Pharmaceutical Development
Analytical Method Development
Presented by: Birgit Schmauser, pharmacist, PhD
Training Workshop on Pharmaceutical Development
|
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
|
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
|
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
|
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
|
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
|
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 “M”s
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
|
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
|
8 with a Focus on Paediatric Medicines / 15-19 October 2007
Accuracy and precision
Accurate
&
precise
Accurate
&
imprecise
Training Workshop on Pharmaceutical Development
|
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
|
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
|
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%
2
174926
148.00 mg/98.66%
3
172933
146.32 mg/97.54%
4
175011
148.08 mg/98.72%
5
179557
151.95 mg/101.30%
6
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
|
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
|
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
|
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
|
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
|
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
|
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
|
18 with a Focus on Paediatric Medicines / 15-19 October 2007
Separation of very closely related analytes
 Specificity
Training Workshop on Pharmaceutical Development
|
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
|
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
|
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
|
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
|
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
|
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
|
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
|
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
|
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
|
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
|
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
|
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
|
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
|
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
|
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
|
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
|
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
|
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
|
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
|
38 with a Focus on Paediatric Medicines / 15-19 October 2007
THANK YOU
Training Workshop on Pharmaceutical Development
|
39 with a Focus on Paediatric Medicines / 15-19 October 2007