1. World Health Organization
Pharmaceutical Development
2 June, 2018
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

2. Pharmaceutical Development
World Health Organization
2 June, 2018
Pharmaceutical Development
Analytical Method Development
Presenter: János Pogány, pharmacist, PhD
WHO expert

3. Analytical Method Development
World Health Organization
2 June, 2018
Analytical Method Development
Outline and Objectives of presentation
Introduction, guidelines
Dossier requirements
Assay
Related substances
Other issues
Main points again

4. Introduction, guidelines
Training Workshop on Pharmaceutical Development with focus on Paediatric Formulations
Introduction, guidelines

5. Interchangeability (IC)
INTERCHANGEABILITY (IC) OF MULTISOURCE FPPs = (ESSENTIAL SIMILARITY WITH INNOVATOR FPP) =
PHARMACEUTICAL EQUIVALENCE (PE) + BIOEQUIVALENCE (BE)
IC = PE + BE

6. Pharmaceutical equivalence
World Health Organization
2 June, 2018
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)

7. 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)

8. 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).  
Validation of analytical procedures: text and methodology Q2(R1) ICH Harmonized Tripartite Guidelines, (2005)

9. World Health Organization
2 June, 2018
General requirements
Qualified and calibrated instruments
Documented methods
Reliable reference standards
Qualified analysts
Sample selection and integrity
Change control
ICH Q2(R1) provides an indication of the data which should be presented in a registration application.
All relevant data collected during validation and formulae used for calculating validation characteristics should be submitted and discussed as appropriate.
… it is important to remember that the main objective of validation of an analytical procedure is to demonstrate that the procedure is suitable for its intended purpose. …
Well-characterized reference materials, with documented purity, should be used throughout the validation study. The degree of purity necessary depends on the intended use.
In accordance with the parent document, and for the sake of clarity, this document considers the various validation characteristics in distinct sections. The arrangement of these sections reflects the process by which an analytical procedure may be developed and evaluated.
In practice, it is usually possible to design the experimental work such that the appropriate validation characteristics can be considered simultaneously to provide a sound, overall knowledge of the capabilities of the analytical procedure, for instance: specificity, linearity, range, accuracy and precision.

10. Measure of variation (spread of data)
World Health Organization
2 June, 2018
Measure of variation (spread of data)
68.26%
95.46%

11. World Health Organization
2 June, 2018
Mean (average) chart
Abnormal variation of process – special causes
USL Upper specification limit
Normal variation due to common causes
average = mean
LSL Lower specification limit
Abnormal variation of process – special causes

12. Capable process
Almost all the measurements of a stable process fall inside the specification limits
USL – LSL Cp
OoS results: .27% ppm
64 ppm ppb

13. NEVIRAPINE – Reference StandardtR
Injection
12466
6.160 1
12311
6.167 2
12432
6.166 3
12530
6.172 4
12457
6.165 5
12479
6.168 6
12446
Mean 74
0.004
STD
0.59%
0.06%
RSD
System suitability requirement:
RSD is NMT 0.85%

14. Training Workshop on Pharmaceutical Development with focus on Paediatric Formulations
Dossier requirements

15. Use of analytical methods - generics
Pharmaceutical
Clinical
At initial phase of pharmaceutical development
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
To develop a stable and reproducible formulation for the manufacture of bioequivalence, dissolution, stability and pilot-scale validation batches
To determine bioavailability in healthy volunteers
At advanced phase of pharmaceutical development
To be robust, transferable, accurate, and precise for specification setting, stability assessment, and QC release of prequalified batches
To optimize, scale-up, and transfer a stable and controlled manufacturing process for the prequalification product
To prove bioequivalence after critical variations to the prequalified dossier

16. Analytical procedure characteristics
World Health Organization
2 June, 2018
Analytical procedure characteristics
Type of characteristic
Identification
Impurities
Quantitative Limit
Assay
Accuracy - +
Precision
Specificity
Detection limit
Quantitation limit
Linearity
Range
Robustness

17. World Health Organization
2 June, 2018
Accuracy - ISO
4. 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.
The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements.
4.1. Repeatability
Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision.
4.2. Intermediate precision
Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc.
4.3. Reproducibility
Reproducibility expresses the precision between laboratories (collaborative studies, usually applied to standardization of methodology).
Source: ISO ISO : Accuracy (Trueness and Precision) of Measurement Methods and Results. ISO, Geneva, Switzerland.

18. Accuracy and precision
World Health Organization
2 June, 2018
Accuracy and precision
Inaccurate and imprecise
Precise
Accurate
Accurate and precise

19. Percent accuracy (hypothetical figures)
RSD %
Recovery %
Nevirapine, mg
LA, %
Sample
Recovered
Added
0.64
99.2
0.495
0.499 50 1
0.31
99.8
0.701
0.703 70 2
0.27
99.9
0.795
0.796 80 3
1.88
100.4
1.005
1.001
100 4
0.38
1.209
1.211
120 5
1.12
1.296
1.299
130 6
0.77
0.917
0.918
Mean
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).

20. Percent accuracy (hypothetical figures)
Red line: LA Green lines: USL and LSL

21. Precision (of any process)
World Health Organization
2 June, 2018
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

22. Repeatability (of any process)
World Health Organization
2 June, 2018
Repeatability (of any process)
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.
Measured mean
REPEATABILITY

23. Repeatability (hypothetical figures)
Imp1
Peak area
Injection
0.301
57935 1
57833 2
0.299
57497 3
0.300
57617 4
57778 5
0.298
57231 6
57649
Mean
0.0013
257
STD
0.4%
RSD
0.0014
270
95% CI
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%.

24. Intermediate Precision and Reproducibility (of any process)
World Health Organization
2 June, 2018
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

25. Intermediate precision (ruggedness)
Assay (mg/5ml)
Sample
52.6
51.7 1
52.1
51.9 2
52.3
53.0 3
52.9
52.5 4
53.2 5
53.1
52.7 6
52.4
Mean
0.44
0.49
STD
0.8%
0.9%
RSD
0.46
0.51
95% CI
Combined values
52.5
Mean
0.48
STD
0.9%
RSD
0.31
95% CI

26. 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.

27. Identification – a special case
World Health Organization
2 June, 2018
Identification – a special case
Diethylene glycol (DEG) in paediatric dosage forms has been implicated as the causative agent in numerous deaths since 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

28. Specificity (hypothetical figures and data)
HPLC chromatograms of (a) API reference standard, (b) FPP and (c) placebo

29. SPECIFICITY – degradants
Purity threshold
Purity angle
A (%) *
Stress
0.280
0.040
100.0
Initial
0.380
0.105
1.630
0.725
0.045
99.3
Acid
0.410
0.120
1.610
1.040
0.270
0.060
99.8
Peroxide
0.360
0.110
1.250
0.690 NA
All others
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 (non-expected) degradants. The peaks are homogeneous and pure. The method is selective, specific and stability-indicating.

30. World Health Organization
2 June, 2018
LOD, LOQ and SNR
Limit of Quantitation (LOQ)
Limit of Detection (LOD)
Signal to Noise Ratio (SNR)
Peak B LOQ
There are no specific criteria set for the Limit of Quantitation (LOQ) and Limit of Detection (LOD) but guidance is available from specifications and pharmacopeias.
The noise is measured by running the instrument at maximum gain with no test being processed. The ripple generated is noise due to the instrument’s electronics, etc. The peak is measured relative to this noise and the ratio is calculated. This is known as the Signal to Noise Ratio (SNR).
Generally:
LOD SNR should be greater than 2:1. Peak A is acceptable for LOD but not for quantitation;
The LOD can be calculated if the standard deviation (SD) of the response (which is standard deviation of the blank) and the slope is determined:
LOD = 3.3 x SD
slope
Similarly LOQ = 10 x SD
The LOQ SNR should generally be above 10:1. Peak B is suitable for quantitation;
Precision as a percentage relative standard deviation should be % at the limit for LOQ.
Peak A LOD
noise
Baseline

31. LOD and LOQ (hypothetical figures)
Impurity 2
Impurity 1
Injection
LOQ
LOD
7892
3497
7235
4176 1
7791
4258
8099
3608 2
8292
3275
7950
4196 3
8050
3464
8166
4303 4
8368
4008
7847
3932 5
8284
4702
8415
5238 6
8113
3867
7952
4242
Mean
238
551
402
548
STD
2.9%
14.3%
5.1%
12.9%
RSD
0.214
0.107
0.171
0.086
Conc. (μg/ml)
0.039
0.019
0.033
0.017
Conc. (%w/w)

32. 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 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 μg/ml (Impurity 1) with a signal:noise ratio of The RSD for six injections of the LOQ solution was ≤2%.

33. Linearity
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.”
Measured Real
mean mean Precision

34. World Health Organization
2 June, 2018
Linearity and range
7. 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 (see section 3) of the analytical procedure. It may be demonstrated directly on the drug substance (by dilution of a standard stock solution) and/or separate weighings of synthetic mixtures of the drug product components, using the proposed procedure. The latter aspect can be studied during investigation of the range.
Linearity should be evaluated by visual inspection of a plot of signals as a function of analyte concentration or content. If there is a linear relationship, test results should be evaluated by appropriate statistical methods, for example, by calculation of a regression line by the method of least squares. In some cases, to obtain linearity between assays and sample concentrations, the test data may need to be subjected to a mathematical transformation prior to the regression analysis. Data from the regression line itself may be helpful to provide mathematical estimates of the degree of linearity.
The correlation coefficient, y-intercept, slope of the regression line and residual sum of squares should be submitted. A plot of the data should be included. In addition, an analysis of the deviation of the actual data points from the regression line may also be helpful for evaluating linearity.
For the establishment of linearity, a minimum of 5 concentrations is recommended. Other approaches should be justified.
8. 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.
Acceptance criterion: correlation coefficient should not be less than

35. 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 = x (R2 = ).
The regression coefficient demonstrates an excellent relationship between peak area and concentration of analyte.
The analyte response is linear across % of the target nevirapine concentration.

36. 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

37. Stability of analytical solution
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.
Stability Measured means

38. Stability of test analytical solution
Impurity-1
Time in hours
Difference
Area
72079
0.7%
71574 1
0.5%
71740 2
0.2%
71960 3
-0.4%
72352 4
71573 5
-0.3%
72322 10
72310 15
72312 20
-0.8%
72670 25
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 analytical solution of Impurity-1 is not a source of variation.

39. Sensitivity and robustness

40. Robustness tR Variation Method parameter Impurity 2 Impurity 1 1.80
0.83 -
STP
1.81
-10%
Flow
1.82
0.84
10%
0.82
-5nm
Wavelength
+5nm
1.89
0.80
-2%
Variation of mobile phase
1.76
+2%
-5oC
Column temperature
+5oC
-0.3 pH
+0.3

41. 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 ≥ ; )
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)

42. 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.

43. World Health Organization
2 June, 2018
THANK YOU