Pharmaceutical Development

Analytical Method Development

Presented by: Birgit Schmauser, pharmacist, PhD

Training Workshop on Pharmaceutical Development

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1 with a Focus on Paediatric Medicines / 15-19 October 2007

Analytical method development

Objectives of the presentation
Originator and multisource generic FPPs
Equivalence (comparability)

Specifications
setting

Stability
assessment

Cleaning validation
Parallel development of analytical methods

Training Workshop on Pharmaceutical Development

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2 with a Focus on Paediatric Medicines / 15-19 October 2007

Interchangeability (IC)
Interchangeability (IC) of multisource generic FPPs
(Essential similarity with Innovator FPP)

Pharmaceutical + Bioequivalence
Equivalence

IC = PE + BE
Training Workshop on Pharmaceutical Development
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3 with a Focus on Paediatric Medicines / 15-19 October 2007

Pharmaceutical equivalence
FPPs meet the same or comparable standards

Same API (chemical and physical equivalence)

Same dosage form and route of administration
Same strength
Comparable labeling

Equivalence in pharmaceutical development

Equivalence in stability
Equivalence in manufacture (WHO-GMP)
Training Workshop on Pharmaceutical Development
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4 with a Focus on Paediatric Medicines / 15-19 October 2007

Prequalification requirements
Validation of analytical methods is a prerequisite for
prequalification of product dossiers
Non-compendial APIs and FPPs are tested with methods
developed by the manufacturer
For compendial APIs and FPPs the applicability of
methods to particular products must be demonstrated
(verification)

Analytical methods must be developed and validated

according to ICH Q2 (R1)
To be used within GLP and GMP environments

Training Workshop on Pharmaceutical Development

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5 with a Focus on Paediatric Medicines / 15-19 October 2007

Use of analytical methods - generics

CLINICAL

PHARMACEUTICAL

METHODS

At initial phase of pharmaceutical development

To determine
bioavailability in
healthy volunteers

To develop a stable and

reproducible formulation for
the manufacture of
bioequivalence, dissolution,
stability and pilot-scale
validation batches

To understand the profile of related

substances and to study stability
To start measuring the impact of
key product and manufacturing
process parameters on consistent
FPP quality

At advanced phase of pharmaceutical development

To prove
bioequivalence after
critical variations to
the prequalified
dossier

To optimise, scale-up and

transfer a stable and
controlled manufacturing
process for the prequalification
product

Training Workshop on Pharmaceutical Development

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6 with a Focus on Paediatric Medicines / 15-19 October 2007

To be robust, transferable, accurate

and precise for specification setting,
stability assessment and QC
release of prequalified product
batches

Prerequisites for validation

Quality fitness for use
ensure by validation
Six Ms

enable by suitable
surroundings

Man (Qualified personel)

Machine (Qualified, calibrated robust instruments)

Methods (Suitable, characterised & documented)

Material (sufficient quality, & Reference standards)
Milieu (Laboratory conditions)

Management
Training Workshop on Pharmaceutical Development
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7 with a Focus on Paediatric Medicines / 15-19 October 2007

Validation characteristics
Identification

Impurities
quantitative

limit

Assay

Accuracy

Precision

Specificity

Detection Limit

Quantitation Limit

Linearity

Range

Robustness

Training Workshop on Pharmaceutical Development

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8 with a Focus on Paediatric Medicines / 15-19 October 2007

Accuracy and precision

Accurate
&
precise

Accurate
&
imprecise

Training Workshop on Pharmaceutical Development

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9 with a Focus on Paediatric Medicines / 15-19 October 2007

Inaccurate
&
precise

Inaccurate & imprecise

Precision
Expresses the closeness of agreement between a series
of measurements obtained from multiple sampling of the
same homogenous sample
Is usually expressed as the standard deviation (S),
variance (S2) or coefficient of variation (RSD) of a series
of measurements
Precision may be considered at three levels
Repeatability (intra-assay precision)
Intermediate Precision (variability within a laboratory)
Reproducibility (precision between laboratories)
Training Workshop on Pharmaceutical Development
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10 with a Focus on Paediatric Medicines / 15-19 October 2007

General consideration
68.26% of measured values
within mean 1 SD
95.46% of measured values
within mean 2 SD
99.73% of measured values
within mean 3 SD

An interval of 3 SD should
be calculated to fully cover variability
Training Workshop on Pharmaceutical Development
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11 with a Focus on Paediatric Medicines / 15-19 October 2007

Mean

Spread of data

1SD
2SD
3SD

Repeatability
Determination of the API in a FPP (tablet):
Six replicate sample preparation steps from a homogenously prepared tablet
mixture (nominal value of API 150 mg)
Injection Peak area Assay
1

173865

147.10 mg/98.06%

174926

148.00 mg/98.66%

172933

146.32 mg/97.54%

175011

148.08 mg/98.72%

179557

151.95 mg/101.30%

176425

149.28 mg/99.52%

Mean

175453

148.45 mg/98.96%

SD

2329

1.98 mg/1.32%

RSD

1.32%

1.32%

Training Workshop on Pharmaceutical Development

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12 with a Focus on Paediatric Medicines / 15-19 October 2007

Mean 3 SD =
Confidence interval of 99.73%
98.96 3x1.32% = 95% - 102.92%

Intermediate Precision
Expresses within-laboratories variations
Different days, different analysts, different equipment etc.
Injection
1
2
3
4
5
6
Mean
SD
RSD

Peak area
analyst 1
173865
174926
172933
175011
179557
176425
175453
2329
1.32%

Peak area
analyst 2
175656
175878
176004
176344
175332
174959
175695
495
0.28%

Peak area
analyst 3
177965
178556
177342
178011
179466
179688
178504
918
0.51%

Training Workshop on Pharmaceutical Development

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13 with a Focus on Paediatric Medicines / 15-19 October 2007

Mean 3 SD: (177252 100%)

Analyst 1: 98.96% 3 x 1.32%
Analyst 2: 99.12% 3 x 0.28
Analyst 3: 100.70% 3 x 0.51

Average of 3 analysts 3SD:

95% - 102.23%

Reproducibility
Expresses the precision between laboratories
Collaborative studies, usually applied to standardisation of
methodology
Transfer of technology
Compendial methods

Training Workshop on Pharmaceutical Development

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14 with a Focus on Paediatric Medicines / 15-19 October 2007

Accuracy
Expresses 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

Training Workshop on Pharmaceutical Development

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15 with a Focus on Paediatric Medicines / 15-19 October 2007

true

mean

Sometimes referred to as TRUENESS

Accuracy
To find out whether a method is accurate:
Drug substance (assay)
Application of the method to an analyte of known purity (e.g. reference
substance)
Comparison of the results of one method with those of a second wellcharacterised method (accuracy known)

Drug product (assay)

Application of the method to synthetic mixtures of the drug product
component to which known quantities of the analyte have been added
Drug product may exceptionally be used as matrix

Drug substance/Drug product (Impurities)

Application of the method to samples spiked with known amounts of
impurities
Training Workshop on Pharmaceutical Development
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16 with a Focus on Paediatric Medicines / 15-19 October 2007

Accuracy
Application of the method to synthetic mixtures of the drug product
component to which known quantities of the analyte have been
added
Recovery reduced
by ~10 15%

Source: Analytical Method

Validation and Instrument
Performance Verification,
Edited by Chung Chow Chan,
Herman Lam, Y.C. Lee,
and Xue-Ming Zhang
ISBN 0-471-25953-5
Wiley & Sons

Training Workshop on Pharmaceutical Development

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17 with a Focus on Paediatric Medicines / 15-19 October 2007

Specificity
Is the ability to assess unequivocally the analyte in the presence of
components which may be expected to be present (impurities,
degradants, matrix)
Identity testing
To ensure the identity of an analyte

Purity testing
To ensure accurate statement on the content of impurities of an analyte

Assay
To allow an accurate statement on the content of an analyte in a sample

Training Workshop on Pharmaceutical Development

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18 with a Focus on Paediatric Medicines / 15-19 October 2007

Separation of very closely related analytes

Specificity

Training Workshop on Pharmaceutical Development

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19 with a Focus on Paediatric Medicines / 15-19 October 2007

Specificity
Overlay chromatogram of an impurity solution with a sample solution

Source: Analytical Method

Validation and Instrument
Performance Verification,
Edited by Chung Chow
Chan, Herman Lam,
Y.C. Lee,
and Xue-Ming Zhang
ISBN 0-471-25953-5
Wiley & Sons

Training Workshop on Pharmaceutical Development

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20 with a Focus on Paediatric Medicines / 15-19 October 2007

Specificity and stability

Stress stability testing to ensure the stability indicating potential of
an analytical method
Apply diverse stress factors to the API
Apply diverse stress factors to the FPP
Stress conditions: e.g. Supplement 2 of Generic Guideline; TRS 929, Annex 5

Assure that the API can be assessed specifically in the presence of

known and unknown (generated by stress) impurities

Assure that known impurities/degradants can be specifically

assessed in the presence of further degradants
By peak purity assessment and (overlay of) chromatograms
Training Workshop on Pharmaceutical Development
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21 with a Focus on Paediatric Medicines / 15-19 October 2007

Limit of Detection (LOD, DL)

The LOD of an analytical procedure is the lowest amount
of analyte in sample which can be detected but not
necessarily quantitated as an exact value
Determination is usually based on
Signal to noise ratio (~3:1) (baseline noise)
or
Standard deviation of response (s) and Slope (S)
3.3 s/S

Training Workshop on Pharmaceutical Development

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22 with a Focus on Paediatric Medicines / 15-19 October 2007

Limit of Quantitation (LOQ, QL)

The LOQ is the lowest amount of analyte in a sample
which can be quantitatively determined with suitable
precision and accuracy.
The quantitation limit is used particularly for the determination
of impurities and/or degradation products

Determination is usually based on

Signal to noise ratio (~10:1) (baseline noise)
or
Standard deviation of response (s) and Slope (S)
10 s/S
Training Workshop on Pharmaceutical Development
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23 with a Focus on Paediatric Medicines / 15-19 October 2007

LOD/LOQ
LOD, LOQ and Signal to Noise Ratio (SNR)
LOQ

Signal to Noise = 10:1

Signal to Noise = 3:1

LOD
Noise

Training Workshop on Pharmaceutical Development

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24 with a Focus on Paediatric Medicines / 15-19 October 2007

LOQ and impurities

In determination of impurities in APIs and FPPs the LOQ
should be determined in the presence of API
LOQ should be NMT reporting level
LOQ should be given relative to the test concentration of API

Specificity of impurity determination should always be

demonstrated in the presence of API at API specification
levels
Spiking of test concentration (API/FPP) with impurities at levels
of their specification range

Training Workshop on Pharmaceutical Development

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25 with a Focus on Paediatric Medicines / 15-19 October 2007

LOQ and impurities

Spiking
API test concentration (normalised)
0.1 mg/ml (100%)
Impurity spiking concentrations
0.001 mg/ml (1%) specification limit
0.0001 mg/ml (0.1%) limit of quantitation (minimum requirement)

Training Workshop on Pharmaceutical Development

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26 with a Focus on Paediatric Medicines / 15-19 October 2007

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
If there is a linear relationship test results should be
evaluated by appropriate statistical methods
Correlation coefficient (R2)
Y-intercept
Slope of regression line
Residual sum of squares
PLOT OF THE DATA
Training Workshop on Pharmaceutical Development
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27 with a Focus on Paediatric Medicines / 15-19 October 2007

Linearity
Usual acceptance criteria for a linear calibration curve
r > 0.999; y-intercept a < 0 to 5% of target concentration; RSD response < 1.5-2%

Source: Analytical Method Validation and Instrument Performance Verification, Edited by Chung ChowChan, Herman Lam,
Y.C. Lee,and Xue-Ming ZhangISBN 0-471-25953-5 Wiley & Sons

Training Workshop on Pharmaceutical Development

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28 with a Focus on Paediatric Medicines / 15-19 October 2007

Range

The range of an analytical procedure is the interval

between the upper and lower concentration (amounts) of
analyte in the sample for which it has been demonstrated
that the analytical procedure has a suitable level of
precision, accuracy and linearity

Training Workshop on Pharmaceutical Development

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29 with a Focus on Paediatric Medicines / 15-19 October 2007

Range
Assay
80 to 120% of test concentration

Content uniformity
70 to 130% of test concentration)

Dissolution
Q-20% to 120%

Impurities
Reporting level 120% of specification limit (with respect to test
concentration of API)

Assay & Impurities

Reporting level to 120% of assay specification
Training Workshop on Pharmaceutical Development
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30 with a Focus on Paediatric Medicines / 15-19 October 2007

Range
Linearity is limited to 150% of
shelf life specification of
impurities
Test concentration can be
used to determine impurities
To determine drug substance
(assay) the test concentration
must be diluted
The range is 0 ~ 150% of
impurity specification

Source: Analytical Method Validation and

Instrument Performance Verification,
Edited by Chung ChowChan, Herman Lam,
Y.C. Lee, and Xue-Ming Zhang
ISBN 0-471-25953-5 Wiley & Sons

Training Workshop on Pharmaceutical Development

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31 with a Focus on Paediatric Medicines / 15-19 October 2007

Robustness
Robustness of an analytical procedure should show
the reliability of an analysis with respect to deliberate
variations in method parameters
The evaluation of robustness should be considered
during the development phase
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 procedure
Training Workshop on Pharmaceutical Development
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32 with a Focus on Paediatric Medicines / 15-19 October 2007

Robustness
Influence of buffer pH and buffer concentration in mobile phase on
retention times of API and impurities
API

Impurity A

Impurity B

Impurity C

As is

10.46

3.86

7.43

8.26

buffer pH 5.9

10.45

3.94

7.51

8.38

buffer pH 6.9

10.46

3.94

7.49

8.34

Buffer conc. 83%

7.84

3.43

6.16

6.66

Buffer conc. 87%

15.26

4.77

9.61

11.18

Conclusion: The buffer composition should be maintained in a range

of 85 0.5%
Missing: Acceptance criterion for maximal deviation of retention time should
be defined unless justified
Training Workshop on Pharmaceutical Development
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33 with a Focus on Paediatric Medicines / 15-19 October 2007

System suitability Testing

Based on the concept that equipment, electronics,
analytical operations and samples to be analysed
constitute an integral system that can be evaluated as
such
System suitability test parameters are established for
each analytical procedure individually
System suitability parameters depend on the type of analytical
procedure

Training Workshop on Pharmaceutical Development

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34 with a Focus on Paediatric Medicines / 15-19 October 2007

Method stability
System suitability over time
Stability of analytical solutions
Sample solution stability
A solution of stavudine is stable for ~ 2 h, then it starts to degrade to
thymine
Impurity-spiked sample solution stability
Cave: A solution containing stavudine spiked with its impurity thymine
does not allow to clearly distinguish between degradation and spike due
to the lower precision at impurity levels
Should be analysed immediately
Training Workshop on Pharmaceutical Development
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35 with a Focus on Paediatric Medicines / 15-19 October 2007

Setting Specifications
Upper and lower specification limits
Process variability
Analytical variability
3 SD and specification acceptance range

Given specification limits/ranges

Assay
Analytical variability
Process variability
Impurities
LOQ and specification limit (e.g. qualification limits NMT 0.15%)
Response factors (LOQ modified by response factor)
Training Workshop on Pharmaceutical Development
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36 with a Focus on Paediatric Medicines / 15-19 October 2007

Methods for cleaning validation

Method for assay and related substances used in stability
studies of API and FPP
Specificity (in samples taken from a cleaning assessment)
Linearity of response (from 50% of the cleaning limit to 10x this
concentration; R2 0.9900)
Precision

Repeatability (RSD 5%)

intermediate precision [ruggedness (USP)]
reproducibility

Limits of detection and quantitation

Accuracy or recovery from rinsate ( 80%), swabs ( 90%), and process
surface ( 70%)
Range (lowest level is at least 2x higher than LOQ)

Training Workshop on Pharmaceutical Development

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37 with a Focus on Paediatric Medicines / 15-19 October 2007

Summary
Analytical procedures play a critical role in
pharmaceutical equivalence and risk
assessment/management
Establishment of product-specific acceptance criteria
Assessment of stability of APIs and FPPs

Validation of analytical procedures should demonstrate

that they are suitable for their intended use
Validation of analytical procedures deserves special
attention during assessment of dossiers for
prequalification
Training Workshop on Pharmaceutical Development
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THANK YOU

Training Workshop on Pharmaceutical Development

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39 with a Focus on Paediatric Medicines / 15-19 October 2007