Method Validation in the Pharmaceutical Industry

Kate Arnot

Analytical Development, PAR&D (AW)

6th November 2002

Overview
The drug development process General recommendations Examples
Assay Dissolution test for an immediate release tablet Identity Identification test for a drug substance Impurities Quantitative test residual solvents in a drug substance Limit test residual solvents in a tablet

DRUG DEVELOPMENT PROCESS

Years 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Start
Clinical Trials
Phase 1
50-100 people

Approval
Phase 2
100-200 people

Phase 3
500-5000 people

Phase 4

Initial research

Drug discovery

Clinical Development

Full Scale Manufacture

Toxicology and Pharmacokinetics Chemical development Pharmaceutical development Sales and Marketing

Cost

2M

150M

300M

10,000 compounds

1 new medicine
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A phased approach to analytical validation

Phase I Limited validation, focussing on key method attributes eg.specificity, limits of quantitation and linearity Phase II Starting to include accuracy and precision data to support specifications 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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General recommendations
Design your validation studies to reflect the way that the method will be applied in routine use as far as possible Ensure that you have considered the specification you want to achieve in designing your studies Know your method before you start to validate!
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Assay - Dissolution test for an immediate release tablet

Dissolution testing is used to evaluate the release of drug substance from tablets Dissolution apparatus 2 (paddles), 50rpm, 37C, aqueous surfactant as dissolution medium, UV analysis, sampling at a single 45 minute timepoint Typical Specification: Q = 80 at 45 minutes (ie each of 6 units should achieve 85% dissolution)
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Dissolution specification USP acceptance table

S1, 6 units Each unit is not less that Q+5% S2, 6 units Average of 12 units (S1+S2) is equal to or greater than Q and no unit is less than Q-15% S3, 12 units Average of 24 units (S1+S2+S3) is equal to or greater than Q, not more than 2 units are less than Q-15%, and no unit is less than Q-25%
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Dissolution apparatus

Typical dissolution profile

% D i s s o l u t i o n
100 90 80 70

60 % dissolved

50

40

30 20

10

0 0 15 30 tim e (m ins) 45 60 75

Time (mins)

Validation parameters evaluated

Specificity Linearity Accuracy and range Precision
Repeatability Intermediate precision

Robustness
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Specificity
Demonstrate absence of interference from excipients and reagents at the monitoring wavelength For example calculate the absorbance of a placebo solution as a percentage of the absorbance of a standard solution Typically results of less that 2% would be considered acceptable Specificity may also be inferred from linearity and accuracy data
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Linearity
Range studied 0 to 150% of nominal (7 solutions, including a blank solution) Linearity experiments conducted in the presence and absence of excipients Determine equation of the line, correlation coefficient, 95% confidence interval of the slope and the intercept. Verify that the 95% confidence interval of the intercept includes zero, or that the result is insignificant in the context of the experiment Check that the response for both experiments is the same calculate the slopes as a % of each other Typically results of 98 to 102% would be considered 12 acceptable

Linearity in the presence of placebo

1.6 1.5

A b s o r b a n c e

1.4

1.3

1.2

1.1

y = 42.983x + 0.004 r = 0.99989 95% CI of slope 0.0730 95% CI of intercept 0.016

Ab 0.9 sor ban 0.8 ce

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 0.018 0.02 0.022 0.024 0.026 0.028 0.03 0.032 0.034 0.036 0.038

concentration (g/100ml)

Concentration (g/100ml)

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Linearity in the absence of placebo

1.6 1.5

A b s o r b a n c e

1.4 1.3 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0

y = 43.033x + 0.006 r = 0.99990 95% CI of slope 0.695 95% CI of intercept 0.009

Absorbance

Comparison of slopes: (42.983/43.033) x 100 = 99.9%

0.002 0.004 0.006 0 .0 08 0.01 0.012 0.014 0.016 0.018 0.02 0.022 0.024 0.026 0.028 0.03 0.032 0 .0 34 0.036 0.038

concentration (g/100m l)

Concentration (g/100ml)

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Accuracy and Range

3 samples at each of 5 different concentration levels were evaluated: 50%, 85%, 100%, 115% and 125% of nominal Samples were analysed against standards as per the method and % recoveries calculated Typically results in the range 95 to 105% would be considered acceptable The overall standard deviation and relative standard deviation may be used as an estimate of precision
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Example Accuracy Data

Experiment 1 50% 97.3 85% 96.8 100% 96.5 115% 96.0 125% 95.8 Mean 96.5 sd 0.7 Experiment 2 50% 99.3 85% 99.4 100% 99.6 115% 98.7 125% 98.9 Mean 99.2 sd 0.5
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Example Accuracy Data continued

Experiment 1
Samples mixed, and allowed to stand overnight

Experiment 2
Samples stirred continuously Time 1 Recovery 98.8% (hours) 4 99.1% 21 99.2% 27 99.3%
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Accuracy/Linearity how do you prepare your samples?

Prepare samples containing drug substance plus an appropriate quantity of ground placebo tablet Prepare samples containing drug substance plus appropriate quantities of an excipient mix Prepare samples by using different weights from a ground active tablet preparation, making a correction for nominal concentration Make sure that you are confident in your sample extraction technique
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Precision - Repeatability
One analyst conducting multiple replicates of the same sample, on the same day using the same equipment, and applying the method as written. For dissolution experiments correcting for tablet weight may be appropriate Determine the mean, the range, the standard deviation and the relative standard deviation.

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Precision Intermediate precision

A second operator conducts the repeatability experiment on a different day using different equipment where possible. Determine the individual data and overall mean, range, standard deviation and relative standard deviation. Consider testing statistically that there is no significant difference between operators before combining the data set.
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Robustness
A number of attributes were evaluated prior to validation: Standard and sample solution stability Effect of pH and the requirement to buffer and/or degass the dissolution medium Reagents from different suppliers were evaluated Sampling tubes, filters and sampling and filtration speeds were evaluated Single point vs profile testing
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Identification Infrared method for a drug substance

Infrared spectroscopy is frequently used in identification testing Sample is dispersed in KBr, analysed using the DRIFTS technique, and the spectrum obtained visually compared with that of a fully characterised reference standard analysed under the same conditions. Typical Specification: Result consistent with reference standard
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Validation parameters evaluated

Specificity
Identification testing should optimally be able to discriminate between compounds of closely related structure which are likely to be present (ICH Q6A) The choice of such potentially interfering materials should be based on sound scientific judgement with a consideration of the interferences that could occur (ICH Q2B)
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Specificity
Demonstrate specificity between drug substance and other potentially interfering compounds eg. starting materials, isolated intermediates and other materials manufactured or formulated in the same plant Consideration should also be given to enantiomers, counter ions/salts, solvates and polymorphs if necessary.

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1.3 1.2 1.1 1.0

1.0 82968I01

Drug substance
Kubelka-Munk

0.9

0.8

0.7

0.9 0.8 Kubelka-Munk 0.7 0.6 0.5 0.4 0.3 0.2

0.1 0.3 0.6

Development compound

0.5

0.4

0.2

0.1 0.0 4000 3500 3000 2500 2000 Wavenumbers (cm-1) 1500 1000 500
3500 3000 2500 2000 1500 1000 500 Wavenumbers (cm-1)

0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 Kubelka-Munk 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 -0.05 4000 3500 3000 2500 2000 Wavenumbers (cm-1) 1500 1000 500 Kubelka-Munk

0.60

Isolated intermediate

0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 -0.00 -0.05 4000

Compound formulated in same plant

3500

3000

2500 2000 Wavenumbers (cm-1)

1500

1000

500

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Impurities Quantitative test for residual solvents in drug substance A range of solvents are used in the manufacture of a drug substance. It is necessary to quantify these, and demonstrate that they comply with ICH Q3C requirements Capillary GC method with flame ionisation detection. Typical Specification: 0.5% w/w maximum
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Validation parameters evaluated

Specificity Linearity Accuracy Precision
Reproducibility Intermediate precision

Range Limits of detection and quantitation Robustness

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Specificity
Demonstrate method is specific for all the solvents used in the registered synthetic process Can use linearity data for solvents in the presence and absence of the drug substance to show that the drug substance does not interfere
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uV 22000 24000 26000 28000 0.0 Triethylamine Ethyl Acetate Propan-2-ol Acetonitrile Toluene 1.0 Tim (m e in) 2.0 Dimethylsulphoxide 3.0 N-Methyl-2-pyrrolidone

Specificity chromatogram

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Linearity
Range studied 0 to 150% of nominal (9 solutions, including a blank solution and a solution at the LOQ) Drug substance required without solvent, or low levels of solvent and then make a correction Determine equation of the line, correlation coefficient, 95% confidence interval of the slope and the intercept. Verify that the 95% confidence interval of the intercept includes zero, or that the result is insignificant in the context of the experiment

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Accuracy
Calculate recoveries from the linearity experiments by analysing the solutions against an external standard as per the method. Typically results in the range 80 to 120% would be considered acceptable The overall standard deviation and relative standard deviation may be used as an estimate of precision
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Table 1

Residual solvents by GC - accuracy of ethyl acetate in the presence of drug substance

Ethyl acetate added theoretical (l/100 ml) 0 1.0 1.5 2.5 5.0 7.5 10.0 12.5 15.0 Ethyl acetate theoretical (mg/100 ml)a Ethyl acetate determined (mg/100 ml) Recovery (% w/w)

Concentration ethyl acetate % nominal (with respect to standard concentration) 0b 10 15 25 50 75 100 125 150 Mean Standard deviation % RSD
a b

0 0.902 1.353 2.255 4.510 6.765 9.020 11.275 13.530

0 0.844 1.289 2.017 4.495 6.874 8.991 11.435 13.616

NA 93.5 95.3 89.4 99.7 101.6 99.7 101.4 100.6 97.7 4.4 4.5

This figure is corrected for the density of ethyl acetate (0.902 g/ml) The 0 level solution represents a test solution of drug substance to which no ethyl acetate was added. A correction for the level of ethyl acetate found in this solution was made for all other test solutions prepared. NA Not applicable

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Intermediate precision - Japanese style

Expt No Operator GC Column 1 1 1 1 2 1 2 2 3 1 2 1 4 1 1 2 5 2 1 1 6 2 2 2 7 2 2 1 8 2 1 2

Experiments are conducted on different days with each analyst using each instrument/column combination in turn Report results, overall mean, overall standard and relative standard deviation, and 95% CI of sd
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Range
Determined from the linearity, accuracy and precision data

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Limit of Quantitation and Detection

Limit of quantitation established as 0.05% w/w Samples at this concentration included in linearity and accuracy experiments Limit of detection estimated as 0.02% w/w by visual inspection of chromatograms

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uV 18000 20000 22000 24000 0.0 0.809 EtOAc 1.0 2.0 Time (min) 2.762 DMSO 3.0 3.061 NMP 1.086 ACN

LOQ chromatogram

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Robustness
A number of attributes were evaluated prior to validation using an experimental design approach: Injector temperature Detector temperature Initial temperature Ramp rate Hold time Split flow Column head pressure
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Impurities Limit test for residual solvent in a tablet

An alcohol is used as a preservative in a purchased film coating concentrate. It is removed during the manufacturing process, but this needs to be demonstrated Fast GC method with flame ionisation detection. Need to demonstrate that tablets comply with requirements for Class 3 38 solvents ie less than 0.5% w/w

Validation parameters evaluated

Specificity For solvents arising from the drug substance and tablet manufacturing process 0.05% w/w 0.05% to 0.5% w/w

Limit of quantitation Linearity

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References
ICH www.ICH.org International Pharmaceutical Product Registration Aspects of Quality, Safety and Efficacy, eds Cartwright and Matthews, Ellis Horwood, 1994, Chapter 8, Analytical validation The validation of analytical methods for drug substances and drug products in UK pharmaceutical laboratories, G S Clarke, J. Pharm. Biomed. Anal., 12. 643 652 (1994) FDA www.FDA.gov Technical review guide: Validation of chromatographic methods
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