Pharmaceutical Development
Training Workshop on
Pharmaceutical Development with
focus on Paediatric Formulations
Protea Hotel
Victoria Junction, Waterfront
Cape Town, South Africa
Date: 16 to 20 April 2007
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April 2007
Pharmaceutical Development
Analytical Method Development
Presenter:
János Pogány, pharmacist, PhD
pogany.janos@chello.hu
WHO expert
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April 2007
Analytical Method Development
Outline and Objectives of presentation
 Introduction, guidelines
 Dossier requirements
– Assay
– Related substances
– Other issues
 Main points again
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April 2007
Training Workshop on
Pharmaceutical Development
with focus on Paediatric
Formulations
Introduction, guidelines
Interchangeability (IC)
INTERCHANGEABILITY (IC) OF MULTISOURCE
FPPs = (ESSENTIAL SIMILARITY WITH
INNOVATOR FPP) =
PHARMACEUTICAL EQUIVALENCE (PE) +
BIOEQUIVALENCE (BE)
IC = PE + BE
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April 2007
Pharmaceutical equivalence
 FPPs meet same or comparable standards (e.g.,
marketing authorization, analytical methods)
–
–
–
–
Same API (chemical and physical equivalence)
Same dosage form and route of administration
Same strength
Comparable labeling
 Pharmaceutical development equivalence
 Stability equivalence
 WHO-GMP (manufacturing equivalence)
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April 2007
Prequalification requirements
 Analytical method validation is required by WHO for the
prequalification of product dossiers. Non-compendial ARV
APIs and FPPs were/are tested with methods developed
by the manufacturer.
 Analytical methods should be used within GMP and GLP
environments, and must be developed using the protocols
and acceptance criteria set out in the ICH guidelines
Q2(R1)
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April 2007
Guidelines used in PQP
 „WHO-GMP 4.11 „It is of critical importance that particular
attention is paid to the validation of analytical test
methods, automated systems and cleaning procedures.”
 Appendix 4. Analytical method validation (in WHO Expert
Committee on Specifications for Pharmaceutical Preparations. 40th
Report. Geneva, WHO, 2006 (WHO Technical Report Series, No.
937). http://whqlibdoc.who.int/trs/WHO_TRS_937_eng.pdf
 Validation of analytical procedures: text and methodology
Q2(R1) ICH Harmonized Tripartite Guidelines, (2005)
http://www.ich.org/LOB/media/MEDIA417.pdf
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April 2007
General requirements
 Qualified and calibrated instruments
 Documented methods
 Reliable reference standards
 Qualified analysts
 Sample selection and integrity
 Change control
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April 2007
Measure of variation (spread of data)
68.26%
95.46%
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April 2007
Mean (average) chart
Abnormal variation of process – special causes
USL
Upper specification limit
average = mean
LSL
Lower specification limit
Abnormal variation of process – special causes
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April 2007
Normal
variation
due to
common
causes
Capable process
Almost all the measurements of a
stable process fall inside the
specification limits
USL – LSL
Cp
6
8
10
1.00 1.33 1.66 2.00
OoS results: .27%.
6 ppm
64 ppm
http://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htm
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April 2007
12
2 ppb
NEVIRAPINE – Reference Standard
Injection
System suitability
requirement:
RSD is NMT 0.85%
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April 2007
tR
A
1
2
3
6.160
6.167
6.166
12466
12311
12432
4
5
6
6.172
6.165
6.168
12530
12457
12479
Mean
STD
RSD
6.166
0.004
0.06%
12446
74
0.59%
Training Workshop on
Pharmaceutical Development
with focus on Paediatric
Formulations
Dossier requirements
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 and 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
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To optimize, scale-up, and transfer a
stable and controlled
manufacturing process for the
prequalification product
April 2007
To be robust, transferable, accurate,
and precise for specification
setting, stability assessment,
and QC release of prequalified
batches
Analytical procedure characteristics
Type of
characteristic
Identification
Impurities
Quantitative
Assay
Limit
Accuracy
-
+
-
+
Precision
-
+
-
+
Specificity
+
+
+
+
Detection limit
-
-
+
-
Quantitation limit
-
+
-
-
Linearity
-
+
-
+
Range
-
+
-
+
Robustness
+
+
+
+
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April 2007
Accuracy - ISO 5725 1-6
Accuracy
Precision
Trueness
Systematic errors
(Random errors)
Intra-assay
variability
Intra-laboratory
variability
Repeatability
Inter-laboratory
variability
Intermediate
precison
Reproducibility
Source: ISO. 1994. ISO 5725 1-6: Accuracy (Trueness and Precision) of Measurement Methods and Results. ISO, Geneva, Switzerland.
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April 2007
Accuracy and precision
Inaccurate &
imprecise
Inaccurate and imprecise
Inaccurate but
precise
Accurate but
imprecise
Precise
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Accurate
April 2007
Accurate and precise
Percent accuracy (hypothetical figures)
Sample
LA, %
Nevirapine, mg
Added
Recovered
Recovery
%
RSD
%
1
2
50
70
0.499
0.703
0.495
0.701
99.2
99.8
0.64
0.31
3
4
5
80
100
120
0.796
1.001
1.211
0.795
1.005
1.209
99.9
100.4
99.8
0.27
1.88
0.38
6
130
Mean
1.299
0.918
1.296
0.917
99.8
99.8
1.12
0.77
The data show that the recovery of analyte in spiked samples met the evaluation
criterion for accuracy (100 ± 2.0% across 50–130% of target concentrations).
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April 2007
% Recovery
Percent accuracy (hypothetical figures)
104
103
102
101
100
99
98
97
96
0
1
2
3
4
5
6
Number of samples
Red line: LA
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April 2007
Green lines: USL and LSL
7
Precision (of any process)
Measured mean
Real mean
The precision (VARIABILITY) of an
analytical procedure is usually
expressed as the standard deviation
(S), variance (S2), or coefficient of
variation (= relative standard
deviation, RSD%.) of a series of
measurements.
The confidence interval (CI) should
be reported for each type of precision
investigated.
PRECISION
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April 2007
Repeatability (of any process)
Measured mean
REPEATABILITY
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April 2007
Repeatability expresses the
precision (spread of the data,
variability) under the same
operating conditions over a short
interval of time. Repeatability is
also termed intra-assay precision.
Repeatability (hypothetical figures)
Injection
Peak area
Imp1
1
57935
0.301
2
57833
0.301
3
57497
0.299
4
57617
0.300
5
57778
0.301
6
57231
0.298
Mean
57649
0.300
STD
257
0.0013
RSD
0.4%
0.4%
270
0.0014
95% CI
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April 2007
The repeatability precision
obtained by one analyst in
one laboratory was 1.25%
RSD for the analyte and,
therefore, meets the
evaluation criterion of RSD
≤2%.
Intermediate Precision and Reproducibility (of any process)
Measured means
Intermediate precision expresses
within-laboratories variations. #1, #2
and #3: different days, different
analysts, different (manufacturing)
equipment, etc.
Reproducibility expresses the
precision between laboratories #1, #2
and #3 (collaborative studies, usually
applied to standardization of
methodology). (Transfer of
technology)
Intermediate precision or
Reproducibility
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April 2007
Intermediate precision (ruggedness)
Sample
1
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Assay (mg/5ml)
51.7
52.6
2
3
4
51.9
53.0
52.5
52.1
52.3
52.9
5
6
Mean
52.3
52.7
52.4
53.2
53.1
52.7
STD
RSD
95% CI
0.49
0.9%
0.51
0.44
0.8%
0.46
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April 2007
Combined values
Mean
STD
RSD
95% CI
52.5
0.48
0.9%
0.31
Specificity (selectivity)
 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 and excipients.
 An example of specificity criterion for an assay method is
that the analyte peak will have baseline chromatographic
resolution of at least 2.0 minutes from all other sample
components
 Stability indicating analytical methods should always be
specific.
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April 2007
Identification – a special case
 Diethylene glycol (DEG) in paediatric dosage forms has been
implicated as the causative agent in numerous deaths since 1937.
The victims were mainly children.
 Illustrative analytical issues of investigation
– IR identity test was able to detect DEG at about 20 %w/w
– Testing of DEG in Glycerol (and in Propylene Glycol) was recommended
with a LOD (sensitivity) of NLT 0.1 %. For detecting DEG at low levels, GC
seemed preferable.
– The assay was the most relevant test (accurate within ± 0.2%)
 Illustrative regulatory issues
– Legislation
– GMP
 Specificity is an essential but not sufficient characteristic of
identification
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April 2007
Specificity (hypothetical figures and data)
HPLC chromatograms of (a) API reference
standard, (b) FPP and (c) placebo
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April 2007
SPECIFICITY – degradants
Stress
A (%) *
Initial
100.0
Acid
99.3
Peroxide
99.8
All others
100.0
Purity angle
0.040
0.105
0.725
0.045
0.120
1.040
0.060
0.110
0.690
NA
Purity threshold
0.280
0.380
1.630
0.280
0.410
1.610
0.270
0.360
1.250
NA
There were no peaks in the placebo chromatogram at the retention times of nevirapine (N),
methylparaben (MP) and propylparaben (PP) peaks.
*Sum of N, MP and PP peak areas. The three ingredients can be assessed in the presence of (nonexpected) degradants. The peaks are homogeneous and pure. The method is selective, specific
and stability-indicating.
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April 2007
LOD, LOQ and SNR
 Limit of Quantitation (LOQ)
 Limit of Detection (LOD)
 Signal to Noise Ratio (SNR)
Peak A
LOD
Baseline
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noise
April 2007
Peak B
LOQ
LOD and LOQ (hypothetical figures)
Impurity 1
Injection
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LOD
Impurity 2
LOQ
LOD
LOQ
1
4176
7235
3497
7892
2
3608
8099
4258
7791
3
4196
7950
3275
8292
4
4303
8166
3464
8050
5
3932
7847
4008
8368
6
5238
8415
4702
8284
Mean
4242
7952
3867
8113
STD
548
402
551
238
RSD
12.9%
5.1%
14.3%
2.9%
Conc. (μg/ml)
0.086
0.171
0.107
0.214
Conc. (%w/w)
0.017
0.033
0.019
0.039
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April 2007
LOD and LOQ
 The limit of detection (LOD) is defined as the lowest concentration of an
analyte in a sample that can be detected, not quantified. It is expressed as a
concentration at a specified signal : noise ratio (SNR), usually between 3
and 2 : 1.
 In this study, the LOD was determined to be 0.086 μg/ml (Impurity 1) with a
signal : noise ratio of 3.6 : 1
 The limit of quantitation (LOQ) is defined as the lowest concentration of an
analyte in a sample that can be determined with acceptable precision and
accuracy under the stated operational conditions of the method. The ICH
has recommended a signal : noise ratio (SNR) of 10:1.
 The LOQ was 0.171 μg/ml (Impurity 1) with a signal:noise ratio of 11.3. The
RSD for six injections of the LOQ solution was ≤2%.
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April 2007
Linearity
Measured
mean
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Slide 33 of 43
Real
mean
April 2007
Precision
Linearity expresses differences
in precision at different points of
a given range.
„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.”
Linearity and range
5000
y = 36.124x - 7.2984
Assay mean
4000
2
R = 0.9998
3000
2000
1000
0
0
20
40
60
80
100
120
140
Concentration, %
Acceptance criterion: correlation coefficient should not be less than 0.9990
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April 2007
Linearity and range
 Concentration range 1.0–1.3 mg/ml (10–130% of the theoretical
concentration in the test preparation, n=3)
 Regression equation was found by plotting the means of peak area
(y) against the analyte concentration (x) expressed in %:
y = 36.124x - 7.2984 (R2 = 0.9998).
 The regression coefficient demonstrates an excellent relationship
between peak area and concentration of analyte.
 The analyte response is linear across 10-130% of the target
nevirapine concentration.
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April 2007
Range (minimum requirements)
 Assay of an API or a FPP: ± 20% of the test concentration.
 Content uniformity: ± 30% of the test concentration (unless a wider
more appropriate range, based on the nature of the dosage form (e.g., metered
dose inhalers), is justified).
 Dissolution testing: ± 20 % over the specified range.
 Impurity: from the reporting level of an impurity to 120% of the
specification. (Unusually potent or toxic impurities, LOD and LOQ should be
commensurate with ICH requirement.)
 If assay and purity are performed together as one test and only a
100% standard is used, linearity should cover the range from the
reporting level of the impurities to 120% of the assay specification
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April 2007
Stability of analytical solution
Stability Measured means
Stability (of the analytical solution)
expresses variation of the
measured mean as a function of
time.
#1 … First measurements
#2, #3, #4, …n Series of
measurements of the same sample
within a relatively short period of time.
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April 2007
Stability of test analytical solution
Time in
hours
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Impurity-1
Area
Difference
0
72079
1
71574
0.7%
2
71740
0.5%
3
71960
0.2%
4
72352
-0.4%
5
71573
0.7%
10
72322
-0.3%
15
72310
20
72312
25
72670
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April 2007
An analytical solution prepared
from Nevirapine 50mg/5ml Oral
Suspension was spiked with
Impurity-1 at specification level
and stored in a capped volumetric
flask on a laboratory bench at
uncontrolled room temperature
under normal lighting conditions
for 25 hours.
Conclusion: the stability of the
-0.3%
analytical solution of Impurity-1 is
-0.3%
not a source of variation.
-0.8%
Sensitivity and robustness
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April 2007
Robustness
Method parameter
STP
Flow
Wavelength
Variation of mobile phase
Column temperature
pH
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April 2007
Variation
-10%
10%
-5nm
+5nm
-2%
+2%
-5oC
+5oC
-0.3
+0.3
tR
Impurity 1
0.83
0.83
0.84
0.82
0.83
0.80
0.84
0.82
0.83
0.83
0.83
Impurity 2
1.80
1.81
1.82
1.81
1.81
1.89
1.76
1.80
1.81
1.81
1.80
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)], and
• 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)
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April 2007
Main Points Again
 Analytical procedures play a critical role in
pharmaceutical equivalence and risk assessment /
management:
– establishment of product-specific acceptance criteria, and
– stability of APIs and FPPs.
 Validation should demonstrate that the analytical
procedure is suitable for its intented purpose.
 HPLC systems and method validation deserves special
attention during the assessment of dossiers for
prequalification.
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April 2007
THANK YOU
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April 2007