatability & Analysis Repeatability) Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision . Intermediate precision Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc. Reproducibility Reproducibility expresses the precision between laboratories (collaborative studies, usually applied to standardization of methodology).

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Variations affecting method reproducibility

Precision Instrument Batches of accessories e.g. chrom. columns same same Intermediate Precision different different Reproducibility different different

Operators
Sample matrices Concentration Batches of material, e.g., reagents Environmental conditions, e.g., temperature Laboratory

same
different different same

different
different different different

different
different different different

same same

different same

different different

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PRECISION - System Repeatability

It will be determined by injecting at least 5 consecutive injections of the same solution . RSD of the response will be evaluated.
Test Type Assay Related Substances Concentration in % w.r.t. nominal sample concentration 100 SL - individual Impurity & API (for unknown)

Content Uniformity
Dissolution Residual Solvent Determination 1 2 3 4 5 % RSD

100
100 SL 100% API 19770367 19748915 19726133 19776942 19847909 0.2 SL (0.5%) of API 99353 99342 99749 99407 99584 0.2
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PRECISION - Analysis Repeatability

Should be assessed using a minimum of 3 concentration levels covering the specified range in presence of sample matrix, each in triplicate (total of 9 determinations). or It will be determined by analyzing at least 6 sample preparations by one person on one instrument in same sample set . Sample should contain all impurities of interest. If single sample does not contain all, multiple samples can be used or spiking can be preferred. RSD of the assay will be evaluated (for active as well as impurities).
Determination 1 2 3 4 5 6 % RSD % API 98.9 99.4 100.2 101.1 99.2 98.7 0.9 % Imp - A 0.08 0.09 0.10 0.10 0.11 0.10 10.7 % Imp - B 0.25 0.28 0.26 0.29 0.25 0.25 6.7
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PRECISION - Intermediate Precision

It will be determined by analyzing at least 6 sample preparations each by two different person, instrument on different day. Sample should contain all impurities of interest. If single sample does not contain all, multiple samples can be used or spiking can be preferred. RSD/Pooled RSD of the assay for all samples will be evaluated (for active as well as impurities) or Absolute/% relative difference between two analyst can be evaluated. Intermediate precision may not be needed if reproducibility is performed.
Active Impurity

Determination
1 2

Analyst 1
98.9 99.4

Analyst 2
100.8 100.3

Analyst 1
0.25 0.28

Analyst 2
0.23 0.26 0.25 0.24 0.22 0.24 7.7
25

3
4 5 6 % RSD

100.2
101.1 99.2 98.7 0.8

101.1
99.8 100.5 100.1

0.26
0.29 0.25 0.25

PRECISION - Reproducibility
It will be determined by analyzing at least 6 sample preparations each by two different laboratories. Sample should contain all impurities of interest. If single sample does not contain all, multiple samples can be used or spiking can be preferred. RSD/Pooled RSD of the assay for all samples will be evaluated (for active as well as impurities) or Absolute/% relative difference between two laboratories can be evaluated.
Active Determination 1 2 3 4 5 6 laboratory 1 98.9 99.4 100.2 101.1 99.2 98.7 laboratory 2 100.8 100.3 101.1 99.8 100.5 100.1 Impurity laboratory 1 0.25 0.28 0.26 0.29 0.25 0.25 laboratory 2 0.23 0.26

0.25
0.24 0.22 0.24

% RSD

0.8

7.7
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DETECTION LIMIT & QUANTITATION LIMIT

DETECTION LIMIT : The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. QUANTITATION LIMIT : The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.
Based on Visual Evaluation Based on Signal-to-Noise Based on the Standard Deviation of the Response and the Slope

Limit of detection and limit of quantitation via signal to noise 27

Quantitation Limit
When impurity is not available 3 separate solutions are prepared containing the API at a reporting threshold concentration. These three solutions can be prepared with 100% placebo. When impurity is available 3 separate solutions are prepared containing the Impurity at a reporting threshold concentration with 100% API and 100% placebo.

Note: Three RT solutions must be prepared from three different stock solutions. These solutions are analysed and the recovery & repeatability is evaluated. First Blank injection is considered for S/N ratio calculation. Quantitation limit: Acceptance Criteria Method type Early Phase (IND/CTA) Late Phase (NDA/MAA) Concentration Range Target QL RT Target QL RT Mean % recovery 50.0 - 150.0 70.0 - 130.0 % RSD (n=3) 25.0 15.0

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LINEARITY
The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. A linear relationship should be evaluated across the range of the analytical procedure at minimum 5 levels. Linearity should be evaluated by visual inspection of a plot of signals as a function of analyte concentration or content. Test results should be evaluated by appropriate statistical methods, for example, by calculation of a regression line by the method of least squares. Correlation coefficient, % RSD of the response factor can be evaluated. Y-intercept, slope of the regression line and residual sum of squares should be submitted .

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LINEARITY
Signal height or peak area as a function of analyte concentration

Divide signal data by their respective concentrations, yielding the relative responses. A graph is plotted with the relative responses on the y-axis and the corresponding concentrations on the xaxis, on a log scale

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RANGE
The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity. The range is normally derived from linearity studies and depends on the intended application of the procedure. It is established by confirming that the analytical procedure provides an acceptable degree of linearity, accuracy and precision.

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ROBUSTNESS
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. The evaluation of robustness should be considered during the development phase and depends on the type of procedure under study. If measurements are susceptible to variations in analytical conditions, the analytical conditions should be suitably controlled or a precautionary statement should be included in the method. Examples of typical variations 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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OTHER ADDITIONAL PARAMATERS

Stability of the Solutions Filtration Study Relative Response factor Automation

Method Equivalency
Carry over

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SYSTEM SUITABILITY
System suitability tests are an integral part of gas and liquid chromatographic methods. They are used to verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis to be done. The tests are based upon the concept that the equipment, electronics, analytical operations, and samples to be analyze d constitute an integral system that can be evaluated as such. System Suitability Test characteristics and limits are recommended as a component of any analytical method and are established to ensure the validity of the analytical method whenever used.
Parameters K R T N Repeatability Recommendations In general k 2.0

R > 2, between the peak of interest and the closest potential interferent (degradant, internal STD, impurity, excipients, etc..)
T2 In general N > 2000 RSD 2.0% (n 5)
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Revalidation & Bracketing Validation

Revalidation is performed under following situations Change in the synthetic route Changes in sample preparation procedure where recovery or sample matrix effect may change Changes in Analyte detection method e.g. Change in UV wavelength, UV to Fluorescence detector etc. Changes in Chromatographic Operational parameters ( Column packing, Separation technique, sample load, etc.) Elucidation of new Impurities or Degradation products.

Only above conditions are not limited. Case to case evaluation is needed !! Bracketing approach for validation Dose Proportional Formulations : Validation can be shown for Lowest strength & additionally intermediate precision or reproducibility shall be done for Highest strength. Non-dose proportional formulations : worst case placebo shall be used.

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Out of Acceptance case studies

Observation Root cause Corrective action

Accuracy passing at 70 & 100% but failing at 130%.

The peak height of sample and standard solution is higher than max linearity range of UV detector.
Impurity was not completely soluble in dilution solvent of the method. One of the excipient is trapping the API. Acidic sample diluent improved the recovery Solubility issue at higher concentration Lower concentration solutions (0.05%) injected after 100% level. Improper homogenization of dissolution media.

Revise the test method reduce the concentration.

to

Accuracy is not meeting the acceptance criteria for specified impurity Accuracy is failing for impurity at quantitation level (0.05%)

Identify the correct dilution solvent in which impurity is soluble. Issue with original diluent. Modified the method prior to revalidation Method validated in 80-120%. Blank injection before lower concentration solution.

Accuracy is not passing for drug Substances at 130%. Linearity test is failing for RSD of Response factor but correlation coefficient is passing. % Dissolution is variable during reproducibility test.

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FDA- Form 483

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. Stress studies conducted as part of method validation do not target a minimum amount of degradation. a standard period of two hours as commonly used for stress studies with no justification Spreadsheets used to calculate linearity, percent recovery, and final assay results for the cleaning validation of were not validated and the data transcribed from chromatographs to the spreadsheets were not checked for accuracy.

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FDA- Warning Letters

On addition to the example of modifying both compendia methods and customer supplied methods, we also observed the use of un-validated in-house methods as well as invalidated modifications to in-house methods. Change control procedures in the laboratory failed to document test method changes to assure accurate, reliable, and reproducible results. The test method did not state whether a helix was to be used during dissolution testing. A was reportedly used during method development, validation and daily method runs, but there is no documentation of a being used in any of the documents.

There is no assurance that qualification or maintenance of the laboratory equipment can

consistently produce valid and accurate analytical results in that numerous examples of test data were invalidated due to instrument malfunction.

Attempts to corroborate data in the validation report with supporting raw data in the laboratory were difficult and frustrating for the FDA personnel conducting the inspection.

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FDA- Warning Letters

OOS accuracy results reported by analyst 3 were never submitted in the final report.

Repeat analysis performed in a different system passed specifications and these

results were submitted in the report. Raw data and calculations were not checked by a second responsible individuals required by your procedures. Inaccurate calculations were noted in the report.

The process validation samples were assays using an HPLC method that had not
been validated. The method validation used for both products did not include a protocol that included specification and acceptance criteria. The method validation was not reviewed and approved until during the current inspection. Lots of both

products were released for distribution prior to completion of the method validation.
Method validation for the product Sennosides is inadequate in that the data does not assess all variables, such as different mobile phase concentrations and analytes, to demonstrate that the method can sustain variance.

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References
ICH Q2 (R1) Validation of Analytical Procedures: Text and Methodology, International Conference on Harmonization. ICH Q3A (R2): Impurities in New Drug Substances, International Conference on Harmonization. ICH Q3B (R2): Impurities in New Drug Products, International Conference on Harmonization. ICH Q6A: Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances, International Conference on Harmonization. ICH Q7: Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients, International Conference on Harmonization.

FDA Draft Guidance for Industry on Analytical Procedures and Methods Validation: Chemistry, Manufacturing, and Controls Documentation. August 2000
Center for Drug Evaluation and Research (CDER) Guidance: Guideline for Submitting Samples and Analytical Data for Methods Validation. February 1987 Center for Drug Evaluation and Research (CDER) Reviewer Guidance: Validation of Chromatographic methods. November 1994 US Pharmacopoeia General chapters: General tests and assays US Pharmacopoeia chapter <1225> Validation of Compendial Procedures US Pharmacopoeia chapter <1226> Verification of Compendial Procedures US Pharmacopoeia chapter <1092> The Dissolution Procedure: Development and Validation

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Summary recommendations
Develop a validation master plan or an operating procedure for method validation For individual method validation projects, develop a validation project plan Define intended use of the method and performance criteria Check all equipment and material for performance and quality Perform validation experiments Summarize the Validation outcome (include the critical method validation observations in the respective methods) Develop an operating procedure for method transfer between laboratories

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Conclusions
Analytical Method Validation is not jus a routine activity. Need to be done in a high level GMP environment Results generated throughout the validation activity needs to be reviewed carefully Successful validation provides Successful Method Transfers & Satisfactory performance

of the Analytical method throughout the Lifecycle

Quality issues if not addressed during method validations may have severe impact during drug development (loss of time, costs, regulatory queries etc.)
Method development

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QUESTIONS ???

Contact details: BM Rao, Ph.D. Director Analytical Development Pharmaceutical Development & Manufacturing Sciences Janssen India pharmaceutical companies of Johnson & Johnson Ltd. Email : drbmrao@hotmail.com

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