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Training Workshop onPharmaceutical Development withfocus on Paediatric Formulations
Protea Hotel
Victoria Junction, Waterfront
Cape Town, South Africa
Date: 16 to 20 April 2007
Pharmaceutical Development
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Pharmaceutical Development
Analytical Method Development
Presenter: Jnos Pogny, pharmacist, PhD
pogany.janos@chello.hu
WHO expert
mailto:pogany.janos@chello.humailto:pogany.janos@chello.hu7/28/2019 Anal Meth Dev
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Analytical Method Development
Outline and Objectives of presentationIntroduction, guidelines
Dossier requirements Assay Related substances Other issues
Main points again
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Training Workshop onPharmaceutical Development
with focus on Paediatric
Formulations
Introduction, guidelines
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Interchangeability (IC)
INTERCHANGEABILITY (IC) OF MULTISOURCEFPPs = (ESSENTIAL SIMILARITY WITHINNOVATOR FPP ) =
PHARMACEUTICAL EQUIVALENCE (PE ) +BIOEQUIVALENCE (BE)
IC = PE + BE
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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 equivalenceStability equivalenceWHO-GMP (manufacturing equivalence)
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Prequalification requirements
Analytical method validation is required by WHO for theprequalification of product dossiers. Non-compendial ARV
APIs and FPPs were/are tested with methods developedby the manufacturer.
Analytical methods should be used within GMP and GLPenvironments, and must be developed using the protocolsand acceptance criteria set out in the ICH guidelinesQ2(R1)
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Guidelines used in PQP
WHO-GMP 4.11 It is of critical importance that particular attention is paid to the validation of analytical testmethods , automated systems and cleaning procedures.
Appendix 4. Analytical method validation (in WHO Expert
Committee on Specifications for Pharmaceutical Preparations. 40thReport. 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
http://whqlibdoc.who.int/trs/WHO_TRS_937_eng.pdfhttp://www.ich.org/LOB/media/MEDIA417.pdfhttp://www.ich.org/LOB/media/MEDIA417.pdfhttp://www.ich.org/LOB/media/MEDIA417.pdfhttp://www.ich.org/LOB/media/MEDIA417.pdfhttp://whqlibdoc.who.int/trs/WHO_TRS_937_eng.pdfhttp://whqlibdoc.who.int/trs/WHO_TRS_937_eng.pdfhttp://whqlibdoc.who.int/trs/WHO_TRS_937_eng.pdf7/28/2019 Anal Meth Dev
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General requirements
Qualified and calibrated instruments
Documented methods
Reliable reference standards
Qualified analysts
Sample selection and integrity
Change control
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Measure of variation (spread of data)
95.46%68.26 %
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Mean (average) chart
Normal
variation due tocommoncauses
USL Upper specification limit
LSL Lower specification limit
Abnormal variation of process
special causes
Abnormal variation of process special causes
average = mean
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Capable process Almost all the measurements of astable process fall inside thespecification limits
USL LSL 6 8 10 12
Cp 1.00 1.33 1.66 2.00
OoS results: .27%. 6 ppm
64 ppm 2 ppb
.htm16 /pmc1 /handbook/pmc/section898http://www.itl.nist.gov/div
http://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htmhttp://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htmhttp://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htmhttp://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htmhttp://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htmhttp://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htmhttp://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htmhttp://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htmhttp://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htmhttp://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htmhttp://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htmhttp://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htmhttp://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htmhttp://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htm7/28/2019 Anal Meth Dev
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NEVIRAPINE Reference Standard
AtR Injection
124666.1601
123116.1672
124326.1663
125306.1724
124576.1655
124796.1686
124466.166Mean
740.004STD
0.59%0.06%RSD
System suitability
requirement:RSD is NMT 0.85%
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Training Workshop onPharmaceutical Development
with focus on Paediatric
Formulations
Dossier requirements
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Use of analytical methods - generics
MethodsPharmaceuticalClinicalAt initial phase of pharmaceutical development
-To understand the profile of related substances and to studystability and start measuring theimpact of key product and manufacturing process
parameters on consistent FPPquality
To develop a stable and reproducibleformulation for the manufactureof bioequivalence, dissolution,stability and pilot-scalevalidation batches
To determine bioavailability inhealthy volunteers
At advanced phase of pharmaceutical developmentTo be robust , transferable , accurate ,
and precise for specificationsetting, stability assessment,and QC release of prequalified
batches
To optimize, scale-up, and transfer a
stable and controlledmanufacturing process for the prequalification product
To prove
bioequivalence after criticalvariations to the
prequalified dossier
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Analytical procedure characteristics
Type of characteristic Identification ImpuritiesQuantitative Limit Assay
Accuracy - + - +
Precision - + - +
Specificity + + + +Detection limit - - + -
Quantitation limit - + - -
Linearity - + - +
Range - + - +
Robustness + + + +
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Accuracy - ISO 5725 1-6
Accuracy
Trueness Precision(Random errors)
Systematic errorsIntra-assayvariability
Intra-laboratoryvariability
Inter-laboratoryvariability
Repeatability Intermediateprecison Reproducibility
Source: ISO. 1994. ISO 5725 1-6: Accuracy (Trueness and Precision) of Measurement Methods and Results. ISO, Geneva, Switzerland.
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Accuracy and precision
Inaccurate &
imprecise
Inaccurate butprecise
Accurate butimprecise Accurate and precisePrecise Accurate
Inaccurate and imprecise
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Percent accuracy (hypothetical figures)
The data show that the recovery of analyte in spiked samples met the evaluationcriterion for accuracy (100 2.0% across 50 130% of target concentrations).
RSD%
Recovery%
Nevirapine, mgLA, %Sample RecoveredAdded
0.6499.20.4950.499501
0.3199.80.7010.703702
0.2799.90.7950.796803
1.88100.41.0051.0011004
0.3899.81.2091.2111205
1.1299.81.2961.2991306
0.7799.80.9170.918Mean
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Percent accuracy (hypothetical figures)
96979899
100101102103104
0 1 2 3 4 5 6 7
Number of samples
% R
e c o v e r y
Red line: LA Green lines: USL and LSL
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Precision ( of any process )
The precision (VARIABILITY ) of ananalytical procedure is usuallyexpressed as the standard deviation(S), variance (S 2), or coefficient of
variation (= relative standarddeviation, RSD%.) of a series of measurements.
The confidence interval (CI) should
be reported for each type of precisioninvestigated.
Measured mean Real mean
PRECISION
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Repeatability ( of any process )
Repeatability expresses theprecision (spread of the data,variability ) under the sameoperating conditions over a shortinterval of time. Repeatability is
also termed intra-assay precision.
REPEATABILITY
Measured mean
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Repeatability (hypothetical figures)
The repeatability precisionobtained by one analyst inone laboratory was 1.25%RSD for the analyte and,
therefore, meets theevaluation criterion of RSD2%.
Imp1Peak areaInjection
0.301579351
0.301578332
0.299574973
0.3005761740.301577785
0.298572316
0.30057649Mean
0.0013257STD
0.4%0.4%RSD
0.001427095% CI
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Intermediate Precision and Reproducibility ( of any process )
Intermediate precision expresseswithin-laboratories variations. #1, #2and #3 : different days, differentanalysts, different (manufacturing ) equipment , etc.
Reproducibility expresses theprecision between laboratories #1, #2and #3 (collaborative studies, usuallyapplied to standardization of methodology). ( Transfer of
technology )
Measured means
Intermediate precision orReproducibility
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Intermediate precision (ruggedness)
Assay (mg/5ml)Sample52.651.71
52.151.92
52.353.03
52.952.54
53.252.35
53.152.76
52.752.4Mean
0.440.49STD
0.8%0.9%RSD
0.460.5195% CI
Combined values52.5Mean
0.48STD
0.9%RSD
0.3195% CI
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Specificity (selectivity)
Specificity is the ability to assess unequivocally theanalyte in the presence of components , which may beexpected to be present. Typically these might includeimpurities, degradants and excipients .
An example of specificity criterion for an assay method isthat the analyte peak will have baseline chromatographicresolution of at least 2.0 minutes from all other samplecomponents
Stability indicating analytical methods should always bespecific.
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Specificity (hypothetical figures and data)
HPLC chromatograms of (a) API referencestandard, (b) FPP and (c) placebo
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SPECIFICITY degradants
Purity thresholdPurity angleA (%) *Stress0.2800.040
100.0Initial 0.3800.1051.6300.7250.2800.045
99.3Acid 0.4100.1201.6101.0400.2700.060
99.8Peroxide 0.3600.1101.2500.690
NANA100.0All 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, specificand stability-indicating.
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Limit of Quantitation (LOQ)
Limit of Detection (LOD)
Signal to Noise Ratio (SNR)
noise
Peak ALOD
Peak B
LOQ
Baseline
LOD, LOQ and SNR
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LOD and LOQ (hypothetical figures)Impurity 2Impurity 1
Injection LOQLODLOQLOD
78923497723541761
77914258809936082
82923275795041963
80503464816643034
83684008784739325
82844702841552386
8113386779524242Mean
238551402548STD
2.9%14.3%5.1%12.9%RSD0.2140.1070.1710.086Conc. (g/ml)
0.0390.0190.0330.017Conc. (%w/w)
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LOD and LOQ
The limit of detection ( LOD) is defined as the lowest concentration of ananalyte in a sample that can be detected, not quantified. It is expressed as aconcentration at a specified signal : noise ratio (SNR) , usually between 3and 2 : 1 .
In this study, the LOD was determined to be 0.086 g/m l (Impurity 1) with asignal : noise ratio of 3.6 : 1
The limit of quantitation ( LOQ ) is defined as the lowest concentration of ananalyte in a sample that can be determined with acceptable precision andaccuracy under the stated operational conditions of the method. The ICHhas recommended a signal : noise ratio (SNR) of 10:1 .
The LOQ was 0.171 g/m l (Impurity 1) with a signal:noise ratio of 11.3 . TheRSD for six injections of the LOQ solution was 2%.
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Linearity
Linearity expresses differencesin precision at different points of a given range.The linearity of an analyticalprocedure is its ability (within agiven range) to obtain test
results, which are directlyproportional to theconcentration (amount) of analyte in the sample.
Measured Realmean mean Precision
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Linearity and range
Acceptance criterion: correlation coefficient should not be less than 0.9990
y = 36.124x - 7.2984
R 2 = 0.9998
0
1000
2000
3000
4000
5000
0 20 40 60 80 100 120 140
Concentration, %
A s s a y m e a n
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Linearity and range
Concentration range 1.0 1.3 mg/ml ( 10 130% of the theoreticalconcentration 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 ( R 2 = 0.9998 ).
The regression coefficient demonstrates an excellent relationshipbetween peak area and concentration of analyte.
The analyte response is linear across 10-130% of the target
nevirapine concentration.
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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., metereddose inhalers), is justified).
Dissolution testing : 20 % over the specified range.
Impurity : from the reporting level of an impurity to 120% of thespecification. (Unusually potent or toxic impurities, LOD and LOQ should becommensurate 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 thereporting level of the impurities to 120% of the assay specification
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Stability of analytical solution
Stability (of the analytical solution)expresses variation of themeasured 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
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Stability of test analytical solution
An analytical solution preparedfrom Nevirapine 50mg/5ml OralSuspension was spiked withImpurity-1 at specification leveland stored in a capped volumetricflask on a laboratory bench atuncontrolled room temperatureunder normal lighting conditionsfor 25 hours.
Conclusion : the stability of theanalytical solution of Impurity-1 isnot a source of variation.
Impurity-1Time in
hours DifferenceArea
720790
0.7%715741
0.5%717402
0.2%719603
-0.4%723524
0.7%715735
-0.3%7232210
-0.3%7231015-0.3%7231220
-0.8%7267025
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Sensitivity and robustness
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Methods for cleaning validation
Method for assay and related substances used in stabilitystudies 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; R 2 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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Main Points Again
Analytical procedures play a critical role inpharmaceutical equivalence and risk assessment /management : establishment of product-specific acceptance criteria , and stability of APIs and FPPs.
Validation should demonstrate that the analyticalprocedure is suitable for its intented purpose.
HPLC systems and method validation deserves specialattention during the assessment of dossiers for prequalification .
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THANK YOU